Advances in Marine Biology, Volume 59

V O L U M E F I F T Y - N I N E ADVANCES IN MARINE BIOLOGY Advances in MARINE BIOLOGY Series Editor MICHAEL LESSER...

Author: Michael Lesser

55 downloads 1006 Views 7MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

V O L U M E F I F T Y - N I N E

ADVANCES IN

MARINE BIOLOGY

Advances in MARINE BIOLOGY Series Editor

MICHAEL LESSER Department of Molecular, Cellular, and Biomedical Sciences University of New Hampshire, Durham, USA Editors Emeritus

LEE A. FUIMAN University of Texas at Austin

CRAIG M. YOUNG Oregon Institute of Marine Biology Advisory Editorial Board

ANDREW J. GOODAY Southampton Oceanography Centre

SANDRA E. SHUMWAY University of Connecticut

V O L U M E F I F T Y - N I N E

ADVANCES IN

MARINE BIOLOGY Edited by

MICHAEL LESSER Department of Molecular, Cellular, and Biomedical Sciences University of New Hampshire, Durham, USA

Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW17BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2011 Copyright # 2011 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. ISBN: 978-0-12-385536-7 ISSN: 0065-2881

For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in UK 11 12 13 14

10 9 8 7 6 5 4 3 2 1

CONTRIBUTORS TO VOLUME 59

€ttger S. Anne Bo Department of Biology, West Chester University, West Chester, PA, USA David Burritt Department of Botany, University of Otago, Dunedin, New Zealand James A. Coyer Department of Marine Benthic Ecology & Evolution, Centre for Ecological and Evolutionary Studies, University of Groningen, Centre for Life Sciences, Groningen, The Netherlands Megan Dethier Friday Harbor Labs, University of Washington, Friday Harbor, WA, USA Steven R. Dudgeon Department of Biology, California State University, Northridge, CA, USA Anneli Ehlers IFM-GEOMAR, Kiel, Germany Britas Klemens Eriksson Department of Marine Benthic Ecology & Evolution, Centre for Ecological and Evolutionary Studies, University of Groningen, Centre for Life Sciences, Groningen, The Netherlands Veijo Jormalainen Section of Ecology, Department of Biology, University of Turku, Turku, Finland Rolf Karez LLUR, Flintbek, Germany Melissa L. Kelley Department of Molecular and Biomedical Sciences and the School of Marine Sciences, University of Maine, Orono, ME, USA Inken Kruse IFM-GEOMAR, Kiel, Germany €bler Janet E. Ku Department of Biology, California State University, Northridge, CA, USA

v

vi

Contributors

Miles Lamare Department of Marine Science, University of Otago, Dunedin, New Zealand Mark Lenz IFM-GEOMAR, Kiel, Germany Kathryn Lister Department of Marine Science, University of Otago, Dunedin, New Zealand, and Department of Botany, University of Otago, Dunedin, New Zealand Markus Molis Biologische Anstalt Helgoland, Alfred-Wegener Institute for Polar and Marine Research, Marine Station Helgoland, Helgoland, Germany Annette F. Muttray Aquatic Ecosystem Protection Research Branch, Environment Canada, Burlington, ON, Canada Jeanine L. Olsen Department of Marine Benthic Ecology & Evolution, Centre for Ecological and Evolutionary Studies, University of Groningen, Centre for Life Sciences, Groningen, The Netherlands Gareth Pearson CCMAR-CIMAR, University of Algarve, Faro, Portugal Sven Rohde University of Oldenburg, Oldenburg, Germany Hendrik Schubert University Rostock, Rostock, Germany W. Kelley Thomas Hubbard Center for Genome Studies, Gregg Hall, University of New Hampshire, Durham, NH, USA Abraham E. Tucker Hubbard Center for Genome Studies, Gregg Hall, University of New Hampshire, Durham, NH, USA Rebecca J. Van Beneden Department of Molecular and Biomedical Sciences and the School of Marine Sciences, University of Maine, Orono, ME, USA Martin Wahl IFM-GEOMAR, Kiel, Germany

Contributors

vii

Charles W. Walker Department of Molecular, Cellular and Biomedical Sciences, Center for Marine Biology and Marine Biomedical Research Group, Rudman Hall, University of New Hampshire, Durham, NH, USA €m Sofia A. Wikstro AquaBiota Water Research, Stockholm, Sweden

SERIES CONTENTS FOR LAST FIFTEEN YEARS*

Volume 31, 1997. Gardner, J. P. A. Hybridization in the sea. pp. 1–78. Egloff, D. A., Fofonoff, P. W. and Onbe´, T. Reproductive behaviour of marine cladocerans. pp. 79–167. Dower, J. F., Miller, T. J. and Leggett, W. C. The role of microscale turbulence in the feeding ecology of larval fish. pp. 169–220. Brown, B. E. Adaptations of reef corals to physical environmental stress. pp. 221–299. Richardson, K. Harmful or exceptional phytoplankton blooms in the marine ecosystem. pp. 301–385. Volume 32, 1997. Vinogradov, M. E. Some problems of vertical distribution of mesoand macroplankton in the ocean. pp. 1–92. Gebruk, A. K., Galkin, S. V., Vereshchaka, A. J., Moskalev, L. I. and Southward, A. J. Ecology and biogeography of the hydrothermal vent fauna of the Mid-Atlantic Ridge. pp. 93–144. Parin, N. V., Mironov, A. N. and Nesis, K. N. Biology of the Nazca and Sala y Gomez submarine ridges, an outpost of the Indo-West Pacific fauna in the eastern Pacific Ocean: composition and distribution of the fauna, its communities and history. pp. 145–242. Nesis, K. N. Goniatid squids in the subarctic North Pacific: ecology, biogeography, niche diversity, and role in the ecosystem. pp. 243–324. Vinogradova, N. G. Zoogeography of the abyssal and hadal zones. pp. 325–387. Zezina, O. N. Biogeography of the bathyal zone. pp. 389–426. Sokolova, M. N. Trophic structure of abyssal macrobenthos. pp. 427–525. Semina, H. J. An outline of the geographical distribution of oceanic phytoplankton. pp. 527–563. Volume 33, 1998. *

The full list of contents for volumes 1–37 can be found in volume 38.

All rights reserved. xi

xii

Series Contents for Last Fifteen Years

Mauchline, J. The biology of calanoid copepods. pp. 1–660. Volume 34, 1998. Davies, M. S. and Hawkins, S. J. Mucus from marine molluscs. pp. 1–71. Joyeux, J. C. and Ward, A. B. Constraints on coastal lagoon fisheries. pp. 73–199. Jennings, S. and Kaiser, M. J. The effects of fishing on marine ecosystems. pp. 201–352. Tunnicliffe, V., McArthur, A. G. and McHugh, D. A biogeographical perspective of the deep-sea hydrothermal vent fauna. pp. 353–442. Volume 35, 1999. Creasey, S. S. and Rogers, A. D. Population genetics of bathyal and abyssal organisms. pp. 1–151. Brey, T. Growth performance and mortality in aquatic macrobenthic invertebrates. pp. 153–223. Volume 36, 1999. Shulman, G. E. and Love, R. M. The biochemical ecology of marine fishes. pp. 1–325. Volume 37, 1999. His, E., Beiras, R. and Seaman, M. N. L. The assessment of marine pollution—bioassays with bivalve embryos and larvae. pp. 1–178. Bailey, K. M., Quinn, T. J., Bentzen, P. and Grant, W. S. Population structure and dynamics of walleye pollock, Theragra chalcogramma. pp. 179–255. Volume 38, 2000. Blaxter, J. H. S. The enhancement of marine fish stocks. pp. 1–54. Bergstro¨m, B. I. The biology of Pandalus. pp. 55–245. Volume 39, 2001. Peterson, C. H. The ‘‘Exxon Valdez’’ oil spill in Alaska: acute indirect and chronic effects on the ecosystem. pp. 1–103. Johnson, W. S., Stevens, M. and Watling, L. Reproduction and development of marine peracaridans. pp. 105–260. Rodhouse, P. G., Elvidge, C. D. and Trathan, P. N. Remote sensing of the global light-fishing fleet: an analysis of interactions with oceanography, other fisheries and predators. pp. 261–303. Volume 40, 2001.

Series Contents for Last Fifteen Years

xiii

Hemmingsen, W. and MacKenzie, K. The parasite fauna of the Atlantic cod, Gadus morhua L. pp. 1–80. Kathiresan, K. and Bingham, B. L. Biology of mangroves and mangrove ecosystems. pp. 81–251. Zaccone, G., Kapoor, B. G., Fasulo, S. and Ainis, L. Structural, histochemical and functional aspects of the epidermis of fishes. pp. 253–348. Volume 41, 2001. Whitfield, M. Interactions between phytoplankton and trace metals in the ocean. pp. 1–128. Hamel, J.-F., Conand, C., Pawson, D. L. and Mercier, A. The sea cucumber Holothuria scabra (Holothuroidea: Echinodermata): its biology and exploitation as beche-de-Mer. pp. 129–223. Volume 42, 2002. Zardus, J. D. Protobranch bivalves. pp. 1–65. Mikkelsen, P. M. Shelled opisthobranchs. pp. 67–136. Reynolds, P. D. The Scaphopoda. pp. 137–236. Harasewych, M. G. Pleurotomarioidean gastropods. pp. 237–294. Volume 43, 2002. Rohde, K. Ecology and biogeography of marine parasites. pp. 1–86. Ramirez Llodra, E. Fecundity and life-history strategies in marine invertebrates. pp. 87–170. Brierley, A. S. and Thomas, D. N. Ecology of southern ocean pack ice. pp. 171–276. Hedley, J. D. and Mumby, P. J. Biological and remote sensing perspectives of pigmentation in coral reef organisms. pp. 277–317. Volume 44, 2003. Hirst, A. G., Roff, J. C. and Lampitt, R. S. A synthesis of growth rates in epipelagic invertebrate zooplankton. pp. 3–142. Boletzky, S. von. Biology of early life stages in cephalopod molluscs. pp. 143–203. Pittman, S. J. and McAlpine, C. A. Movements of marine fish and decapod crustaceans: process, theory and application. pp. 205–294. Cutts, C. J. Culture of harpacticoid copepods: potential as live feed for rearing marine fish. pp. 295–315. Volume 45, 2003.

xiv

Series Contents for Last Fifteen Years

Cumulative Taxonomic and Subject Index. Volume 46, 2003. Gooday, A. J. Benthic foraminifera (Protista) as tools in deep-water palaeoceanography: environmental influences on faunal characteristics. pp. 1–90. Subramoniam, T. and Gunamalai, V. Breeding biology of the intertidal sand crab, Emerita (Decapoda: Anomura). pp. 91–182 Coles, S. L. and Brown, B. E. Coral bleaching—capacity for acclimatization and adaptation. pp. 183–223. Dalsgaard J., St. John M., Kattner G., Mu¨ller-Navarra D. and Hagen W. Fatty acid trophic markers in the pelagic marine environment. pp. 225–340. Volume 47, 2004. Southward, A. J., Langmead, O., Hardman-Mountford, N. J., Aiken, J., Boalch, G. T., Dando, P. R., Genner, M. J., Joint, I., Kendall, M. A., Halliday, N. C., Harris, R. P., Leaper, R., Mieszkowska,N., Pingree, R. D., Richardson, A. J., Sims, D. W., Smith, T., Walne, A. W. and Hawkins, S. J. Long-term oceanographic and ecological research in the western English Channel. pp. 1–105. Queiroga, H. and Blanton, J. Interactions between behaviour and physical forcing in the control of horizontal transport of decapod crustacean larvae. pp. 107–214. Braithwaite, R. A. and McEvoy, L. A. Marine biofouling on fish farms and its remediation. pp. 215–252. Frangoulis, C., Christou, E. D. and Hecq, J. H. Comparison of marine copepod outfluxes: nature, rate, fate and role in the carbon and nitrogen cycles. pp. 253–309. Volume 48, 2005. Canfield, D. E., Kristensen, E. and Thamdrup, B. Aquatic Geomicrobiology. pp. 1–599. Volume 49, 2005. Bell, J. D., Rothlisberg, P. C., Munro, J. L., Loneragan, N. R., Nash, W. J., Ward, R. D. and Andrew, N. L. Restocking and stock enhancement of marine invertebrate fisheries. pp. 1–358. Volume 50, 2006. Lewis, J. B. Biology and ecology of the hydrocoral Millepora on coral reefs. pp. 1–55.

Series Contents for Last Fifteen Years

xv

Harborne, A. R., Mumby, P. J., Micheli, F., Perry, C. T., Dahlgren, C. P., Holmes, K. E., and Brumbaugh, D. R. The functional value of Caribbean coral reef, seagrass and mangrove habitats to ecosystem processes. pp. 57–189. Collins, M. A. and Rodhouse, P. G. K. Southern ocean cephalopods. pp. 191–265. Tarasov, V. G. EVects of shallow-water hydrothermal venting on biological communities of coastal marine ecosystems of the western Pacific. pp. 267–410. Volume 51, 2006. Elena Guijarro Garcia. The fishery for Iceland scallop (Chlamys islandica) in the Northeast Atlantic. pp. 1–55. JeVrey, M. Leis. Are larvae of demersal fishes plankton or nekton? pp. 57–141. John C. Montgomery, Andrew Jeffs, Stephen D. Simpson, Mark Meekan and Chris Tindle. Sound as an orientation cue for the pelagic larvae of reef fishes and decapod crustaceans. pp. 143–196. Carolin E. Arndt and Kerrie M. Swadling. Crustacea in Arctic and Antarctic sea ice: Distribution, diet and life history strategies. pp. 197–315. Volume 52, 2007. Leys, S. P., Mackie, G. O. and Reiswig, H. M. The Biology of Glass Sponges. pp. 1–145. Garcia E. G. The Northern Shrimp (Pandalus borealis) Offshore Fishery in the Northeast Atlantic. pp. 147–266. Fraser K. P. P. and Rogers A. D. Protein Metabolism in Marine Animals: The underlying Mechanism of Growth. pp. 267–362. Volume 53, 2008. Dustin J. Marshall and Michael J. Keough. The Evolutionary Ecology of Offspring Size in Marine Invertebrates. pp. 1–60. Kerry A. Naish, Joseph E. Taylor III, Phillip S. Levin, Thomas P. Quinn, James R. Winton, Daniel Huppert, and Ray Hilborn. An Evaluation of the Effects of Conservation and Fishery Enhancement Hatcheries on Wild Populations of Salmon. pp. 61–194. Shannon Gowans, Bernd Wu¨ rsig, and Leszek Karczmarski. The Social Structure and Strategies of Delphinids: Predictions Based on an Ecological Framework. pp. 195–294.

xvi

Series Contents for Last Fifteen Years

Volume 54, 2008. Bridget S. Green. Maternal Effects in Fish Populations. pp. 1–105. Victoria J. Wearmouth and David W. Sims. Sexual Segregation in Marine Fish, Reptiles, Birds and Mammals: Behaviour Patterns, Mechanisms and Conservation Implications. pp. 107–170. David W. Sims. Sieving a Living: A Review of the Biology, Ecology and Conservation Status of the Plankton-Feeding Basking Shark Cetorhinus Maximus. pp. 171–220. Charles H. Peterson, Kenneth W. Able, Christin Frieswyk DeJong, Michael F. Piehler, Charles A. Simenstad, and Joy B. Zedler. Practical Proxies for Tidal Marsh Ecosystem Services: Application to Injury and Restoration. pp. 221–266. Volume 55, 2008. Annie Mercier and Jean-Franc¸ois Hamel. Introduction. pp. 1–6. Annie Mercier and Jean-Franc¸ois Hamel. Gametogenesis. pp. 7–72. Annie Mercier and Jean-Franc¸ois Hamel. Spawning. pp. 73–168. Annie Mercier and Jean-Franc¸ois Hamel. Discussion. pp. 169–194. Volume 56, 2009. Philip C. Reid, Astrid C. Fischer, Emily Lewis-Brown, Michael P. Meredith, Mike Sparrow, Andreas J. Andersson, Avan Antia, Nicholas R. Bates, Ulrich Bathmann, Gregory Beaugrand, Holger Brix, Stephen Dye,Martin Edwards, Tore Furevik, Reidun Gangst, Hja´lmar Ha´tu´n, Russell R. Hopcroft, Mike Kendall, Sabine Kasten, Ralph Keeling, Corinne Le Que´re´, Fred T. Mackenzie, Gill Malin, ´ lafsson, Charlie Paull, Eric Rignot, Koji Cecilie Mauritzen, Jo´n O Shimada, Meike Vogt, Craig Wallace, Zhaomin Wang and Richard Washington. Impacts of the Oceans on Climate Change. pp. 1–150. Elvira S. Poloczanska, Colin J. Limpus and Graeme C. Hays. Vulnerability of Marine Turtles to Climate Change. pp. 151–212. Nova Mieszkowska, Martin J. Genner, Stephen J. Hawkins and David W. Sims. Effects of Climate Change and Commercial Fishing on Atlantic Cod Gadus morhua. pp. 213–274. Iain C. Field, Mark G. Meekan, Rik C. Buckworth and Corey J. A. Bradshaw. Susceptibility of Sharks, Rays and Chimaeras to Global Extinction. pp. 275–364. Milagros Penela-Arenaz, Juan Bellas and Elsa Va´zquez. Effects of the Prestige Oil Spill on the Biota of NW Spain: 5 Years of Learning. pp. 365–396.

Series Contents for Last Fifteen Years

xvii

Volume 57, 2010. GeraintA. Tarling, Natalie S. Ensor, Torsten Fregin, William P. GoodallCopestake and Peter Fretwell. An Introduction to the Biology of Northern Krill (Meganyctiphanes norvegica Sars). pp. 1–40. Tomaso Patarnello, Chiara Papetti and Lorenzo Zane. Genetics of Northern Krill (Meganyctiphanes norvegica Sars). pp. 41–58. Geraint A. Tarling. Population Dynamics of Northern Krill (Meganyctiphanes norvegica Sars). pp. 59–90. John I. Spicer and Reinhard Saborowski. Physiology and Metabolism of Northern Krill (Meganyctiphanes norvegica Sars). pp. 91–126. Katrin Schmidt. Food and Feeding in Northern Krill (Meganyctiphanes norvegica Sars). pp. 127–172. Friedrich Buchholz and Cornelia Buchholz. Growth and Moulting in Northern Krill (Meganyctiphanes norvegica Sars). pp. 173–198. Janine Cuzin-Roudy. Reproduction in Northern Krill. pp. 199–230. Edward Gaten, Konrad Wiese and Magnus L. Johnson. Laboratory-Based Observations of Behaviour in Northern Krill (Meganyctiphanes norvegica Sars). pp. 231–254. Stein Kaartvedt. Diel Vertical Migration Behaviour of the Northern Krill (Meganyctiphanes norvegica Sars). pp. 255–276. Yvan Simard and Michel Harvey. Predation on Northern Krill (Meganyctiphanes norvegica Sars). pp. 277–306. Volume 58, 2010 A. G. Glover, A. J. Gooday, D. M. Bailey, D. S. M. Billett, P. Chevaldonne, A. Cola¸co, J. Copley, D. Cuvelier, D. Desbruyeres, V. Kalogeropoulou, M. Klages, N. Lampadariou, C. Lejeusne, N. C. Mestre, G. L. J. Paterson, T. Perez, H. Ruhl, J. Sarrazin, T. Soltwedel, E. H. Soto, S. Thatje, A. Tselepides, S. Van Gaever, and A. Vanreusel. Temporal Change in Deep-Sea Benthic Ecosystems: A Review of the Evidence From Recent Time-Series Studies. pp. 1–96 Hilario Murua. The Biology and Fisheries of European Hake, Merluccius merluccius, in the North-East Atlantic. pp. 97–154 Jacopo Aguzzi and Joan B. Company. Chronobiology of Deep-Water Decapod Crustaceans on Continental Margins. pp. 155–226 Martin A. Collins, Paul Brickle, Judith Brown, and Mark Belchier. The Patagonian Toothfish: Biology, Ecology and Fishery. pp. 227–300

C H A P T E R O N E

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology Charles W. Walker*,1, Rebecca J. Van Benedeny, €ttgerx, Melissa L. Kelleyy, Annette F. Muttrayz, S. Anne Bo Abraham E. Tucker{ and W. Kelley Thomas{

Contents 1. 2. 3. 4.

Introduction p53 in Vertebrate Cancer and Stress Biology Structure of p53 Family Member Proteins in Bivalves Data Mining the Molluscan Genome for p53 Upstream Regulatory and Downstream Transcriptional Targets 5. Bivalve Molluscs as Models for Animal Cancer and Stress Biology 5.1 Haemocyte cancer in the soft shell clam and the involvement of the mitochondrial hsp70 protein (mortalin) in cytoplasmic sequestration of p53 5.2 Transcriptional induction of apoptosis by soft shell clam p53 5.3 Non-transcriptional induction of apoptosis by soft shell clam p53 5.4 Haemocyte cancer in other bivalves 5.5 Germinomas in bivalves 5.6 Environmental stressors and potential links to bivalve haemocyte and germinoma cancers 6. Concluding Remarks 6.1 Bivalves as models for human cancer and stress biology 6.2 In vitro and in vivo models for bivalve tumour cells 6.3 Use of bivalves as bioindicator species—understanding the signalling pathways involved Acknowledgements References

3 6 7 12 14 14

16 16 18 19 21 24 24 25 26 27 29

* Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH, USA y Department of Molecular and Biomedical Sciences and the School of Marine Sciences, University of Maine, Orono, ME, USA z Aquatic Ecosystem Protection Research Branch, Environment Canada, Burlington, ON, Canada x Department of Biology, West Chester University, West Chester, PA, USA { Hubbard Center for Genome Studies, Gregg Hall, University of New Hampshire, Durham, NH, USA

1

Corresponding author.

Advances in Marine Biology, Volume 59 ISSN 0065-2881, DOI 10.1016/B978-0-12-385536-7.00001-7

# 2011 Elsevier LTD. All rights reserved.

1

2

Charles W. Walker et al.

Abstract The human p53 tumour suppressor protein is inactivated in many cancers and is also a major player in apoptotic responses to cellular stress. The p53 protein and the two other members of this protein family (p63, p73) are encoded by distinct genes and their functions have been extensively documented for humans and some other vertebrates. The structure and relative expression levels for members of the p53 superfamily have also been reported for most major invertebrate taxa. The functions of homologous proteins have been investigated for only a few invertebrates (specifically, p53 in flies, nematodes and recently a sea anemone). These studies of classical model organisms all suggest that the gene family originally evolved to mediate apoptosis of damaged germ cells or to protect germ cells from genotoxic stress. Here, we have correlated data from a number of molluscan and other invertebrate sequencing projects to provide a framework for understanding p53 signalling pathways in marine bivalve cancer and stress biology. These data suggest that (a) the two identified p53 and p63/73-like proteins in soft shell clam (Mya arenaria), blue mussel (Mytilus edulis) and Northern European squid (Loligo forbesi) have identical core sequences and may be splice variants of a single gene, while some molluscs and most other invertebrates have two or more distinct genes expressing different p53 family members; (b) transcriptional activation domains (TADs) in bivalve p53 and p63/73-like protein sequences are 67–69% conserved with human p53, while those in ecdysozoan, cnidarian, placozoan and choanozoan eukaryotes are 33% conserved; (c) the Mdm2 binding site in the transcriptional activation domain is 100% conserved in all sequenced bivalve p53 proteins (e.g. Mya, Mytilus, Crassostrea and Spisula) but is not present in other non-deuterostome invertebrates; (d) an Mdm2 homologue has been cloned for Mytilus trossulus; (e) homologues for both human p53 upstream regulatory and transcriptional target genes exist in molluscan genomes (missing are ARF, CIP1 and BH3 only proteins) and (f) p53 is demonstrably involved in bivalve haemocyte and germinoma cancers. We usually do not know enough about the molecular biology of marine invertebrates to address molecular mechanisms that characterize particular diseases. Understanding the molecular basis of naturally occurring diseases in marine bivalves is a virtually unexplored aspect of toxicoproteomics and genomics and related drug discovery. Additionally, increases in coastal development and concomitant increases in aquatic pollutants have driven interest in developing models appropriate for evaluating potential hazardous compounds or conditions found in the aquatic environment. Data reviewed in this study are coupled with recent developments in our understanding the molecular biology of the marine bivalve p53 superfamily. Taken together, they suggest that both structurally and functionally, bivalve p53 family proteins are the most highly conserved members of this gene superfamily so far identified outside of higher vertebrates and invertebrate chordates. Marine bivalves provide some of the most relevant and best understood models currently available for experimental studies by biomedical and marine environmental researchers.

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

3

1. INTRODUCTION In humans, the p53 gene family includes three isoforms, p53, p63 and p73 that are located on different chromosomes (p53, chromosome 17p12.1; p63, chromosome 3q27-29; p73, chromosome 1p36.3). Distinct p53-, 63- and 73-related genes are also found in other mammalian and nonmammalian vertebrates (Le Bras etal., 2003). All identified family members produce splice variants that are uniquely expressed in different cell types, both spatially and temporally (Murray-Zmijewski et al., 2006; Khoury and Bourdon, 2010). While p53-related genes are absent from plant and fungal genomes, both p53- and p63/73-like genes have been detected throughout the non-vertebrate animal world (Wahl and Carr, 2001; Nedelcu and Tan, 2007; Pankow and Bamberger, 2007; Rutkowski etal., 2010). Distinct p53 and p63/73-like family members can be identified in completed genome projects for invertebrate deuterostomes, lophotrochozoan, ecdysozoan, cnidarian, placozoan and choanozoan eukaryotes, some of which include splice variants (Fig. 1.1). Comparative evidence from available eukaryotic genome projects suggests that vertebrate p53, p63 and p73 genes form a monophylogenetic group distinct from invertebrate p53 superfamily members (Nedelcu and Tan, 2007; Fernandes and Atchley, 2008; Rutkowski et al., 2010). These studies also indicate that an ancient p63-like gene was ancestral to all p53- and p73-related genes in the major lines of animal evolution and existed from 500 million to 1 billion years ago (Belyi et al., 2010). This ancestral p63 gene developed in the germ line and subsequently gave rise to the alternate forms of genes in the p53 superfamily now found in both germ and somatic cells of widely separated phylogenetic taxa (Lu and Abrams, 2006; Nedelcu and Tan, 2007; Belyi et al., 2010; Dotsch et al., 2010; Rutkowski et al., 2010). Functional data for several nematodes and flies indicate that a p53-related protein maintains genomic stability after genotoxic challenge and prevents germ cell proliferation upon induced expression (Akdemir et al., 2007; Belyi et al., 2010). Additionally, p63 protects the mammalian female germ line during meiotic arrest (Suh et al., 2006). Similar results have been obtained for a p63/73-like protein in the sea anemone Nematostella vectensis that is involved in selective germ cell death (Akdemir et al., 2007; Pankow and Bamberger, 2007). With the exception of the sea anemone, genes in the cellular senescence, DNA editing and repair pathways governed by the transcriptional activation domain (TAD) are not present in these organisms, perhaps because their adult somatic cells do not divide by mitosis and their TAD is only 2% (Drosophila) and 33% (Caenorhabditis) conserved respectively relative to human p53. In the vertebrates, transcriptional target genes for the p53 protein are focused in four areas: down-regulation of p53, cellular senescence, DNA editing/repair and apoptosis. These functions are important in both

4

Charles W. Walker et al.

[(Figure_1)TD$IG]

Figure 1.1 Phylogenetic tree of the p53 superfamily. Sequences corresponding to the conserved DNA binding domain were used to generate an Unrooted Phylogenetic Tree using the Splits Tree 4 program (Huson and Bryant, 2006). Abbreviations used are Ag, Anopheles gambiae; Ap, Acyrthosiphon pisum; Bf, Branchiostoma floridae (Amphioxus); Cbr, Caenorhabditis brenneri; Cbri, Caenorhabditis briggsae; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Co, Capsaspora owczarzaki; Cp, Capitella sp.; Cq, Culex quinquefasciatus; Cre, Caenorahbditis remanei; Cs, Ciona savignyi; Dp, Daphnia pulex; Dm, Drosophila melanogaster; Dr, Danio rerio (Zebra fish); Hr, Helobdella robusta; Hs, Homo sapiens; Lf, Loligo forbesi; Lg, Lottia gigantea; Ma, Mya arenaria; Mb, Monosiga brevicollis; Me, Mytilus edulis; Nav, Nasonia vitripenes; Nv, Nematostella vectensis; Ph, Pediculus humanus; Pp, Pristionchus pacificus (Elephant shark); Le, Leuconraja erinacea (Little skate); Sp, Strongylocentrotus purpuratus; Ta, Trichoplax sp.; Tc, Tribolium castaneum; Tp, Trichonella sp.; TET, tetramerization domain; SAM, sterile alpha motif; TS, tetramerization domain and SAM domains. Reprinted from Rutkowski et al. (2010) with permission from CSH Press. (See Plate no.1 in the Color Plate Section).

vertebrate cancer and stress biology, but the roles of homologous p53 pathways in invertebrates have scarcely been investigated. Data mining molluscan genomes indicates that (1) conserved genes corresponding to human p53 upstream regulatory and downstream transcriptionally regulated cancer and stress pathways can be identified and (2) these genes are homologues for functional components in all four of the p53-related vertebrate transcriptional outcomes.

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

5

Despite the wide taxonomic distribution of p53-like family member genes in both vertebrate and invertebrate animals, virtually nothing is known about the activity of proteins resulting from the transcriptional activity of these genes in most invertebrates, especially those marine species for which sequences have recently become available. Expression data are available for several marine bivalves demonstrating correlations between mRNA levels and fluctuations in environmental parameters or experimental treatments ( Jessen-Eller et al., 2002; Butler et al., 2004; Muttray etal., 2008; Holbrook etal., 2009). These studies do not provide an understanding of what p53-related proteins are actually doing as a result of these changes. Functions of p53 proteins have been more thoroughly investigated in the soft shell clam Mya arenaria in cancerous clam haemocytes (CCH) (Kelley et al., 2001; Walker et al., 2006; B€ ottger et al., 2008; Walker and B€ ottger, 2008) and in germinomas (Van Beneden etal., 1997; Olberding et al., 2004). In earlier literature, CCH have also been described as clam leukaemia or systemic disseminated neoplasia (Barber, 2004); germinomas have been called gonadal neoplasias or gonadal cancers. The former disease is characterized by cytoplasmic sequestration and inactivation of clam p53 protein mediated by over-expression of the clam mitochondrial hsp70 protein, mortalin (Kelley et al., 2001; Walker et al., 2006; B€ ottger etal., 2008; Walker and B€ ottger, 2008). The germinoma is characterized by lower levels of p53 protein in gonadal tissues relative to non-tumour bearing gonads (Olberding etal., 2004). With the exception of CCH and clam germinoma, no other invertebrate models have been shown to involve p53 family members in normal or abnormal adult somatic cell functions related to cancer. Little molecular evidence regarding stress biology and relationships to human biology is available in relation to the function of p53 superfamily members in bivalves. Experimental results for M. arenaria reflect those in the human cancers undifferentiated neuroblastoma and colorectal adenocarcinoma and suggest that apoptosis can be induced both transcriptionally (by nuclear-directed p53) and non-transcriptionally (by mitochondriadirected p53) following treatment with agents that generate genotoxic stress (DNA-damage inducing) and non-genotoxically by competing with p53 for mortalin binding (Kelley et al., 2001; Walker et al., 2006, 2009; B€ ottger et al., 2008; Walker and B€ ottger, 2008). While we are at an early stage in our understanding of the function of the p53 gene superfamily in bivalve cancer and stress biology in particular, even less information exists for invertebrates in general. In this review, we combine the results of gene-specific cDNA and genomic cloning of p53 family genes from the bivalves, M. arenaria (the soft shell or longneck clam) and Mytilusedulis (the blue mussel) with information obtained from data mining to provide a framework for understanding p53 pathways in marine bivalves. The major focus of this review is the p53 gene family and

6

Charles W. Walker et al.

its structure, function and involvement in the cancer and stress biology of marine bivalves and the importance of these organisms as models for human cancer and environmental stress biology. In particular, we emphasize human health and disease-related issues and the highly neglected disciplines of marine bivalve cancer and environmental stressors.

2. P53 IN VERTEBRATE CANCER AND STRESS BIOLOGY Function of the p53 protein was originally thought to rely exclusively on its action as a transcription factor. Many transcriptional targets for p53 have been identified that yield proteins involved in downregulation of p53, cellular senescence, DNA editing/repair and apoptosis (Horvath et al., 2007; Jegga et al., 2008; Riley et al., 2008). In approximately 60% of human cancers, transcription of p53 target sequences is perturbed by mutations in the DNA binding domain and can result in disregulation of all four of these transcriptional outcomes (Yee and Vousden, 2005). Importantly, the apoptotic function is retained when mutant p53 proteins lacking the transcriptional activation domain are employed (Vaseva and Moll, 2008). Efforts to account for this result have revealed an additional non-transcriptional function involving a cytoplasmic pool of wild-type p53 that can also participate in a mitochondrial pathway driving apoptosis (Talos et al., 2005). This pathway is normally induced by severe stress (Moll and Zaika, 2001; Vaseva and Moll, 2008) and initially results in translocation of wild-type p53 to the mitochondria where it inactivates multi-domain antiapoptotic Bcl-2 proteins resulting in apoptosis (Tomita et al., 2006). Variations in mutational levels and differential expression, phosphorylation and acetylation of p53 are among the factors responsible for p53 malfunction (Greenblatt et al., 1994; Shieh et al., 1997). Although cytoplasmic sequestration has not yet been recorded for human leukaemias or lymphomas, a subset of naturally occurring human cancers do retain p53 in the cytoplasm (e.g. undifferentiated neuroblastoma, breast, retinoblastoma (Rb), colorectal adenocarcinoma and glioblastoma cancers) and this phenotype can be induced in mouse NIH 3T3 and human HeLa cells (Wadhwa et al., 2002). In all of these cases, wild-type p53 is inactivated because it is retained in the cytoplasm. A variety of molecular mechanisms have been linked to cytoplasmic sequestration in these human cancers, including truncation of the nuclear localization motif receptor protein importin a (Kim et al., 2000), overactive nuclear export by an Mdm2dependent pathway (Rodriguez-Lopez et al., 2001) and cytoplasmic tethering by foreign viral (Zhao and Liao, 2003) or local cytoplasmic proteins (Wadhwa et al., 1998; Nikolaev et al., 2003; Kaul et al., 2007). In human undifferentiated neuroblastoma and colorectal adenocarcinoma cells,

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

7

mortalin protein is responsible for cytoplasmic tethering when the latter protein is over-expressed (Dundas et al., 2005; Walker et al., 2006). Dundas et al. (2005) also indicate that high levels of mortalin expression are correlated with poor clinical outcome for colorectal cancer. A recent study of primary and secondary glioblastomas suggests that mortalin and possibly other tethering molecules (e.g. cullin 7 or PARC) may also be responsible for cytoplasmic sequestration in these cancers (Nikolaev et al., 2003; Nagpal et al., 2006). The effects of environmental stressors on the biology of human cancer are addressed in a number of studies (Meisner et al., 1993; Nawrot et al., 2006; Soliman et al., 2007; Kampa and Castas, 2008). We will emphasize those that have parallels in marine bivalve haemocyte and germinoma cancers.

3. STRUCTURE OF P53 FAMILY MEMBER PROTEINS IN BIVALVES Approximately 17 kb of M. arenaria genomic DNA indicate that both soft shell clam p53 and p63/73 proteins are encoded by a single gene (GenBank Accession number FJ041332). Clam p53 (earlier called Map53 by Kelley et al., 2001) consists of 10 exons and clam p63/73 (earlier called Map73 by Kelley etal., 2001) is comprised of 12 exons. Of these, the first nine exons encode the core sequence for both proteins (Fig. 1.2a and b). The molecular organization of the clam gene is similar to its vertebrate counterparts: (a) exon 1 is non-coding with the translation initiation site contained within exon 2; (b) the presence of a relatively large intron at the 50 end of the gene, located between exons 3 and 4 and (c) intron/exon boundaries are similar between clam p63/73 and other p53 gene family members. The distinction between p53- and p63/73-like sequences is based on the presence of a SAM (sterile alpha motif) domain in the carboxy terminus of the latter (Thanos and Bowie, 1999; Nedelcu and Tan, 2007). In at least three molluscs, cDNAs for both proteins have been cloned and core sequence for the predicted p53- and p63/73-like proteins is virtually identical (Fig. 1.2b). In all three of these cases, the core sequence includes the following highly conserved regions: the transcriptional activation domain (the clam TAD is 67% conserved with human p53; it is only 42% conserved with human p73 and only 33% conserved with human p63), Mdm2 binding site (including all essential Mdm2 binding residues—F, L, W, L), ATM phosphorylation site (S15), proline-rich domain, DNA binding domains (DBDs II–V), nuclear import (3 NLSs) and export (1 NES) signals and the tetramerization domain. In the soft shell clam, the core sequence is a structural mosaic of the corresponding human proteins, with the TAD closely resembling human p53

8

Charles W. Walker et al.

[(Figure_2)TD$IG]

Figure 1.2 (a) Genomic structure of p53/63/73 in M. arenaria. Boxes represent exons and V indicates intron regions (not drawn to scale). The 50 -UTR is grey, the core sequence for both clam p53 and p63/73 proteins is green, the p53 extension is blue and the p63/73 extension is red. The yellow arrow denotes the location of a predicted DN isoform; (b) amino acid sequences for clam p53 and p63/73 with intron/exon boundaries marked (!, clam p53; r, human p53). Functional domains are indicated by horizontal lines above the sequence. Conserved amino acids implicated in Mdm2 binding are underlined. Sequence highlighted in yellow denotes the predicted DN isoform. Genomic DNA was purified from flash-frozen gill tissue of M. arenaria as previously described (Barker et al., 1997). DNA template was prepared using the Universal Genome Walker Kit according to the manufacturer’s instructions (Clontech). Clam sequence-specific PCR primers were designed based on the clam p53 and p63/73 coding sequences (GenBank Accession numbers AF253323 and AF253324, respectively) and used in conjunction with adapter primers (Clontech). Amplified products were cloned into the pCRII vector (Invitrogen) and sequenced in both directions at the University of Maine DNA Sequencing Facility. Contiguous sequences were assembled using Sequencher version 4.1.2 software (Gene Codes Corp., Ann Arbor, MI) and the open reading frames identified; sequence for genomic DNA was deposited under GenBank Accession number FJ041332 and (c) sequence for M. edulis DNp63/73 (DQ865151.1) was aligned to FJ041332 using Spidey (http://www.ncbi.nlm.nih.gov/spidey/) to elucidate a sequence and intron/exon boundaries for a predicted DN isoform in M. arenaria, and was found to be 87% identical to M. edulis at the nucleic acid level and 100% at the protein level and located in intron 3. (See Plate no.2 in the Color Plate Section).

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

9

(>67%) and the DBDs resembling human p73 (75–88%). Clam proteins have either short (p53) or long (p63/73) C-terminal extensions. Clam genomic DNA sequence demonstrates that unlike humans, where p53, p63 and p73 proteins are generated from distinct genes on different chromosomes, clam p53 and p63/73 mRNAs result from splice variants of a single gene. M. edulis and Loligoforbesi p53- and p63/73-like proteins also have identical core sequences and may be splice variants of one gene as in M. arenaria (Fig. 1.2). However, Mytilus trossulus has distinct genes that code for unique p53 and p63/73-like sequences (Muttray etal., 2008; Vassilenko et al., 2010). In vertebrates the DeltaNotchp63/73-like (DN) splice variant of p73 is commonly expressed. The distinction between p73 and DeltaNotch p73-like (DN) sequences is based on the replacement of the long TA domain with a truncated ‘DN’ domain (Ishimoto et al., 2002). Among invertebrates, only M. trossulus and M. edulis have been found to express DNp63/73-like mRNAs (Muttray et al., 2007). Unfortunately, the lack of genomic p53 sequence data from Mytilus sp. limits our understanding of how this DNp63/73 isoform may be spliced from the gene. However, given the overall sequence homology between M. arenaria and Mytilus p63/73 isoforms, some inferences can be made by aligning the M. arenaria genomic p53 sequence with the Mytilus DNp63/73 mRNA sequence. It appears that the nucleotide sequence encoding the truncated DN domain from Mytilus is present in the long intron located between exons 3 and 4 of the M. arenaria genomic sequence and is 87% identical between Mya and Mytilus (Fig. 1.2c). It is therefore likely that an additional exon encoding a DN isoform may also exist in Mya and that the DN isoform could be expressed under conditions that have not been explored by isoform cloning. Expression levels for the DN isoform are generally low in normal mussel haemocytes and variable in cancerous mussel haemocytes (Muttray etal., 2010). The function of the DN isoform in mussels is unknown. Four residues (F19, L22, W23 and L26) that are critical for hydrophobic binding interactions with Mdm2 are located in the TAD of human p53 (Kussie et al., 1996; Dobbelstein and Roth, 1998; Holbrook et al., 2009). Protein modeling predicted homologous Mdm2 binding sites in Mya/Mytilus p53 (Kelley etal., 2001; Muttray etal., 2005; Holbrook etal., 2009) (Figs. 1.3 and 1.4). These residues are also present in p53 sequences of the Pacific oyster Crassostrea gigas (Farcy et al., 2008) and the surf clam Spisula solidissima (Cox et al., 2003). In molluscs, the putative Mdm2 binding site is identical for both p53- and p63/73-like proteins. An Mdm2 homologue has also been cloned from the mussel, M. trossulus (GenBank Accession number ADF80285) and shown to bind p53 protein although no functional data are available (Muttray et al., 2010). While Mdm2 homologues have also been identified from the genomes of the

10

Charles W. Walker et al.

[(Figure_3)TD$IG]

Figure 1.3 Structural analysis of M. arenaria p53 shows conservation of a putative Mdm2 binding site. Representation of a portion of the human Mdm2 protein (ribbon structure) bound to the minimal binding site of the human p53 protein (stick structure). Modified from Kussie et al. (1996) using RasMol software. Four residues critical for hydrophobic binding interactions (F19, L22, W23 and L26, shown in green) are conserved between human p53 and clam p53. Two potentially important changes are the substitutions in M. arenaria p53 of glutamic acid and tyrosine, for S20 (red) and D21 (blue) in human p53, respectively (Holbrook et al., 2009) (See Plate no.3 in the Color Plate Section).

placozoan,Trichoplaxadhaerens and the northern deer tick, Ixodesscapularis, p53 in these species does not have a conserved Mdm2 binding site (Lane et al., 2010a,b) (Figs. 1.3 and 1.4). Given the conservation of key domains in human and clam p53 proteins (Kelley et al., 2001), the hypothesis that the two proteins function in a similar manner was addressed experimentally (Holbrook et al., 2009). Plasmids expressing either human p53 or M. arenaria p53 were introduced by transient transfection into the p53-null H1299 cell line derived from human liver. Functionality was assessed by monitoring the p53/mdm2 feedback loop and expression of p53-mediated downstream markers of growth arrest (e.g. CIP1) under non-stressed conditions. Expression of human p53 spontaneously induced expression of the cyclin/CDK binding protein CIP1 while clam p53 expression did not (Fig. 1.5). Expression of M. arenaria p53 transfected into the human p53-null H1299 cell line induced expression of human Mdm2 to approximately

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

11

[(Figure_4)TD$IG]

Figure 1.4 Species tree comparing p53 family members in sequenced genomes for invertebrates and humans (Homo sapiens). Comparisons include from left to right (a) percent similarity of amino acids in the TADs of invertebrate p53 genes compared to human p53 TAD, (b) presence or absence of Mdm2 binding site homologous with that of human p53, (c) number and nature of p53 family member genes present; (d) existence or absence of core sequence and (e) taxonomy. Human and M. arenaria p53-like sequences were used to retrieve homologous gene models from the following genome projects located at the JGI Eukaryotic Genomics Web site (http://genome.jgipsf.org/euk_cur1.html for Capitella sp.; D. pulex; C. intestinalis; B. floridae; M. brevicollis; Trichoplax adhaerans and N. vectensis) and at WormBase (http://www.wormbase.org/ db/searches/blast_blat) for C. elegans; at Flybase (http://flybase.org/blast/) for D. melanogaster and at (http://www.spbase.org/SpBase/wwwblast/blast.php) for S. purpuratus. Protein alignment was determined with ClustalX software. The species tree was constructed using PhyloWidget (Jordan and Piel, 2008) based on topology of Dunn et al. (2008).

the same level as did human p53, suggesting that Mya p53 is also able to participate in an Mdm2 feedback loop. Conversely, the addition of human Mdm2 RNAi to H1299 cells induced the expression of human p53 and Mya p53 (Holbrook et al., 2009). The M. trossulus Mdm2 protein also binds Mytilus p53 as shown in two independent assays, the yeast twohybrid assay and immunoprecipitation (Muttray etal., 2010). An obvious limitation of this in vitro assay is that it was done at 37 C, much higher than ambient temperatures typically experienced by the clam, which may have a significant effect on protein folding and protein/protein interactions. Taken together, these data suggest that the M. arenaria and Mytilus p53 proteins share some functional similarity with human p53 as well as with other invertebrates, positioning the molluscs at a critical juncture in evolution of this gene family.

12

Charles W. Walker et al.

[(Figure_5)TD$IG]

Figure 1.5 Human p53 elicited expression of CIP1in p53-null H1299 cells, but M. arenaria p53 did not. Representative Western blot and summary densitometry data show that human p53 expressed in H1299 cells (black bars) elicited a robust CIP1 response that remains elevated over the 48-h observation period. Neither M. arenaria p53 (grey bars) nor vector alone (pcDNA3.1/His, Invitrogen; white bars) altered CIP1 expression. There are no significant differences in CIP1 expression over time within any treatment group (P > 0.05; N = 3). Values were pooled for betweentreatment comparisons; different letters denote significant differences; M, marker lane (after Holbrook et al., 2009).

4. DATA MINING THE MOLLUSCAN GENOME FOR P53 UPSTREAM REGULATORY AND DOWNSTREAM TRANSCRIPTIONAL TARGETS The study by Nedelcu and Tan (2007) compares the DNA binding domains of invertebrate p53 family members, but does not consider the TADs or p53-related regulatory or transcriptional targets for these species. Since the TAD of soft shell clams and mussels is 67–69% conserved with that of humans it is possible that clam p53- and p63/73-like proteins may share transcriptional activating functions with corresponding human proteins (Fig. 1.4). Considerable experimental evidence supports this hypothesis for the soft shell clam and will be discussed below (Kelley et al., 2001; Walker et al., 2006; B€ ottger et al., 2008; Walker and B€ ottger, 2008; Holbrook et al., 2009). Three genome sequencing projects have been completed for molluscs (for the archeogastropod Lottia gigantea; http://genome.jgi-psf.org/ Lotgi1/Lotgi1.home.html; for the sea hare, Aplysia californica: http:// genome.ucsc.edu/cgi-bin/hgGateway?hgsid +155184841&clade=other&org=Sea+hare&db=0 and very recently for the oyster, C.gigas, by the Oyster Genome Sequence Map Project, China, unpublished). Undoubtedly, completion of bivalve genomes and particularly functional studies of p53-related proteins will be of great advantage

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

13

in developing a more thorough understanding of the biology the p53 family of genes in cancer and stress biology. In this chapter, our analysis of genes in the limpet (Lottia gigantea) genome that are either upstream regulators or are transcriptional target genes for molluscan p53 provides important information critical for designing experiments to determine the extent of functional similarity between bivalve and human p53related proteins in their respective normal or cancerous cytoplasmic and nuclear environments (Fig. 1.6). Upstream regulatory oncogene and stress-induced genes important in human biology that are also present in the molluscan genome (Fig. 1.6) include Rb and E2F1 and ATM, ATR, ras and Chk2. Certain genes are missing (e.g. ARF, alternate reading frame), although HES1 and HEY1

[(Figure_6)TD$IG]

Figure 1.6 Comparison of genes present in H. sapiens and genomic DNA for L. gigantea p53 upstream regulatory and downstream transcriptionally regulated cellular senescence, DNA editing and repair and apoptosis pathways. Lottia genome searches were performed using BLAST for human sequences for p53 regulatory and transcriptional target genes from the following genome projects located at the JGI Eukaryotic Genomics Web site (http://genome.jgi-psf.org/euk_cur1.html for Capitella sp.; D. pulex; C. intestinalis; B. floridae; M. brevicollis; T. adhaerans and N. vectensis) and at WormBase (http://www.wormbase.org/db/searches/blast_blat) for C. elegans; at Flybase (http://flybase.org/blast/) for D. melanogaster and at (http://www.spbase.org/SpBase/wwwblast/blast.php) for S. purpuratus. Protein alignment was determined with ClustalX software.

14

Charles W. Walker et al.

that behave in an analogous manner by blocking Mdm2 are highly conserved (Huang et al., 2004). The ubiquitin ligases, Mdm2, COP1 and Pirh2 are all present in the molluscan genome as is the ubiquitin-specific peptidase HAUSP. Unrelated p53 binding proteins such as mortalin, PARC, cullin 7 and Bcl-xl also occur. Genes are also found in the molluscan genome that are transcriptional targets for p53 in humans and are associated with outcomes in cellular senescence (e.g. CDKN1A, SFN, TP53I3, CDC25C), DNA editing and repair (e.g. DDB2, DDIT4, GADD45a, TRIM22, ribonucleotide reductase) and apoptosis (e.g. FAS, Bcl-2, Bcl-xl, Mcl-1, BAX, BAK, procaspases, 3, 7 and 9, APAF1, ICAD and CAD). Some genes important for mammals in regulation (e.g. CIP1) or apoptosis (e.g. all BH3-only proteins) are absent from the molluscan genome (Fig. 1.6). The inability of clam p53 to up-regulate CIP1 in the H1299 cell line described above (Holbrook etal., 2009) might be related to the absence of CIP1 from the molluscan genome (as is also the case for all non-chordate genomes). Clam p53 protein expressed in human cells might not recognize the promoter for the human CIP1 gene and therefore cannot initiate its expression.

5. BIVALVE MOLLUSCS AS MODELS FOR ANIMAL CANCER AND STRESS BIOLOGY 5.1. Haemocyte cancer in the soft shell clam and the involvement of the mitochondrial hsp70 protein (mortalin) in cytoplasmic sequestration of p53 The soft shell clam, M. arenaria, naturally develops a diffuse cancer of its haemocytes at high incidence (1–20% of populations) (Barber, 1996; Walker et al., 2009). This fatal disease is found in clams at multiple sites along the coasts of all states between Maryland and Maine as well as in eastern Canada (B€ ottger et al., in preparation a). Cancerous clam haemolymph contains 100% cancerous haemocytes (5 108 cells ml1) with an average diameter of 7–10 mm and a mean nuclear to cytoplasmic volume ratio of 1:1. These nearly round, mitotic haemocytes have a monotonous appearance, lack large pseudopodia, attach only loosely with thin cytoplasmic projections to plastic or glass and are not motile nor phagocytic (Fig. 1.7a). They contain membrane bound vacuoles (5–15/cell) that stain intensely for lipid with oil red O (Fig. 1.7b). Protocols for maintaining cancerous haemocytes from the soft shell clam in vitro and for amplifying them in vivo have been developed and are described in detail by Walker et al. (2009). These were the first successful

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

15

[(Figure_7)TD$IG]

Figure 1.7 Clam haemocyte cancer: (a) interference image of freshly collected cancerous clam haemocytes; notice dumb-bell shaped haemocyte involved in cytokinesis near the centre; (b) Cytospin of CCH stained with oil red O; notice mitotic division figure on the left of the image; (c) Quantum-dot immunocytochemical localization in cytoplasmically sequestered p53 in an individual CCH; (d) quantum-dot immunocytochemical localization in cytoplasmic mortalin in the same CCH; (e) dual quantum-dot immunocytochemical localization of cytoplasmically sequestered p53 and mortalin in the same CCH–yellow indicates co-localization of the two proteins (scale bar in (a) and (b) ¼ 20 mm and in (c–e) ¼ 10 mm) and (f) coimmunoprecipitation of clam p53 and mortalin in normal (NCH) and CCH. Std, protein standard; C1 and C2, first and second elute from CCH lysates; N1 and N2, first and second elude from NCH lysates; NA, first elude from gel loaded with NCH and CCH lysate mix (negative control); Q, first elude from quenched gel loaded with NCH and CCH lysate mix (negative control); Ccyt, cytoplasmic protein extract from CCH and Cnuc, nuclear protein extract from CCH. (See Plate no.4 in the Color Plate Section)

methods at maintaining malignant cells from any invertebrate in vitro. They provide ready experimental access to simultaneous in vitro and in vivo versions of this naturally occurring cancer model from the same organism for use by both biomedical and environmental researchers (see Appendix A of this review for a signi¢cantly updated version of the clam culture medium originally presented in Walker et al., 2009). Cancerous clam haemocytes express a highly conserved homologue for human wild-type p53 protein (GenBank Accession number

16

Charles W. Walker et al.

AF253323) that is rendered non-functional through sequestration in the cytoplasm by mortalin protein (the mitochondrial Hsp70), when the latter is over-expressed (Fig. 1.7c–e). Expression of clam mortalin is 1634 times higher in CCH than in normal clam haemocytes (NCH) as determined by quantitative PCR (B€ ottger et al., 2008). Cytoplasmic sequestration does not occur in NCH (Kelley et al., 2001; Walker et al., 2006). A similar phenotype involving cytoplasmic sequestration of wildtype p53 has been observed in an unrelated group of human cancers, including undifferentiated neuroblastoma and colorectal adenocarcinoma (Moll et al., 1992, 1995, 1996; Kelley et al., 2001; Dundas et al., 2005; Walker et al., 2006, 2009; Walker and B€ ottger, 2008). These were the first structural and functional data for p53 and p63/73 mRNAs and gene products in a naturally occurring, non-mammalian disease model for human cancers and also for any bivalve mollusc (Kelley et al., 2001). Based on the substantial homology of the TAD in these proteins (65–69% identity), it was suggested that clam p53- and p63/73-like proteins might both share transcriptional functions with human proteins. Subsequent studies have indicated that as in humans, both transcriptional and non-transcriptional p53-dependent mechanisms are involved in soft shell clam haemocyte cancer (Kelley et al., 2001; Walker et al., 2006; B€ ottger et al., 2008; Walker and B€ ottger, 2008; Holbrook et al., 2009). These will be discussed in detail in the following sections.

5.2. Transcriptional induction of apoptosis by soft shell clam p53 Genotoxic stress induced by treatment of cancerous clam haemocytes with etoposide promotes denovo transcription of wild-type p53 (602-fold increase over untreated CCH), translocation of p53 protein to the nucleus (Fig. 1.8a and b), followed by DNA damage (Fig. 1.8b and c) and apoptosis (B€ ottger et al., 2008). The most attractive interpretation of these data is that treatment with etoposide elevates p53 levels in the cytoplasm of CCH and that de novo p53 protein overwhelms mortalin tethering, resulting in nuclear translocation of some p53 and apoptosis. After treatment with etoposide, transcriptional targets of p53 would be active in evaluating and repairing DNA, or failing that induction of the apoptosis of CCH that we have observed.

5.3. Non-transcriptional induction of apoptosis by soft shell clam p53 Treatment of CCH with the nuclear pore blocker wheat germ agglutinin (WGA) demonstrated that clam p53 is found only in the cytoplasm

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

17

[(Figure_8)TD$IG]

Figure 1.8 Transcriptional initiation of apoptosis by soft shell clam p53. Distribution of p53 before and after treatment of CCH with etoposide; (a) and (b) show the distribution of p53 using an M. arenaria polyclonal antibody (Kelley et al., 2001). (a) Zero time control for CCH in which clam p53 protein is sequestered in the cytoplasm with a perinuclear distribution (notice the clear nuclei, n), scale bar ¼ 10 mm; (b) cancerous clam haemocytes from the same individual as Fig. 1.7a in which clam p53 protein has re-localized to the nucleus following treatment with 0.075 mM etoposide for 18 h, scale bar ¼ 10 mm; (c) comet assay showing no evidence of DNA damage in control normal clam haemocytes after 18 h following sham injection with carrier minus etoposide; (d) comet assay showing extensive DNA damage in leukaemic clam haemocytes from the same individual as in part (a) following treatment with 0.5 mm etoposide for 18 h.

(Fig. 1.9a), with some mitochondrial localization (Fig. 1.9d). When treated with MKT-077 (a chemotherapeutic rhodocyanin dye) only (no pre-treatment with WGA), clam p53 was initially found in the cytoplasm, but was also translocated into the nucleus at 6 h (Fig. 1.9b) with very little accumulation at the mitochondria (Fig. 1.9e). Pre-treatment of CCH with WGA followed by post-treatment with MKT-077 demonstrated that clam p53 is found only in the cytoplasm (Fig. 1.9c), with increased mitochondrial localization of p53 over the results from treatment with MKT-077 alone (Fig. 1.9f). Treatment with MKT-077 induced apoptosis, as detected by the TUNEL assay (results not shown). The most attractive interpretation of our results is that MKT-077 competes with

18

Charles W. Walker et al.

[(Figure_9)TD$IG]

Figure 1.9 Non-transcriptional initiation of apoptosis by soft shell clam p53. Distribution of p53 in cytoplasmic, nuclear and mitochondrial protein extracts from CCH using an M. arenaria polyclonal antibody (Kelley et al., 2001) following treatment with MKT-077 for 6 h. Treatment with MKT-077 occurred both with and without WGA pre-treatment. Pre-treatment of CCH with WGA to block nuclear import employed FITC-labelled WGA (Sigma) transfected into CCH using the Chariot protein delivery system (Active Motif). WGA was allowed to couple with the Chariot compound for 30 min at room temperature to form complexes that were incubated for 1 h with CCH suspended in culture medium at concentrations of 6 105 CCH/100 ml medium. Cytoplasmic, nuclear and mitochondrial protein extracts from CCH were treated as follows: for nuclear/cytoplasmic extracts— (a) WGA only, (b) 3.5 mM MKT-077 only, (c) pre-treatment with WGA followed post-treatment with 3.5 mM MKT-077; and for mitochondrial extracts— (d) WGA only, (e) with 3.5 mM MKT-077 only and (f) with 3.5 mM MKT-077 following pre-treatment with WGA. Treatment with WGA blocks nuclear access for p53 and results in its translocation to the mitochondrion where apoptosis of CCH follows. When normal clams are treated identically, p53 is transported to the mitochondria, but apoptosis does not result. Std, protein standard; nuc, nuclear protein; cyt, cytoplasmic protein.

p53 for the mortalin p53 binding site. As a result unbound p53 is available to be transported to the nucleus or mitochondria. Since nuclear access is blocked, p53 is only transported to the mitochondria where it binds to and inactivates antiapoptotic Bcl-2 proteins in the mitochondrial membrane. Pro-apoptotic Bax proteins from the cytoplasm are then recruited to the mitochondria and participate in mitochondrial catastrophy.

5.4. Haemocyte cancer in other bivalves In some locations, the blue and bay mussels, M.edulis and M.trossulus, also develop a cancer-like, proliferative disease of their haemocytes at high prevalence that is morphologically similar to that described above for M. arenaria (Bower, 1989; Barber, 2004). While mussel haemocyte cancer has received attention as a tool for environmental monitoring (Muttray et al., 2008; Pantzartzi et al., 2010), studies on the molecular basis for the disease have only recently begun (Muttray et al., 2005). The elucidation of

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

19

the structure of mussel p53 and p63/73 genes (Muttray et al., 2007) has provided the basis for future research into the function of their gene products in mussel haemocyte cancer. M. arenaria and Mytilus spp. p53 and p63/73 share 69 and 57% sequence similarity at the protein level, respectively, and it is possible that the genes and gene products of the two species also share some of the functional properties that contribute to haemocyte cancer. Thus far, expression analysis has shown that p53 as well as an Mdm2-like gene product are expressed at higher levels in cancerous mussel haemocytes when compared to normal haemocytes (Muttray et al., 2010). The increase in p53 also results in an increase in transcription of Mdm2, similar to what has been reported for mammalian p53–Mdm2 feedback interactions. Indeed, Mytilus p53 and Mdm2 proteins interact, as has been described above. In addition, the DNp63/73 isoform was expressed at higher levels in mussel haemocyte cancer. This is similar to some human cancers where it has been suggested as a biomarker of poor prognosis (Muttray et al., 2008). Because p53 was expressed at higher levels in cancerous haemocytes that escape apoptosis, the hypothesis that p53 may have acquired inactivating mutations was tested. Although specific single nucleotide polymorphisms (SNPs) were detected in cancerous mussel haemocytes, they were silent and thought unlikely to affect protein function (Vassilenko et al., 2010). However, variations in mRNA secondary structure and hence rates of transcription, and a potential change in splicing patterns may affect protein levels.

5.5. Germinomas in bivalves Bivalve germinomas (gonadal cancer; germ cell cancers) were first described over four decades ago in hard shell clams (Mercenaria spp.) (Yevich and Barry, 1969). Early stages of the disease exhibit clusters of neoplastic cells that appear to originate basally in gonadal follicles (Rhodes and Van Beneden, 1996). As the disease progresses, density of the stromal extracellular matrix increases, accompanied by infiltrating phagocytic cells. In late stages, neoplastic cells fill the gonadal lumen, eventually obliterating the follicular structure (Fig. 1.10a–d). Unlike haemocyte cancer, the germinoma does not appear to be fatal. The drastic morphological changes and the apparent loss of germ cells suggest that there could be a significant impact on reproductive output at both the individual and population levels (Barber, 1996). Investigation of the molecular basis of the gonadal cancer in M. arenaria has focused on the effects of dioxin, aryl hydrocarbon (AhR) and the herbicide 2,4-D and molecular mechanisms that might mediate their toxicity (Butler et al., 2001, 2004; Van Beneden, 2005). Aberrant regulation of p53 gene has also been investigated. The level of p53 protein under feedback control by Mdm2, is normally elevated in

20

Charles W. Walker et al.

[(Figure_0)TD$IG]

Figure 1.10 Comparison of non-tumour and tumour-bearing gonadal tissue in female M. arenaria. (a) Typical gross appearance of mature gonad; note large, creamy appearance (arrow); (b) clam with advanced germinoma; note brown mottling (arrow); (c) histological section showing normal ovarian follicle containing mature eggs (H&E stain), scale bar = 10 mm; (d) histological section showing typical small, undifferentiated cells of advanced germinoma (H&E stain), scale bar = 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this chapter.)

response to cellular stress. In human cervical cancers, abnormal degradation of p53 is mediated by the E3 ubiquitin ligase (Huibregtse et al., 1991). Since altered levels of the clam E3 homologue mRNA were detected earlier in laboratory exposure studies; the E3 cDNA sequence was determined (GenBank Accession number AF154109) (Kelley and Van Beneden, 2000). Protein expression levels of the clam homologues of E3 and p53 were examined in tumour- and non-tumour-bearing clams by Western blot analysis (Kelley and Van Beneden, 2000; Olberding et al., 2004). E3 protein expression was found to be significantly higher in tumour-bearing females compared to non-germinomabearing females (Fig. 1.11). Conversely, the level of p53 protein was found to be significantly lower in germinoma-bearing females compared to non-germinoma-bearing individuals (Fig. 1.11). These findings supported the hypothesis that unscheduled E3-mediated degradation of p53 releases normal cell cycle arrest at G1, allowing abnormal cell proliferation and tumour formation. However, the direct interaction of p53 and E3 was not confirmed by protein/protein binding studies (Olberding et al., 2004).

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

21

[(Figure_1)TD$IG]

Figure 1.11 Clam germinoma tissue exhibited significantly higher levels of E3 protein concomitant with significantly lower levels of p53 protein as compared to normal gonadal tissues. Summary graphs represent densitometry scans from the Western blots normalized to actin. Non-tumorigenic samples consisted of both male and female tissue; there were no statistically significant gender differences for either MaE3 or Map53 protein levels. Germinoma proteins were isolated from females; no male germinoma tissue was available for analysis. * significant difference; P < 0.05; error bars represent SD) (after Olberding et al., 2004).

5.6. Environmental stressors and potential links to bivalve haemocyte and germinoma cancers Linkages between environmental toxins and cancer are often difficult to demonstrate for lack of an appropriate model system (Van Beneden et al., 1997; Van Beneden, 2005). Bivalve molluscs, however, are excellent candidates for such models. First, they are long-lived and sessile and filter feeding exposes them to both suspended and soluble compounds. Production of reactive oxygen species (ROS) is also a common response to environmental stressors in many marine invertebrates including M. arenaria (Abele et al., 2002; Lesser, 2006) and Mytilus spp. (Winston et al., 1996). All other parameters being equal, an increase in antioxidant capacity is often associated with higher exposures to xenobiotics (Frenzilli et al., 2004). 5.6.1. Contaminants: field studies Based on limited, highly site-specific data, several environmental contaminants have been circumstantially linked to haemocyte cancer in M. arenaria (Brown, 1981). Reports show significant correlation with oil (Yevich and Barry, 1969; Barry and Yevich, 1975; Brown et al., 1977; Lowe and More, 1978; Yevich and Barszcz, 1978; Walker et al., 1981), polyaromatic hydrocarbons (PAHs) (Lowe and More, 1978), polychlorinated biphenyls (PCBs) (Reinisch et al., 1984; B€ ottger et al., in preparation b), industrial and municipal waste (Walker et al., 1981; Reinisch et al., 1984; St-Jean et al., 2005), pesticides (Kagley, 2003) and heavy metal pollution (Walker et al., 1981; Kagley, 2003) (Fig. 1.12).

22

Charles W. Walker et al.

[(Figure_2)TD$IG]

Figure 1.12 Prevalence of haemocyte cancer in M.arenaria correlated with different contaminants. Results were compiled and averaged for oil (Yevich and Barry, 1969; Barry and Yevich, 1975; Brown et al., 1977; Lowe and More, 1978; Yevich and Barszcz, 1978; Walker et al., 1981), PAHs and PCBs combined (B€ ottger et al., in preparation b; Reinisch et al., 1984), industrial and municipal waste (Walker et al., 1981; Reinisch et al., 1984; St-Jean et al., 2005) and heavy metal pollution (Walker et al., 1981; Kagley, 2003). Data reflect frequencies for haemocyte cancer and different contaminants averaged from literature values for a number of sites.

Germinomas in the soft-shell clam, M. arenaria, were first observed following the March 1971 oil spill at Long Cove, ME (Dow and Hurst, 1975). In the aftermath of the spill, it was speculated that they might be linked to exposure to petroleum hydrocarbons. Since both haemocyte and germinoma cancers have also been detected in unpolluted sites, it is not clear if any of these factors or any group of them is responsible for onset or progression of these diseases. 5.6.2. Contaminants: laboratory studies Despite the overwhelming number of reports linking carcinogenic chemicals in the environment and clam haemocyte cancer, it has been difficult to show clear cause–effect relationships in the laboratory (Harsbarger, 1967). Haemocyte cancer did develop in Uniopictorum exposed to PAHs/ PCBs (Khudolei and Sirenko, 1978) but did not following the same treatment in M. edulis (Rasmussen, 1986), M. arenaria (MacCallum et al., 2003) and Macoma baltica (Tay, 2003). Laboratory exposure studies under the direction of George Gardner at the EPA Laboratory (Narragansett, RI) were conducted in an attempt to

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

23

mimic the 1971 Maine oil spill. Adult M. arenaria were exposed to sediments from Long Cove, ME, contaminated with petroleum hydrocarbons; exposures, however, failed to induce germinomas. A companion study (Gardner et al., 1991), however, reported that oysters (Crassostrea virginica) exposed to Black Rock Harbor sediments heavily contaminated with PAHs and PCBs developed a variety of neoplastic lesions, predominantly kidney lesions (9.3%) and germinomas (1.3%). The potential link of germinoma and environmental contaminants has been further investigated in a series of controlled laboratory exposure studies (Rhodes and Van Beneden, 1996; Butler etal., 2004). Van Beneden and Gardner tested the hypothesis that either dioxin or herbicides may induce the germinoma in M.arenaria (Rhodes and Van Beneden, 1996; Butler etal., 2004). Clams were exposed to either (a) 3H-TCDD (2,3,7,8-tetrachlorodibenzo-pdioxin) in the presence or absence of the initiating carcinogen diethylnitrosamine (DEN) or (b) 2,4-D (Kelley and Van Beneden, 2000). Further investigation revealed that laboratory exposure to dioxin elicited early changes (<2 weeks post-exposure) in gene expression patterns that included induction of biosynthetic pathways, signal transduction cascades and ubiquitin-mediated proteolytic pathways (Rhodes and Van Beneden, 1996, 1997; Kelley and Van Beneden, 2000). Using differential display PCR (a differential mRNA expression screen, technology which preceded today’s microarrays), Rhodes and Van Beneden reported differentially expressed mRNA sequences in TCDD-exposed animals compared to unexposed controls (Rhodes and Van Beneden, 1997; Kelley and Van Beneden, 2000). So far, laboratory studies to directly link contaminants with gonadal tumour induction have been largely unsuccessful. In summary, laboratory exposure to potential carcinogenic chemicals does not reproduce observations made in the field. 5.6.3. Viral involvement Experimental evidence also exists for viral induction of cancer in clam haemocytes (Oprandy, 1981; Oprandy and Chang, 1983). A virus similar to a B-type retrovirus was isolated from M. arenaria and CCH were encountered following injection of this virus from CCH into healthy clams (Oprandy and Chang, 1983). ‘Virus-like particles’ have been identified by electron microscopy (Elston et al., 1992) and by reverse transcriptase activity from CCH, but are absent from NCH, suggesting retroviral involvement in the disease (House et al., 1998; AboElkhair et al., 2009). Haemocyte cancer detected in M. arenaria in the field has been attributed to viral induction in isolated hotspots (Farley et al., 1986, 1991) and to species specificity of the disease (Kent etal., 1991; Muttray etal., 2009). In the laboratory, clam haemocyte cancer is most effectively transferred to normal clams after inoculation of normal clams with lysed CCH in diseased clam haemolymph, although haemolymph alone will also initiate

24

Charles W. Walker et al.

the disease in normal clams (Walker et al., 2009). Similar results have been obtained for Cerastoderma edule, following inoculation of normal cockles with haemolymph from diseased cockles. These results certainly suggest that a virus may be involved in the aetiology of bivalve haemocyte cancers (Collins and Mulcahy, 2003). However, irrefutable proof of the involvement of either environmental contaminants or viral agents or both in the aetiology of these diseases remains unavailable. These results point out that the aetiology of these diseases is complex and likely involves multiple causes including environmental, viral and genetic factors. Infectious and environmental aetiologies are not mutually exclusive and future research should focus on probing interactions between different disease factors. The use of haemocyte cell culture (Appendix A) and efforts to identify the source of haemocyte stem cells may be crucial in determining the aetiology of this disease.

6. CONCLUDING REMARKS Marine bivalves are ecologically and commercially important organisms that provide excellent models for both biomedical and environmental researchers. For biomedical research, studies of naturally occurring cancers analogous to human diseases should indicate molecular mechanisms that are held in common. As we have shown above, clam haemocyte cancer provides excellent in vitro and in vivo models for transcriptional and non-transcriptional outcomes reflecting those seen in similar human cancers under stress and with similar malfunctions in p53 functionality. Bivalves are simultaneously good models for the effects of toxic compounds, since they have low monooxygenase activity and thus, compounds slowly accumulate and tissue burdens can serve as integrative indices of bioavailable contaminants (Van Beneden, 2005). Ultimately, the naturally occurring, widely accessible and inexpensive-to-maintain clam haemocyte and germinoma cancer models may help to identify genes that are susceptible to environmental stress. Understanding the molecular basis of bivalve cancer and stress biology should significantly increase the usefulness of bivalve models as a sentinel species for sophisticated ecotoxicological studies and as environmental indicator species.

6.1. Bivalves as models for human cancer and stress biology Analysing the effects of genotoxic compounds on malignancy are currently limited to highly regulated vertebrate cancer models where tumours are induced (e.g. the mouse). The CCH and germinoma models are more similar to outbreeding, human clinical populations than are those generated from inbred mouse strains intentionally exposed to

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

25

known tumour viruses (Kovalchuk etal., 2000). Fly and worm models for human cancers involving mortalin do not exist principally because adult somatic cells in these organisms do not divide, so an invivo version of the model is unavailable. Also, existing fly and worm models have highly derived homologues for p53 only (p63 and p73 are lacking) (Brodsky et al., 2000; Jin et al., 2000; Derry et al., 2001; Schumacher et al., 2001). Additionally, very few members of the Bcl-2 family of proteins exist in either fly or worm models (Igaki and Miura, 2004; Brumby and Richardson, 2005; Lu and Abrams, 2006) and these genes are far less conserved than are those found in molluscs like the soft shell clam. Haemocyte cancer in clams is an example of a transplantable tumour that is not hampered by two of the characteristics that limit the applicability of mouse transplantable tumour models (Ostrand-Rosenberg, 2004; Walker et al., 2009). For example, histocompatibility is not an issue since clams have only innate immunity (Medzhitov and Biron, 2003) and tumour cell inoculation is not subcutaneous as it is in mouse models, but occurs in an anatomically appropriate site for the tumour (the clam circulatory system) (Walker et al., 2009). Cancerous clam haemocytes provide excellent in vivo and in vitro models for human and other animal cancers displaying cytoplasmic sequestration of the p53 tumour suppressor by mortalin tethering. Clam haemocyte cancer is the only nonmammalian model sufficiently characterized to provide both in vitro and in vivo versions of the model from the same individuals for simultaneous use in studies of cytoplasmic sequestration and inactivation of p53 proteins and their subsequent reactivation by treatments with genotoxic and nongenotoxic agents.

6.2. In vitro and in vivo models for bivalve tumour cells The limited lifespan of human cells in culture has lead to the use of treatments that allow cells to achieve more passages. The lifespan of human cell lines can be extended by transfection with viral genes that produce products that sequester tumour suppressor molecules like p53 and Rb. In these cultures some cells will occasionally acquire mutations that make them immortal and even tumorigenic (Masters, 2000). The intriguing feature of CCH is that although they do not necessarily appear to be immortalized, they are already tumorigenic. While CCH maintained invitro are healthy and mitotic, refinements of our culture medium may be necessary to increase growth rates and to develop an immortalized cell line from CCH. However, if a virus is involved in this disease, the lifespan of CCH may already be extended via suppression of p53 transcription. Viral modification of the functions of CCH might be directly or indirectly involved in suppressing the activities of wild-type p53 in complexes with over-expressed mortalin that sequester p53 in the cytoplasm.

26

Charles W. Walker et al.

If this is the case, CCH may already be an immortalized cell line as defined for human cells in suspension culture (Masters, 2000; Walker etal., 2009).

6.3. Use of bivalves as bioindicator species—understanding the signalling pathways involved Several recent studies of Mytilus sp. and other molluscs (e.g. the abalone, Haliotistuberculata) have emphasized using changes in mRNA and protein levels for p53, heat-shock family member proteins (especially Hsp70 and Hsp90) and other cell stress-related gene products (e.g. SOD, superoxide dismutase and MT, metallothionein) as biosensors for environmental monitoring (Dondero et al., 2006a,b; Grattarola et al., 2006; Kourtidis et al., 2006a,b; Farcy et al., 2007, 2008; Banni et al., 2009; Pantzartzi et al., 2009, 2010). For example, many heat-shock family proteins are inducibly expressed in response to environmental stressors and functionally integrate proliferative, survival and cell death signalling events (Beere, 2001). In the case of clam haemocyte cancer, where the mitochondrial heat-shock protein mortalin is over-expressed, a response to stressors in the environment might imply a direct line from the environment to a well-studied molecular cancer mechanism that immortalizes molluscan cells (Kelley et al., 2001; Walker et al., 2006, 2009). Since mortalin is not heatshock inducible, its over-expression must rely upon a different stressor. Environmental monitoring of appropriate gene expression profiles is potentially very useful to emphasize changes in the immediate environment of bivalves and other members of marine communities. As a result, these issues can be addressed locally. However, the impact of potentially harmful environmental factors at the molecular level may be very complex. A recent paper by Pantzartzi et al. (2009) suggests that two cytoplasmic Hsp90 genes from Mytilusgalloprovincialis contain promoters with p53 binding sites, suggesting the possibility that expression of these genes is transcriptionally regulated by p53. It has been shown that both Hsp70 and Hsp90 participate with other proteins in multicomponent complexes that mediate translocation of cytoplasmic p53 to the nucleus using the molecular motor dynein (Galigniana et al., 2004; Giannakakou et al., 2000, 2002). The response of bivalve cells to increases in Hsp90 resulting from stress-induced p53 transcription might drive changes in expression of mortalin, blocking apoptosis and immortalizing clam haemocytes. Even more intriguing is how stress can lead to mitochondrial translocation of p53 and non-transcriptional induction of apoptosis in CCH. Certainly the dynein-dependent complex just described cannot alone be involved, since many mitochondria are distributed peripherally in the cytoplasm and are nowhere near the nucleus. While it is unknown even for human cancers what alternative multicomponent or other complexes involving mortalin and perhaps kinesin are involved in

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

27

mitochondrial translocation, recent studies have implicated both the DNAJ-like protein Tid 1 and loss of hyperubiquination in this process for several human colon carcinoma and breast cancer cell lines (Marchenko et al., 2007; Ahn et al., 2010; Kampinga and Craig, 2010). Despite our current understanding that changes in the expression of several stress-related genes can serve as excellent biosensors, the complex interactions in the normal signalling mechanism following a specific suite of environmental perturbations are virtually unknown.

ACKNOWLEDGEMENTS This research was supported by National Cancer Institute grants (CA71008-01 and CA104112-01), UNH Sea Grant (R/FMD-166) and UNH Hatch Grant (353) to CWW and grants from the NIH (R010RR08774/ES012066), Maine Sea Grant (R-02-03 to RJVB and S. Lindsay) and the Maine Agriculture and Forestry Experiment Station (ME08509) to RJVB. This is MAFES publication number 3089.

28

Charles W. Walker et al.

Appendix A Walker’s Clam Haemocyte Culture Medium (Updated Formulation after Walker et al., 2009)

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

29

REFERENCES Abele, D., Heise, K., Portner, H. O., and Puntarulo, S. (2002). Temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria. J. Exp. Biol. 205, 1831–1841. AboElkhair, M., Synard, S., Siah, A., Pariseau, J., Davidson, J., Johnson, G., Greenwood, S. J., Casey, J. W., Berthe, F. C., and Cepica, A. (2009). Reverse transcriptase activity in tissues of the soft shell clam Myaarenaria affected with haemic neoplasia. J.Invertebr. Pathol. 102, 133–140. Ahn, B. Y., Trinh, D. L., Zajchowski, L. D., Lee, B., Elwi, A. N., and Kim, S. W. (2010). Tid1 is a new regulator of p53 mitochondrial translocation and apoptosis in cancer. Oncogene 29, 1155–1166. Akdemir, F., Christich, A., Sogame, N., Chapo, J., and Abrams, J. M. (2007). p53 directs focused genomic responses in Drosophila. Oncogene 26, 5184–5193. Banni, M., Negri, A., Rebelo, M., Rapallo, F., Boussetta, H., Viarengo, A., and Dondero, F. (2009). Expression analysis of the molluscan p53 protein family mRNA in mussels (Mytilus spp.) exposed to organic contaminants. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 149, 414–418. Barber, B. (1996). Effects of gonadal neoplasms on oogenesis in softshell clams, Mya arenaria. J. Invert Pathol. 67, 161–168. Barber, B. (2004). Neoplastic diseases of commercially important marine bivalves. Aquat. Living Resour. 17, 449–466. Barker, C. M., Calvert, R. J., Walker, C. W., and Reinisch, C. L. (1997). Detection of mutant p53 in clam leukemia cells. Exp. Cell Res. 232, 240–245. Barry, M. M., and Yevich, P. P. (1975). The ecological, chemical and histopathological evaluation of an oil spill site: Part III. Histopathological studies. Mar. Pollut. Bull. 6, 171–173. Beere, H. M. (2001). Stressed to death: regulation of apoptotic signaling pathways by the heat shock proteins. Sci. STKE 2001, re1. Belyi, V. A., Ak, P., Markert, E., Wang, H., Hu, W., Puzio-Kuter, A., and Levine, A. J. (2010). The origins and evolution of the p53 family of genes. Cold Spring Harb. Perspect. Biol. 2, a001198. B€ ottger, S., Jerszyk, E., Low, B., and Walker, C. (2008). Genotoxic stress-induced expression of p53 and apoptosis in leukemic clam hemocytes with cytoplasmically sequestered p53. Cancer Res. 68, 777–782. B€ ottger, S. A., Taraska, N., Lock, N., and Walker, C. W. (in preparation a). Frequencies of neoplasia in Mya arenaria along the East Coast of North America. Dis. Aquat. Org.. B€ ottger, S. A., Taraska, N., and Walker, C. W. (in preparation b). Neoplasia development and growth linked to different environmental conditions in Maine and New Hampshire. Environ.Toxocol. Bower, S. M. (1989). The summer mortality syndrome and haemic neoplasia in the blue mussels (Mytilus edulis) from British Columbia. Can.Tech. Rep. Fish. Aquat. Sci. 1703, 1–65. Brodsky, M. H., Nordstrom, W., Tsang, G., Kwan, E., Rubin, G. M., and Abrams, J. M. (2000). Drosophila p53 binds a damage response element at the reaper locus. Cell 101, 103–113. Brown, R. S. (1981). The value of the multidisciplinary approach to research on marine pollution effects as evidenced in a three-year study to determine the etiology and pathogenesis of neoplasia in the soft-shell clam, Mya arenaria. Rapports et ProcesVerbaux des Reunions, Conseil International pour L’Exploration scienti¢que de la Mer Medeterranee Monaco 179, 125–128.

30

Charles W. Walker et al.

Brown, R. S., Wolke, R. E., Saila, S. B., and Brown, C. W. (1977). Prevalence of neoplasia in 10 New England populations of the soft-shell clam (Mya arenaria). Ann. N.Y. Acad. Sci. 298, 522–534. Brumby, A. M., and Richardson, H. E. (2005). Using Drosophila melanogaster to map human cancer pathways. Nat. Rev. Cancer 5, 626–639. Butler, R. A., Kelley, M. L., Olberding, K. E., Gardner, G. R., and Van Beneden, R. J. (2004). Aryl hydrocarbon receptor (AhR)-independent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on softshell clam (Mya arenaria) reproductive tissue. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 138, 375–381. Butler, R. A., Kelley, M. L., Powell, W. H., Hahn, M. E., and Van Beneden, R. J. (2001). An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding. Gene 278, 223–234. Collins, C. M., and Mulcahy, M. F. (2003). Cell-free transmission of a haemic neoplasia in the cockle Cerastoderma edule. Dis. Aquat. Organ. 54, 61–67. Cox, R. L., Stephens, R. E., and Reinisch, C. L. (2003). p63/73 homologues in surf clam: novel signaling motifs and implications for control of expression. Gene 320, 49–58. Derry, W. B., Putzke, A. P., and Rothman, J. H. (2001). Caenorhabditiselegans p53: role in apoptosis, meiosis, and stress resistance. Science 294, 591–595. Dobbelstein, M., and Roth, J. (1998). The large T antigen of simian virus 40 binds and inactivates p53 but not p73. J. Gen.Virol. 79(Pt 12), 3079–3083. Dondero, F., Dagnino, A., Jonsson, H., Capri, F., Gastaldi, L., and Viarengo, A. (2006a). Assessing the occurrence of a stress syndrome in mussels (Mytilus edulis) using a combined biomarker/gene expression approach. Aquat.Toxicol. 78(Suppl. 1), S13–S24. Dondero, F., Piacentini, L., Marsano, F., Rebelo, M., Vergani, L., Venier, P., and Viarengo, A. (2006b). Gene transcription profiling in pollutant exposed mussels (Mytilus spp.) using a new low-density oligonucleotide microarray. Gene 376, 24–36. Dotsch, V., Bernassola, F., Coutandin, D., Candi, E., and Melino, G. (2010). p63 and p73, the ancestors of p53. Cold Spring Harb. Perspect. Biol. 2, a004887. Dow, R. L., and Hurst, J. W. (1975). The ecological, chemical and histopathological evaluation of an oil spill site. Part I. Ecological studies. Mar. Pollut. Bull. 6, 164–166. Dundas, S. R., Lawrie, L. C., Rooney, P. H., and Murray, G. I. (2005). Mortalin is overexpressed by colorectal adenocarcinomas and correlates with poor survival. J. Pathol. 205, 74–81. Dunn, C. W., Hejnol, A., Matus, D. Q., Pang, K., Browne, W. E., Smith, S. A., Seaver, E., Rouse, G. W., Obst, M., and Edgecombe, G. D., et al., (2008). Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452, 745–749. Elston, R. A., Moore, J. D., and Brooks, K. (1992). Disseminated neoplasia of bivalve molluscs. Rev. Aquat. Sci. 6, 405. Farcy, E., Fleury, C., Lalong, C., Dubos, M. P., Voiseux, C., Fievet, B., and Lebel, J. M. (2008). Molecular cloning of a new member of the p53 family from the Pacific oyster Crassostrea gigas and seasonal pattern of its transcriptional level. Mar. Environ. Res. 66, 300–308. Farcy, E., Serpentini, A., Fievet, B., and Lebel, J. M. (2007). Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotistuberculata: transcriptional induction in response to thermal stress in hemocyte primary culture. Comp.Biochem.Physiol. B Biochem. Mol. Biol. 146, 540–550. Farley, C. A., Otto, S. V., and Reinisch, C. L. (1986). New occurence of epizootic sarcoma in Chesapeake Bay soft shell clams, Mya arenaria. Fish. Bull. 84, 851–857. Farley, C. A., Plutschak, D. L., and Scott, R. F. (1991). Epizootiology and distribution of transmissible sarcoma in Maryland softshell clams, Mya arenaria, 1984–1988. Environ. Health Perspect. 90, 34–41.

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

31

Fernandes, A. D., and Atchley, W. R. (2008). Biochemical and functional evidence of p53 homology is inconsistent with molecular phylogenetics for distant sequences. J. Mol. Evol. 67, 51–67. Frenzilli, G., Bocchetti, R., Pagliarecci, M., Nigro, M., Annarumma, F., Scarcelli, V., Fattorini, D., and Regoli, F. (2004). Time-course evaluation of ROS-mediated toxicity in mussels, Mytilus galloprovincialis, during a field translocation experiment. Mar. Environ. Res. 58, 609–613. Galigniana, M. D., Harrell, J. M., O’Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004). Hsp90-binding immunophilins link p53 to dynein during p53 transport to the nucleus. J. Biol. Chem. 279, 22483–22489. Gardner, G. R., Yevich, P. P., Harsbarger, J. C., and Malcolm, A. R. (1991). Carcinogenicity of Black Rock Harbor sediment to the eastern oyster and trophic transfer of Black Rock Harbor carcinogens from the blue mussel to the winter flounder. Environ. Health Perspect. 90, 53–66. Giannakakou, P., Nakano, M., Nicolaou, K. C., O’Brate, A., Yu, J., Blagosklonny, M. V., Greber, U. F., and Fojo, T. (2002). Enhanced microtubule-dependent trafficking and p53 nuclear accumulation by suppression of microtubule dynamics. Proc. Natl. Acad. Sci. U. S. A. 99, 10855–10860. Giannakakou, P., Sackett, D. L., Ward, Y., Webster, K. R., Blagosklonny, M. V., and Fojo, T. (2000). p53 is associated with cellular microtubules and is transported to the nucleus by dynein. Nat. Cell Biol. 2, 709–717. Grattarola, M., Carloni, M., Dondero, F., Viarengo, A., and Vergani, L. (2006). Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20. Mol. Biol. Rep. 33, 265–272. Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C. (1994). Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855–4878. Harsbarger, J. C. (1967). Responses of invertebrates to vertebrate carcinogens. Fed. Proc. 26, 1693–1697. Holbrook, L. A., Butler, R. A., Cashon, R. E., and Van Beneden, R. J. (2009). Soft-shell clam (Mya arenaria) p53: a structural and functional comparison to human p53. Gene 433, 81–87. Horvath, M. M., Wang, X., Resnick, M. A., and Bell, D. A. (2007). Divergent evolution of human p53 binding sites: cell cycle versus apoptosis. PLoS Genet. 3, e127. House, M. L., Kim, C. H., and Reno, P. W. (1998). Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity. Dis. Aquat. Org. 34, 187–192. Huang, Q., Raya, A., DeJesus, P., Chao, S. H., Quon, K. C., Caldwell, J. S., Chanda, S. K., Izpisua-Belmonte, J. C., and Schultz, P. G. (2004). Identification of p53 regulators by genome-wide functional analysis. Proc. Natl. Acad. Sci. U. S. A. 101, 3456–3461. Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1991). A cellular protein mediates association of p53 with the E6 oncoprotein of human papilloma virus types 16 or 18. EMBOJ. 10, 4129–4135. Huson, D. H., and Bryant, D. (2006). Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23, 254–267. Igaki, T., and Miura, M. (2004). Role of Bcl-2 family members in invertebrates. Biochim. Biophys. Acta 1644, 73–81. Ishimoto, O., Kawahara, C., Enjo, K., Obinata, M., Nukiwa, T., and Ikawa, S. (2002). Possible oncogenic potential of DeltaNp73: a newly identified isoform of human p73. Cancer Res. 62, 636–641.

32

Charles W. Walker et al.

Jegga, A. G., Inga, A., Menendez, D., Aronow, B. J., and Resnick, M. A. (2008). Functional evolution of the p53 regulatory network through its target response elements. Proc. Natl. Acad. Sci. U. S. A. 105, 944–949. Jessen-Eller, K., Kreiling, J. A., Begley, G. S., Steele, M. E., Walker, C. W., Stephens, R. E., and Reinisch, C. L. (2002). A new invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls. Environ.Health Perspect. 110, 377–385. Jin, S., Martinek, S., Joo, W. S., Wortman, J. R., Mirkovic, N., Sali, A., Yandell, M. D., Pavletich, N. P., Young, M. W., and Levine, A. J. (2000). Identification and characterization of a p53 homologue in Drosophila melanogaster. Proc. Natl. Acad.Sci. U. S.A. 97, 7301–7306. Jordan, G. E., and Piel, W. H. (2008). PhyloWidget: web-based visualizations for the tree of life. Bioinformatics 24, 1641–1642. Kagley, A. N. (2003). Assessment of seasonal variability of cytochemical responses to contaminant exposure in the blue mussel Mytilus edulis (complex). Arch. Environ. Contam.Toxicol. 44, 43–52. Kampa, M., and Castas, E. (2008). Human health effects of air pollution. Environ. Poll. 151, 362–367. Kampinga, H. H., and Craig, E. A. (2010). The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat. Rev. Mol. Cell Biol. 11, 579–592. Kaul, S. C., Deocaris, C. C., and Wadhwa, R. (2007). Three faces of mortalin: a housekeeper, guardian and killer. Exp. Gerontol. 42, 263–274. Kelley, M. L., and Van Beneden, R. J. (2000). Identification of an E3 ubiquitin-protein ligase in the softshell clam (Mya arenaria). Mar. Environ. Res. 50, 289–293. Kelley, M. L., Winge, P., Heaney, J. D., Stephens, R. E., Farell, J. H., Van Beneden, R. J., Reinisch, C. L., Lesser, M. P., and Walker, C. W. (2001). Expression of homologues for p53 and p73 in the softshell clam (Mya arenaria), a naturally-occurring model for human cancer. Oncogene 20, 748–758. Kent, M. L., Wilkenson, M. T., Drum, A. S., and Elston, R. A. (1991). Failure of transmission of hemic neoplasia of bay mussels, Mytilus trossulus, to other bivalve species. J. Invert. Pathol. 57, 435–436. Khoury, M. P., and Bourdon, J. C. (2010). The isoforms of the p53 protein. Cold Spring Harb. Perspect. Biol. 2, a000927. Khudolei, V. V., and Sirenko, O. A. (1978). Tumor induction by N-nitroso compounds in bivalve mollusks. Can. Lett. 4, 349–354. Kim, I. S., Kim, D. H., Han, S. M., Chin, M. U., Nam, H. J., Cho, H. P., Choi, S. Y., Song, B. J., Kim, E. R., and Bae, Y. S., et al., (2000). Truncated form of importin alpha identified in breast cancer cell inhibits nuclear import of p53. J. Biol. Chem. 275, 23139–23145. Kourtidis, A., Drosopoulou, E., Nikolaidis, N., Hatzi, V. I., Chintiroglou, C. C., and Scouras, Z. G. (2006a). Identification of several cytoplasmic HSP70 genes from the Mediterranean mussel (Mytilus galloprovincialis) and their long-term evolution in Mollusca and Metazoa. J. Mol. Evol. 62, 446–459. Kourtidis, A., Drosopoulou, E., Pantzartzi, C. N., Chintiroglou, C. C., and Scouras, Z. G. (2006b). Three new satellite sequences and a mobile element found inside HSP70 introns of the Mediterranean mussel (Mytilusgalloprovincialis). Genome 49, 1451–1458. Kovalchuk, A. L., Qi, C. F., Torrey, T. A., Taddesse-Heath, L., Feigenbaum, L., Park, S. S., Gerbitz, A., Klobeck, G., Hoertnagel, K., and Polack, A., et al., (2000). Burkitt lymphoma in the mouse. J. Exp. Med. 192, 1183–1190. Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996). Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 274, 948–953.

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

33

Lane, D. P., Cheok, C. F., Brown, C., Madhumalar, A., Ghadessy, F. J., and Verma, C. (2010a). Mdm2 and p53 are highly conserved from placozoans to man. Cell Cycle 9, 540–547. Lane, D. P., Cheok, C. F., Brown, C. J., Madhumalar, A., Ghadessy, F. J., and Verma, C. (2010b). The Mdm2 and p53 genes are conserved in the Arachnids . Cell Cycle 9, 748–754. Le Bras, M., Bensaad, K., and Soussi, T. (2003). Data mining the p53 pathway in the Fugu genome: evidence for strong conservation of the apoptotic pathway. Oncogene 22, 5082–5090. Lesser, M. P. (2006). Oxidative stress in marine environments: biochemistry and physiological ecology. Annu. Rev. Physiol. 68, 253–278. Lowe, D. M., and More, M. N. (1978). Cytology and quantitative cytochemistry of a proliferative atypical hemocytic condition in Mytilusedulis (Bivalvia, Mollusca). J.Nat. Can. Inst. 60, 1455–1459. Lu, W. J., and Abrams, J. M. (2006). Lessons from p53 in non-mammalian models. Cell Death Di¡er. 13, 909–912. MacCallum, G. S., McGladdery, S. E., and Davidson, J. (2003). Factors, risks and significance of emergent neoplasia in cultured and wild soft-shell clams (Mya arenaria) in Atlantic Canada. AQUANET AP-18 Report, 32. Marchenko, N. D., Wolff, S., Erster, S., Becker, K., and Moll, U. M. (2007). Monoubiquitylation promotes mitochondrial p53 translocation. EMBO J. 26, 923–934. Masters, J. R. (2000). Human cancer cell lines: fact and fantasy. Nat. Rev. Mol. Cell Biol. 1, 233–236. Medzhitov, R., and Biron, C. A. (2003). Innate immunity. Curr. Opin.Immunol. 15, 2–4. Meisner, L. F., Roloff, B. D., and Belluck, D. A. (1993). In vitro effects of N-nitrosoatrazine on chromosome breakage. Arch. Environ. Contam.Toxicol. 24, 108–112. Moll, U. M., LaQuaglia, M., Benard, J., and Riou, G. (1995). Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc. Natl. Acad. Sci. U. S. A. 92, 4407–4411. Moll, U. M., Ostermeyer, A. G., Haladay, R., Winkfield, B., Frazier, M., and Zambetti, G. (1996). Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage. Mol. Cell Biol. 16, 1126–1137. Moll, U. M., Riou, G., and Levine, A. J. (1992). Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc. Natl. Acad. Sci. U. S. A. 89, 7262–7266. Moll, U. M., and Zaika, A. (2001). Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett. 493, 65–69. Murray-Zmijewski, F., Lane, D. P., and Bourdon, J. C. (2006). p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress. Cell Death Di¡er. 13, 962–972. Muttray, A. F., Cox, R. L., Reinisch, C. L., and Baldwin, S. A. (2007). Identification of DeltaN isoform and polyadenylation site choice variants in molluscan p63/p73-like homologues. Mar. Biotechnol. 9, 217–230. Muttray, A. F., Cox, R. L., St-Jean, S., van Poppelen, P., Reinisch, C. L., and Baldwin, S. A. (2005). Identification and phylogenetic comparison of p53 in two distinct mussel species (Mytilus). Comp. Biochem. Physiol. CToxicol. Pharmacol. 140, 237–250. Muttray, A. F., O’Toole, T. F., Morrill, W., Van Beneden, R. J., and Baldwin, S. A. (2010). An invertebrate mdm homolog interacts with p53 and is differentially expressed together with p53 and ras in neoplastic Mytilus trossulus haemocytes. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 156, 298–308.

34

Charles W. Walker et al.

Muttray, A. F., Schulte, P. M., and Baldwin, S. A. (2008). Invertebrate p53-like mRNA isoforms are differentially expressed in mussel haemic neoplasia. Mar.Environ.Res. 66, 412–421. Muttray, A. F., St-Jean, S., Bishay, R., van Poppelen, P., and Baldwin, S. A. (2009). Comparison of tumor-suppressor gene expression in two mussel species with different susceptibilities to haemic neoplasia, a cancer of the haemolymph. Aquatic Toxicity Workshop, La Malbaie. Nagpal, J., Jamoona, A., Gulati, N. D., Mohan, A., Braun, A., Murali, R., and JhanwarUniyal, M. (2006). Revisiting the role of p53 in primary and secondary glioblastomas. Anticancer Res. 26, 4633–4639. Nawrot, T., Plusquin, M., Hogervorst, J., Roels, H. A., Celis, H., Thijs, L., Vangronsveld, J., Van Hecke, E., and Staessen, J. A. (2006). Environmental exposure to cadmium and risk of cancer: a prospective population-based study. Lancet Oncol. 7, 119–126. Nedelcu, A. M., and Tan, C. (2007). Early diversification and complex evolutionary history of the p53 tumor suppressor gene family. Dev. Genes Evol. 217, 801–806. Nikolaev, A. Y., Li, M., Puskas, N., Qin, J., and Gu, W. (2003). Parc: a cytoplasmic anchor for p53. Cell 112, 29–40. Olberding, K. E., Kelley, M. L., Butler, R. A., and Van Beneden, R. J. (2004). A HECT E3 ubiquitin-protein ligase with sequence similarity to E6AP does not target p53 for degradation in the softshell clam (Mya arenaria). Mutat. Res. 552, 61–71. Oprandy, J. J. (1981). Isolation of a viral agent causing hematopoietic neoplasia in the softshell clam, Mya arenaria. J. Invert. Pathol. 38, 45–51. Oprandy, J. J., and Chang, P. W. (1983). 5-bromodeoxyuridine induction of hematopoietic neoplasia and retrovirus activation in the soft-shell clam, Mya arenaria. J. Invert. Pathol. 42, 196–206. Ostrand-Rosenberg, S. (2004). Animal models of tumor immunity, immunotherapy and cancer vaccines. Curr. Opin. Immunol. 16, 143–150. Pankow, S., and Bamberger, C. (2007). The p53 tumor suppressor-like protein nvp63 mediates selective germ cell death in the sea anemone Nematostellavectensis. PLoSOne 2, e782. Pantzartzi, C., Drosopoulou, E., Yiangou, M., Drozdov, I., Tsoka, S., Ouzounis, C. A., and Scouras, Z. G. (2010). Promoter complexity and tissue-specific expression of stress response components in Mytilus galloprovincialis, a sessile marine invertebrate species. PLoSComput. Biol. 6, e1000847. Pantzartzi, C. N., Kourtidis, A., Drosopoulou, E., Yiangou, M., and Scouras, Z. G. (2009). Isolation and characterization of two cytoplasmic hsp90s from Mytilus galloprovincialis (Mollusca: Bivalvia) that contain a complex promoter with a p53 binding site. Gene 431, 47–54. Rasmussen, L. P. D. (1986). Occurence, prevalence and seasonality of neoplasia in the marine mussel Mytilus edulis from three sites in Denmark. Mar. Biol. 92, 59–64. Reinisch, C. L., Charles, A. M., and Stone, A. M. (1984). Epizootic neoplasia in soft shell clams collected from New Bedford Harbor. Hazard.Waste 1, 73–81. Rhodes, L. D., and Van Beneden, R. J. (1996). Application of differential display polymerase chain reaction to the study of neoplasms of feral marine bivalves. Mar.Environ. Res. 42, 81–85. Rhodes, L. D., and Van Beneden, R. J. (1997). Gene expression analysis in aquatic animals using differential display polymerase chain reaction. In ‘‘Techniques in Aquatic Toxicology’’ (G. K. Ostrander ed.), pp. 161–183. Lewis Publishers, CRC Press, Boca Raton, FL. Riley, T., Sontag, E., Chen, P., and Levine, A. (2008). Transcriptional control of human p53-regulated genes. Nat. Rev. Mol. Cell Biol. 9, 402–412.

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

35

Rodriguez-Lopez, A. M., Xenaki, D., Eden, T. O., Hickman, J. A., and Chresta, C. M. (2001). MDM2 mediated nuclear exclusion of p53 attenuates etoposide-induced apoptosis in neuroblastoma cells. Mol. Pharmacol. 59, 135–143. Rutkowski, R., Hofmann, K., and Gartner, A. (2010). Phylogeny and function of the invertebrate p53 superfamily. Cold Spring Harb. Perspect. Biol. 2, a001131. Schumacher, B., Hofmann, K., Boulton, S., and Gartner, A. (2001). The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis. Curr. Biol. 11, 1722–1727. Shieh, S. Y., Ikeda, M., Taya, Y., and Prives, C. (1997). DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325–334. Soliman, A. S., Lo, A. C., Banerjee, M., El-Ghawalby, N., Khaled, H. M., Bayoumi, S., Seifeldin, I. A., Abdel-Aziz, A., Abbruzzese, J. L., and Greenson, J. K., et al., (2007). Differences in K-ras and p53 gene mutations among pancreatic adenocarcinomas associated with regional environmental pollution. Carcinogenesis 28, 1794–1799. St-Jean, S. D., Stephens, R. E., Courtenay, S. C., and Reinisch, C. L. (2005). Detecting p53 family proteins in leukemia cells of Mytilus edulis from Pictou Harbour, Nova Scotia. Can. J. Fish. Aquat. Sci. 62, 2055–2066. Suh, E. K., Yang, A., Kettenbach, A., Bamberger, C., Michaelis, A. H., Zhu, Z., Elvin, J. A., Bronson, R. T., Crum, C. P., and McKeon, F. (2006). p63 protects the female germ line during meiotic arrest. Nature 444, 624–628. Talos, F., Petrenko, O., Mena, P., and Moll, U. M. (2005). Mitochondrially targeted p53 has tumor suppressor activities in vivo. Cancer Res. 65, 9971–9981. Tay, K. -L. (2003). Histopathologic and histochemical biomarker responses of Baltic clam, Macoma balthica, to contaminated Sydney Harbour sediment, Nova Scotia, Canada. Environ. Health Perspect. 111, 273–280. Thanos, C. D., and Bowie, J. U. (1999). p53 Family members p63 and p73 are SAM domain-containing proteins. Protein Sci. 8, 1708–1710. Tomita, Y., Marchenko, N., Erster, S., Nemajerova, A., Dehner, A., Klein, C., Pan, H., Kessler, H., Pancoska, P., and Moll, U. M. (2006). WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization. J. Biol. Chem. 281, 8600–8606. Van Beneden, R. J. (2005). Molecular pathology of contaminant-associated neoplasia in bivalves. Proceedings of the American College of Veterinary Pathologists/American Society for Veterinary Clinical Pathology Annual Meeting, Boston, MA. Van Beneden, R. J., Walker, C. W., and Laughner, E. S. (1997). Characterization of gene expression of a p53 homologue in the sift-shell clam (Mya arenaria). Mol. Mar. Biol. Biotechnol. 6, 116–122. Vaseva, A. V., and Moll, U. M. (2008). The mitochondrial p53 pathway. Biochim.Biophys. Acta Bioenerget. 1787, 414–420. Vassilenko, E. I., Muttray, A. F., Schulte, P. M., and Baldwin, S. A. (2010). Variations in p53-like cDNA sequence are correlated with mussel haemic neoplasia: a potential molecular-level tool for biomonitoring. Mutat. Res. 701, 145–152. Wadhwa, R., Sugihara, T., Hasan, M. K., Taira, K., Reddel, R. R., and Kaul, S. C. (2002). A major functional difference between the mouse and human ARF tumor suppressor proteins. J. Biol. Chem. 277, 36665–36670. Wadhwa, R., Takano, S., Robert, M., Yoshida, A., Nomura, H., Reddel, R. R., Mitsui, Y., and Kaul, S. C. (1998). Inactivation of tumor suppressor p53 by mot-2, a hsp70 family member. J. Biol. Chem. 273, 29586–29591. Wahl, G. M., and Carr, A. M. (2001). The evolution of diverse biological responses to DNA damage: insights from yeast and p53. Nat. Cell Biol. 3, E277–E286. Walker, C. W., and B€ ottger, S. A. (2008). A naturally occurring cancer with molecular connectivity to human diseases. Cell Cycle 7, 2286–2289.

36

Charles W. Walker et al.

Walker, C., B€ ottger, S., and Low, B. (2006). Mortalin-based cytoplasmic sequestration of p53 in a nonmammalian cancer model. Am. J. Pathol. 168, 1526–1530. Walker, C., B€ ottger, S. A., Mulkern, J., Jerszyk, E., Litvaitis, M., and Lesser, M. (2009). Mass culture and characterization of tumor cells from a naturally occurring invertebrate cancer model: applications for human and animal disease and environmental health. Biol. Bull. 216, 23–39. Walker, H. A., Lorda, E., and Saila, S. B. (1981). A comparison of the incidence of five pathological conditions in soft-shell clams, Mya arenaria, from environments with various pollution histories. Mar. Environ. Res. 5, 109–123. Winston, G. W., Moore, M. N., Kirchin, M. A., and Soverchia, C. (1996). Production of reactive oxygen species by heyocytes from the marine mussel, Mytilus edulis: lysosomal localization and effect of xenobiotics. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 113, 221–229. Yee, K. S., and Vousden, K. H. (2005). Complicating the complexity of p53. Carcinogenesis 26, 1317–1322. Yevich, P. P., and Barry, M. M. (1969). Ovarian tumors in the quahog Mercenaria mercenaria. J. Invertebr. Pathol. 14, 266–267. Yevich, P. P., and Barszcz, C. A. (1978). Neoplasia in soft-shell clams (Mya arenaria) collected from oil-impacted sites. Ann. N.Y. Acad. Sci. 298, 409–426. Zhao, L. Y., and Liao, D. (2003). Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis. J.Virol. 77, 13171–13181.

C H A P T E R T W O

Stress Ecology in FUCUS: Abiotic, Biotic and Genetic Interactions Martin Wahl*,1, Veijo Jormalaineny, Britas Klemens Erikssonz, James A. Coyerz, Markus Molisx, Hendrik Schubert{, Megan Dethierjj, Rolf Karez#, Inken Kruse*, Mark Lenz*, €mzz Gareth Pearson**, Sven Rohdeyy, Sofia A. Wikstro and Jeanine L. Olsenz

Contents 1. Introduction 1.1 The concept of stress 1.2 Background studies and the constraints of monodisciplinary research 2. Methods 2.1 Data search 2.2 Data acquisition 2.3 Groupings 2.4 Effect sizes 3. The Genus Fucus 3.1 Global distribution and diversification 3.2 Life history and demography 3.3 Latitudinal and zonational shifts 3.4 Genomic introductions and invasions

39 39 41 42 42 43 43 43 57 57 58 61 62

*

IFM-GEOMAR, Kiel, Germany Section of Ecology, Department of Biology, University of Turku, Turku, Finland Department of Marine Benthic Ecology & Evolution, Centre for Ecological and Evolutionary Studies, University of Groningen, Centre for Life Sciences, Groningen, The Netherlands x Biologische Anstalt Helgoland, Alfred-Wegener Institute for Polar and Marine Research, Marine Station Helgoland, Helgoland, Germany { Institute of Biosciences, Ecology Department, University Rostock, Rostock, Germany jj Friday Harbor Labs, University of Washington, Friday Harbor, WA, USA # LLUR, Flintbek, Germany ** CCMAR-CIMAR, University of Algarve, Faro, Portugal yy Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl-von-Ossietzky University, Oldenburg, Germany zz AquaBiota Water Research, Stockholm, Sweden y z

1

Corresponding author.

Advances in Marine Biology, Volume 59 ISSN 0065-2881, DOI 10.1016/B978-0-12-385536-7.00002-9

# 2011 Elsevier LTD. All rights reserved.

37

38

Martin Wahl et al.

4. The Stressful Environment 4.1 Abiotic stress 4.2 Biotic stress 4.3 Protection against abiotic and biotic stresses 4.4 Modulation of stresses 5. Genetic Levels of Stress Response 5.1 Sensitivity versus genetic diversity of a population: stress from the evolutionary perspective 5.2 Indirect detection of adaptation to stress on relevant spatial scales 5.3 Direct detection of adaptation to abiotic and biotic stress 6. Conclusions 7. Prospects 7.1 Experiments and modelling 7.2 Next generation molecular ecology 7.3 How will climate change affect fucus? 7.4 Open questions References

64 64 71 76 77 81 82 83 84 86 87 88 89 89 91 91

Abstract Stress regimes defined as the synchronous or sequential action of abiotic and biotic stresses determine the performance and distribution of species. The natural patterns of stress to which species are more or less well adapted have recently started to shift and alter under the influence of global change. This was the motivation to review our knowledge on the stress ecology of a benthic key player, the macroalgal genus Fucus. We first provide a comprehensive review of the genus as an ecological model including what is currently known about the major lineages of Fucus species with respect to hybridization, ecotypic differentiation and speciation; as well as life history, population structure and geographic distribution. We then review our current understanding of both extrinsic (abiotic/biotic) and intrinsic (genetic) stress(es) on Fucus species and how they interact with each other. It is concluded that (i) interactive stress effects appear to be equally distributed over additive, antagonistic and synergistic categories at the level of single experiments, but are predominantly additive when averaged over all studies in a meta-analysis of 41 experiments; (ii) juvenile and adult responses to stress frequently differ and (iii) several species or particular populations of Fucus may be relatively unaffected by climate change as a consequence of pre-adapted ecotypes that collectively express wide physiological tolerences. Future research on Fucus should (i) include additional species, (ii) include marginal populations as models for responses to environmental stress; (iii) assess a wider range of stress combinations, including their temporal fluctuations; (iv) better differentiate between stress sensitivity of

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

39

juvenile versus adult stages; (v) include a functional genomic component in order to better integrate Fucus’ ecological and evolutionary responses to stress regimes and (vi) utilize a multivariate modelling approach in order to develop and understand interaction networks.

1. INTRODUCTION The shifting of environmental variables in the course of ongoing global climate change is expected to impact the performance, and ultimately the distribution, of numerous species in marine coastal systems (reviewed in Harley et al., 2006; Parmesan, 2006; IPCC Climate Change, 2007). Many shallow coastal habitats, however, are defined by a physically demanding environment with steep abiotic gradients and drastic environmental fluctuations at small spatial and temporal scales. Marine organisms living in these intertidal or shallow subtidal habitats are regularly exposed to strong water motion and subjected to extreme fluctuations in temperature, pH, irradiance, salinity or nutrient availability, and the amplitude of these fluctuations far exceeds climate changes predicted for the coming decades (e.g. Thomsen and Melzner, 2010). Although organisms in these habitats cope with ambient abiotic stresses at least to the point of transient tolerance, they must also contend with stressful biotic interactions including competition, epibiosis, parasitism and herbivory, all of which have the potential to modulate the abiotic stresses (e.g. Wahl, 2008b). Understanding how organisms in harsh and fluctuating habitats cope with single and multiple stresses is essential to clarifying and evaluating the risks of global change. Foundation species in the intertidal/shallow subtidal regions of northern hemisphere temperate coasts frequently include members of the algal genus Fucus, which typically consists of three to four zoned species across the intertidal-shallow subtidal gradient (Fig. 2.1). Their ecology, physiology and genetics have been the subjects of intense research during the past four decades, although some species have been studied since the mid-1800s. Thus, results of these many studies provide insights into stress ecology and may form the basis for understanding how global climate change will affect northern intertidal/subtidal habitats.

1.1. The concept of stress Stress is defined here as the impact of any set of abiotic and/or biotic factors that adversely affects individual ‘performance’ and ultimately impairs population growth rate through reduced individual survival, growth and/or reproduction (Grime, 1989; Vinebrooke et al., 2004).

40

Martin Wahl et al.

[(Figure_1)TD$IG]

Figure 2.1 Zonation patterns characteristic of Fucus species along (a) intertidal and (b) atidal shores of the Baltic Sea.

In this sense, stress is ubiquitous and widespread, particularly at the margins of species’ distributions (outside of which population growth remains negative) and at isolated locations within a distributional range. Stress remains a contentious concept despite its widely recognized importance. Whether or not a factor is considered stressful depends on the target organism; the intensity, duration and recurrence of the stress; and on the various interactions among stresses. As stresses typically cooccur, it is crucial to determine whether a set of stresses is likely to act additively, antagonistically or synergistically, which in turn, will determine whether the combined impact will be minor or detrimental. Understanding interactions and feedbacks is one of the fundamental challenges for understanding ecological dynamics at a variety of scales (Green and Sadedin, 2005; Christensen et al., 2006; Gamfeldt and Kallstrom, 2007) and is a necessary prerequisite for any prediction of responses to changing stress regimes. Nevertheless, most studies of stress have focused on the physiological responses of organisms to an unfavourable variable or set of variables (Crain et al., 2008; Darling and Cote, 2008; Schiel, 2009) without linking those responses to the biotic interactions among the species being stressed or to the underlying phenotypes (Davis et al., 2005; Hughes et al., 2008). Stresses act as selective agents for stress tolerance. Adaptability of a population therefore is determined by the interaction between the extrinsic components of stress—abiotic and biotic factors—and the intrinsic

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

41

component of stress (Bijlsma and Loeschcke, 2005) estimated as the standing genetic variation. If this evolutionary potential is compromised (i.e. in small, isolated populations or at range margins), then an extrinsic stress will be further magnified. It is now recognized that the temporal differential between ecological and genetic changes can be small or negligible (Spielman et al., 2004). Thus, stress regimes can have a significant and nearly simultaneous impact in both ecological and evolutionary time scales. Understanding this coupling is the major challenge of ecological and evolutionary genomics (EEG) in natural communities (Tautz et al., 2010).

1.2. Background studies and the constraints of monodisciplinary research General reviews of intertidal/shallow subtidal ecology include BenedettiCecchi (2000), Connell (1972), Gruner et al. (2008), Menge (1995); for intertidal physiological ecology, see Helmuth et al. (2002); for intertidal community genetics, see Schmidt et al. (2008) and for environmental change impact on marine ecosystems, see Harley (2006), Helmuth et al. (2006), Hillebrand et al. (2010), Korpinen et al. (2007a), Parmesan (2006), Schiel et al. (2004) and Thompson et al. (2002). Although fucoids and other macrophytic algae are sometimes included in these reviews, most reviews are overwhelmingly focused on sessile invertebrates (but see Davison and Pearson, 1996). Research on the genus Fucus has produced >1500 publications (excluding purely taxonomic contributions) over the past decades. Although the genus provides a unique system for examining individual, population, species and community level processes, these hierarchical levels have fostered persistent splits in research approaches (see review by Tomanek and Helmuth, 2002). For example, physiological ecologists are interested in testing thermal limits, photosynthetic capacity, desiccation tolerance and osmoregulation of species as the basis for upperintertidal zonation or geographical range limits. Reviews of physiological stress and photobiology in algae sensu lato include Bischof et al. (2002), Chapman (1995), Davison and Pearson (1996), Dethier and Williams (2009), Dethier et al. (2005), Schiel and Foster (2006), Wiencke et al. (1992, 2006). Community ecologists, however, focus on biotic interactions related to competition, epibiosis, predation and defence strategies against the former, as well as on phenology, reproduction, recruitment and life-history traits as the key factors shaping populations and their inter-specific interactions in a community. Specific reviews have been published on the ecology of brown seaweeds in general (Schiel and Foster, 2006), on Laminariales and Fucales (Bartsch et al., 2008), and on the Fucales (Chapman, 1995). Last but not least, molecular ecologists

42

Martin Wahl et al.

(who entered the field in the 1990s) focus on the role of genetic variation, fitness, gene flow and selection as the determinant processes leading to adaptation. Consequently, a large body of research has accumulated on the roles of various types of stress on Fucus spp. but relatively little integration or synthesis has emerged. Over the past 15 years physiological and community ecology studies have greatly expanded and become more rigorous, and when combined with advancing molecular approaches, have the potential to elucidate the complex pathways and networks of stress effects. Chapman (1995) focused his review of the genus Fucus on the years 1970–1993; our review focuses on subsequent studies. We begin our review with an overview of Fucus and its ecological importance. We then turn to a review of studies on abiotic stresses, both single and multiple, and their reported impacts at different ontogenetic stages of Fucus life history. Next we introduce biotic interactions, how they can be shifted by abiotic stresses, and how these shifts may increase or buffer stress effects. Woven throughout this part of the review we also provide the results of a meta-analysis of stress combination studies. We then turn to the genetic level and review what is known about genetic stress and the potential for adaptation in the new perspective of ecological and evolutionary genomics. In the final section, we discuss our conclusions and prospects, aiming to inspire the research agenda for the coming years.

2. METHODS In addition to a review of individual stress studies, a meta-analysis of multi-stress experiments was performed in an attempt to elucidate general effects of stress combinations on Fucus.

2.1. Data search We searched for articles in Science Citation Index Expanded (ISI Web of Science) that conducted factorial experiments with at least two stresses using the following search parameters: (1) ts = (stress* or salinity or temperature or UV or irradiation or irradiance or desiccation or emersion or pollution or eutrophication or ‘nutrient enrichment’ or disturbance or osmoregulation or ‘wave exposure’ or ‘wave energy’ or ‘water motion’ or ‘wave action’ or ‘ice scour’ or competition or herbivor* or grazing or epibiot* or epiphyt* or fouling or pathogen* or parasit*), (2) ts = (interact* or antagonis* or synerg* or additiv* or non-additiv* or multiple or combined or factorial or experiment*), (3) ts = Fucus or fucoid* or fucales or fucaceae. The search yielded a total of 488 papers, but only 41 experiments with fully crossed factorial experiments, adequate replication

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

43

and response to stress (measured as a quantitative performance variable) were chosen for our analysis (Table 2.1).

2.2. Data acquisition Figures were scanned from the original articles and the data (means and standard deviations for the different stress treatments) extracted using GRABIT v. 1.7.2 XP add-in for Microsoft Excel (Datatrend Software, USA). Some papers included multiple experiments from which data could be extracted yielding a final meta-analysis data set with 41 entries. The full data set is available in spreadsheet format in Appendix S1. Note that for biological stresses (which often use an exclusion treatment to examine the effects of herbivory and competition), the exclusion treatment without the biological stress becomes the stress-free control in our meta-analysis.

2.3. Groupings All groupings represent trade-offs, especially with respect to the number of studies available. Given the relatively small number of studies meeting our criteria and the large number of stresses tested, we opted for broad categories (Table 2.2). Data were divided into subsets that included two-way interactions between treatments (i.e. abiotic abiotic, biotic biotic, abiotic biotic, abiotic nutrient enrichment and biotic nutrient enrichment). Three-way (or higher) interaction experiments were divided into separate two-way treatment combinations. In some cases, a single experiment could be divided and used in several analyses, thus the number of experiments used is larger than the number of articles listed in Table 2.1 and the number of comparisons used in any one grouping differed among tests depending on the combinations. For example, abiotic stress nutrient enrichment and abiotic stress biotic stress involve 15 and 12 comparisons, respectively. The interaction between nutrient enrichment 1 nutrient enrichment 2 was not analysed because only two experiments were found.

2.4. Effect sizes Calculation of effect sizes followed Hedges et al. (1999). For each subset of the data we calculated the log response (effect) ratio (LRR), defined as the dimensionless ratio of the treatment over the control response value (effect = ln(stress treatment/control)). Thus, a LRR of 0.69 corresponds to a 100% increase in performance of the response variable, and a LRR of 0.69 corresponds to a 50% decrease. Each treatment and combination

44

Table 2.1

Summary of studies exploring interactions including those articles used for the meta-analysis Focal species

Performance trait

Quality of the interactiona

Methodb

Reference

Used for metaanalysis

Abiotic–abiotic Nutrient enrichment wave action

F. serratus, F. vesiculosus

Growth

M

Kraufvelin (2006)

Y

UVR salinity

F. vesiculosus

Photosynthesis

M

Nygard and Ekelund (2006)

Y

UVR temperature

F. gardneri

Germination rate, germling cell number

L

Hoffman et al. (2003)

N

Light temperature

F. vesiculosus

Microbial fouling

X: no interaction of nutrient enrichment and wave action An: low-salinity stress decreased the sensitivity of F. vesiculosus towards UV An: negative effects of UVR on germination are reduced under high temperatures. Ad: negative effects of UVR and low temperature on cell division An: low light counteracts the microfouling– enhancing impact of warming

L

Wahl et al. (2010)

N

Martin Wahl et al.

Interaction

Table 2.1

(continued) Performance trait

Quality of the interactiona

Methodb

Reference

Used for metaanalysis

Light desiccation

F. serratus

Photoinhibition

F

Huppertz et al. (1990)

N

Salinity temperature

F. vesiculosus

Germination

L

Maczassek and Wahl (in prep.)

N

Salinity nutrients

F. vesiculosus

Growth

L

Nygard and Dring (2008)

N

Desiccation wave action

F. distichus

Survival adults

L, F

Haring et al. (2002)

N

Nitrate enrichment phosphate enrichment

F. vesiculosus

Germination

L

Bergstr€ o m et al. (2003)

Y

F. vesiculosus

Biomass production

An: desiccation reduces the photoinhiting effects of high irradiation S: hyposalinity impact was enhanced by increasing temperature Ad: low nutrients enhanced the effect of low salinity Sc: desiccated thalli experienced a higher waveinduced mortality S: negative effect of phosphates enhanced by nitrate addition Ad: shading decreases biomass

F

Eriksson et al. (2006)

Y

45

(continued )

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

Focal species

Interaction

46

Table 2.1

(continued)

Interaction

Focal species

Performance trait

Nutrient enrichment shade

Parental temperature embryonic temperature

F. vesiculosus

Survival, growth

Salinity temperature

F. vesiculosu, F. distichus, F. virusoides

Growth of germlings

Quality of the interactiona

Reference

Used for metaanalysis

L

Li and Brawley (2004)

Y

L

Munda and Kremer (1977)

Y

Martin Wahl et al.

production, nutrients have no consistent effects but tend to vary among habitats of different complexity S: parental exposure to increased temperature enhanced heatshock resistance of embryos Ad-And: hypo salinities and unfavourable temperature (high and low) affect germlings negatively and the combined effect is

Methodb

Table 2.1

(continued)

Salinity temperature

Biotic–biotic Competition grazing Competition grazing

Focal species

Performance trait

Quality of the interactiona

F. vesiculosus, F. spiralis

Survival of embryos

typically less than the sum of single stressor effects An: hypersalinity minimized negative heatshock effects on survival

F. evanescens

Growth rate

F. vesiculosus

Colonization success

Ad: competition by red algal canopy and grazing impair growth rate Ad to (S) depending on grazer community. Negative effect of competition tends to be accentuated by gastropod grazing

Methodb

Reference

Used for metaanalysis

L

Li and Brawley (2004)

Y

F

Worm and Chapman (1998)

Y

F

Korpinen and Jormalainen (2008a)

Y

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

Interaction

(continued )

47

48

Table 2.1

(continued) Performance trait

Quality of the interactiona

Methodb

Reference

Used for metaanalysis

Epibiotism grazing

F. vesiculosus

Growth rate

M

Jormalainen et al. (2008b)

Y

Consumption parasitism

F. distichus

Endophytism

F

Parker and Chapman (1994)

N

Grazing grazing

F. vesiculosus

Defence induction

L

Yun etal. (2010), Long et al. (2007)

N

Competition grazing

F. vesiculosus, F. serratus

Cover

Ad: epibiotism and grazing reduce growth An: grazing by littorinids and gamarids reduces endophyte infection An: grazing by one consumer species reduces the impact of a second consumer species Ad: in F. serratus, competition by other canopyforming algae decreased cover, limpet grazing had no effect. (S): in F. vesiculosus, grazing tended to amplify the negative effect of competition

F

Jenkins et al. (1999)

Y

Martin Wahl et al.

Focal species

Interaction

Table 2.1

(continued)

Competition grazing

Abiotic–biotic Warming competition

Emersion epibiotism

Focal species

Performance trait

Quality of the interactiona

Methodb

Reference

Used for metaanalysis

F. serratus, F. vesiculosus

Cover, recruitment

Ad: competition by canopy algae and grazing reduce recruitment, sometimes to a degree that the interactive effect becomes An because there is no room for additive decrease as cover hits zero

F

Cervin et al. (2005)

N

F. vesiculosus

Cover

F

Allison (2004)

Y

F. vesiculosus

Fouling resistance

(Ad): heat-shock decreases cover development, no effect of interspecific competition Anc: emersion reduces fouling (by enhancing anti-fouling

L

Brock et al. (2007)

N

49

(continued )

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

Interaction

50

Table 2.1

(continued)

Interaction

Focal species

Performance trait

F. vesiculosus

Growth

Wave exposure grazing

F. vesiculosus

Cover

Wave exposure grazing

F. vesiculosus

Vulnerability to grazing

Increasing depth competition

F. serratus

Abundance

Irradiance grazing

F. vesiculosus

Palatability

metabolite concentration) Anc: warming reduces grazing by driving a shift from a voracious to the less voracious consumer Sd: exposure promotes negative effect of grazing An: exposure reduces isopod grazing by toughening of thallus Anc: competition with F. vesiculosus relaxes in deeper depth An: light limitation decreases mannitol content and thus attractiveness of

Methodb

Reference

Used for metaanalysis

F

Moore et al. (2007)

N

F

Jonsson et al. (2006)

Y

F

Nietsch (2009)

N

C

Malm and Kautsky (2003)

N

L

Weinberger et al. (2011)

N

Martin Wahl et al.

Warming grazing

Quality of the interactiona

Table 2.1

(continued) Focal species

Performance trait

F. vesiculosus

Growth rate

F. vesiculosus

Growth rate

F. vesiculosus

Anti-grazer defence

Desiccation grazing

F. distichus

Growth, reproduction, survival

Emersion grazing

F. distichus

Growth, reproduction

Wave exposure grazing competition

F. vesiculosus

Grazing loss

Increasing depthe grazing Increasing depthe epibiotism Irradiance grazing

Quality of the interactiona

Reference

Used for metaanalysis

F

Jormalainen and Ramsay (2009) Rohde et al. (2008)

N

L

Rohde et al. (2004)

Y

F, M

Dethier et al. (2005)

Y

C

Dethier and Williams (2009)

N

M

Engkvist et al. (2004)

N

F

Y

51

Fucus to isopod grazers S: negative effect of grazing increases with deeper depth Ad: epibiotism and increasing depth reduce growth rate Ad: light limitation decreases antigrazer defences Ad: desiccation and grazing impair growth and reproduction Anc: emersion reduces grazing pressure (but this was dependent on season) S: grazing pressure shifts from F. serratus to F. vesiculosus under increased exposure

Methodb

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

Interaction

(continued )

52

Table 2.1

(continued) Focal species

Performance trait

Quality of the interactiona

Methodb

Reference

Used for metaanalysis

Wave exposure grazing

F. vesiculosus, F. serratus

Grazing loss

M

Engkvist et al. (2004)

N

Nutrient enrichment grazing Nutrient enrichment grazing

F. vesiculosus

Colonization success

F

Colonization success

Korpinen and Jormalainen (2008a) Worm et al. (2001)

N

F. vesiculosus

Nutrient enrichment epibiotism

F. vesiculosus

Growth rate

M

Jormalainen et al. (2003)

Y

Nutrient enrichment grazing

F. vesiculosus

Growth rate

(Ad): negative effect of grazing but no effect of exposure on overall grazing loss (S to An): depending on the grazer community And: grazers counteract negative (indirect) effects of nutrient enrichment S: nutrient enrichment enhances negative effect of epibiotism An: gastropod grazing counteracts the negative effect (due to increased epibiotism) of

M

Ra berg and Kautsky (2008)

Y

Interaction

F

N

Martin Wahl et al.

(continued)

Interaction

Focal species

Performance trait

Nutrient enrichment inter-specific competition

F. vesiculosus

Colonization success

Nutrient enrichment temperature inter-specific competition Nutrient enrichment temperature intra-specific competition

F. serratus, F. evanescens

Germling growth, germling survival

F. serratus, F. evanescens

Germling growth, germling survival

Quality of the interactiona

nutrient enrichment S: nutrient enrichment enhanced the negative effect of competition S: warming and nutrient enrichment enhance competition effects Ad: temperature increased competition and reduced growth. S: in F. serratus, negative effect of competition on survival increased with warming and

Methodb

Reference

Used for metaanalysis

F

Korpinen and Jormalainen (2008a)

Y

L

Steen (2004)

N

L

Steen and Scrosati (2004)

Y

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

Table 2.1

53

(continued )

54

Table 2.1

(continued)

Interaction

Focal species

Performance trait

F. vesiculosus

Germination

Nutrient enrichment grazing

F. vesiculosus

Colonization success

Nutrient enrichment grazing shade

F. vesiculosus

Number of recruits

Nutrient enrichment grazing

F. vesiculosus

Palatability

nutrient concentration S: eutrophication favours competing annual algae more in spring than in autumn An: negative grazing effect is dampened by negative effect of nutrient enrichment Ad: low light, nutrient enrichment and grazing reduce recruitment Sc: nutrient enrichment enhances grazing

Methodb

Reference

Used for metaanalysis

F

Berger et al. (2004)

N

F

Korpinen et al. (2007b)

Y

F

Eriksson et al. (2007)

Y

L

Hemmi and Jormalainen (2002)

N

Martin Wahl et al.

Season eutrophication competition

Quality of the interactiona

Table 2.1

(continued) Performance trait

Quality of the interactiona

Methodb

Reference

Used for metaanalysis

Nutrient enrichment grazing

F. vesiculosus

Growth (apical tip divisions)

M

Hemmi et al. (2005)

Y

Nutrient enrichment grazing Nutrient enrichment competition grazing

F. serratus

Palatability

M

Kraufvelin et al. (2006)

N

F. vesiculosus

Biomass

(S): nutrient enrichment tended to increase grazing losses Sc: nutrient enrichment enhances grazing An: nutrients enhance competition (when grazers are present)

F

Worm et al. (2001)

Y

Ad, additive (sum = A + B), no significant interaction; S, synergetic (sum > A + B), positive interaction; An, antagonistic (sum < A + B), negative interaction; (), indicate a non-significant tendency (0.05 < P < 0.1) and X, no interaction. b L, lab experiment; M, flow-through outdoor mesocosm experiment; F, field experiment and C, correlative field data. c Interactive effect on algal performance interpreted from non-factorial designs where algal performance was not directly measured. d Interaction suggested, but statistical test lacking. e Light availability was the main abiotic factor (decreasing with increasing depth). a

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

Focal species

Interaction

55

56

Table 2.2

Martin Wahl et al.

Stress groupings used in the meta-analysis

Abiotic stress

Biotic stress Nutrient enrichment

Depth Desiccation Irradiance Salinity Shading Temperature Wave action Competition Herbivory Nitrogen and phosphorous enrichment

was compared separately against a ‘stress free’ control (Elser et al., 2007). For each separate experiment we calculated the natural LRRs using the following equations: LRRmain effect stress A ¼ ðln MeanA1B0 Þ ðln MeanA0B0 Þ

ð1Þ

LRRmain effect stress B ¼ ðln MeanA0B1 Þ ðln MeanA0B0 Þ

ð2Þ

LRRcombined effect of stress A and B ¼ ðln MeanA1B1 Þ ðln MeanA0B0 Þ; ð3Þ where A and B refer to the different stresses in the two-way interaction. A1 and B1 refer to the treatments with the stresses added, and A0 and B0 refer to the unstressed controls. In order to account for differences in replication and precision associated with individual studies, we calculated average effects of experimental treatments as weighted averages of the natural log response ratios (LRRs) obtained in Equations (1)–(3) (LRR*; Hedges et al., 1999). Statistical significance was tested by calculating the confidence interval around the weighted average (Hedges et al., 1999). An effect was considered significant if the confidence interval didnot cross the zero line. Given two stresses, an additive interaction is the sum of the two stresses. While the basic analysis provides an indication as to whether there is a synergistic or antagonistic trend, as compared to a simple additive combined effect, it does not explicitly test for an interaction. Therefore, we conducted a second analysis using the equations developed in Gruner et al. (2008). Note that in this analysis we used unweighted effect sizes. In this approach, an interaction effect is additive if the interaction LRR does not differ from zero (i.e. the confidence interval does cross the zero line) and thereby generates a true statistical test of whether

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

57

the interaction effect differs from additivity. Given two stresses, synergistic interactions are indicated by a negative value and antagonistic interactions by positive interaction LRRs.

3. THE GENUS FUCUS We describe stress ecology using Fucus because (1) the genus includes a set of ecologically important foundation species with a wide longitudinal and latitudinal distribution, (2) most Fucus species occur in stressful habitats of the intertidal or shallow subtidal and (3) some species (notably Fucus vesiculosus) are already experiencing losses and gains in their geographic distributions.

3.1. Global distribution and diversification Fucus spp. commonly occur along protected and exposed rocky intertidal and shallow subtidal shores (Fig. 2.1), as well as in tidal marshes, over a wide latitudinal gradient within the North Pacific Ocean, North Atlantic Ocean and Baltic Sea (L€ uning, 1990). The genus is rare in the Mediterranean, where a single species is found only in the northern Adriatic Sea. Only one species, the cold-adapted Fucus distichus (see Coyer et al., 2006a for taxonomic notation used in this review) is found in both the Pacific and Atlantic oceans, ranging from Japan and Alaska in the North Pacific to Svalbard, northern Norway, the White Sea, Iceland and northern Scotland in the North Atlantic (with recent introductions to the North and Kattegat Sea and Bergen Harbor (Wikstr€ o m et al., 2006; Sjøtun, personal communication). Other species are found only in the North Atlantic (the Pacific occurrence of Fucus spiralis likely is an introduction; Coyer et al., 2011a and references therein), where they range from northern Canada and northern Norway/White Sea to the Iberian peninsula, which is the southern limit for many but not all species. For example, while the distributions of Fucus serratus/high shore F. spiralis and F. vesiculosus end in northern and southern Portugal, respectively, the southern form of F. spiralis occurs in southern Portugal, the Canary Islands, the Azores and northern Morocco (Coyer et al., 2003; Billard et al., 2010; Zardi et al., unpubl.). In the North Atlantic, most Fucus species are characterized by hotspots of genetic and species diversity in southwestern Ireland and the Brittany peninsula of France or NW Iberia, all putative glacial refugia during the Last Glacial Maximum (Coyer et al., 2003; Hoarau et al., 2007; Coyer et al., 2011c; Neiva et al., 2010). On the other hand, genetic diversity is markedly reduced in populations of F. vesiculosus along the North American Atlantic coast a likely signature of postglacial recolonization

58

Martin Wahl et al.

from Europe (Muhlin and Brawley, 2009) and in F.serratus at the edges of its southern distribution on the Iberian Peninsula (Coyer et al., 2003). Rapid ecotypic diversification is reflected in patterns of zonation along the shore, as well as morphological variability, hybridization and polyploidy. The ecotypic attributes have resulted in >150 described species, subspecies and forms (www.AlgaeBase.org, Guiry and Guiry, 2010), although recent molecular phylogenetic studies suggest the number of taxa to be <10 (Serr~ao et al., 1999a; Coyer et al., 2006a; F. Canovas, C. Mota, E. Serrao and G. Pearson, unpubl. data). The discovery of sibling and/or cryptic species by molecular methods is relevant to both ecological and evolutionary questions regarding stress, because smaller bounds need to be established on the distributions of some species. For example, recent work has demonstrated conclusively that Fucus radicans is not an ecotypic variant of F. vesiculosus (Bergstr€ o m et al., 2005), but is a separate species exclusively confined to the northeastern and northwestern Baltic and has evolved in the past 400–2000 years (Pereyra et al., 2009). Other recent studies have revealed that the highand low-shore form of F. spiralis are reproductively isolated (Billard et al., 2010) and allopatric F. spiralis found in southern Portugal and North Africa constitutes a new species (Coyer et al., 2011a; Zardi et al., unpubl.). Hybridization and introgression, which further complicate our understanding of the species’ distribution, are well documented between F. vesiculosus and F. spiralis (Wallace et al., 2004; Billard et al., 2005a,b, 2010; Engel et al., 2005; Coyer et al., 2011a), as well as between Fucusceranoides and F. vesiculosus (Neiva et al., 2010), and F. serratus and F. distichus (Coyer et al., 2002a,b, 2007). Some hybrids (F. vesiculosus F. spiralis) or polyploidy variants (F.vesiculosus, Wallace et al., 2004; Coyer et al., 2006b) commonly occur, while other hybrids are less fit (F. serratus F. distichus) (Coyer et al., 2007). In conclusion, a confusing taxonomy combined with complex processes of hybridization and rapid ecotypic differentiation make it advisable to genetically identify Fucus entities at the outset of any proposed study. With few exceptions (e.g. F.serratus, high shore F.spiralis, F.vesiculosus with vesicles), traditional (visual) taxonomy is unreliable.

3.2. Life history and demography Fucoid life histories are monophasic with a diploid adult (van den Hoek et al., 1995). Haploid sperm and eggs are produced in numerous conceptacles within each of several large receptacles on apical tips of thallus branches. Reproduction is iteroparous, with both dioecious and hermaphroditic species. Eggs are fertilized close to (or on) the female and the diploid zygotes typically settle and mature within meters of the female

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

59

parent (Serr~ao et al., 1997; Pearson and Serr~ao, 2006; Schiel and Foster, 2006, and references therein). Limited dispersal of gametes, together with synchronous production of eggs and sperm, increases the probability of selfing and inbreeding in hermaphroditic species (Coleman and Brawley, 2005; Perrin et al., 2007) and possibly breeding among closely related parents in dioecious species. Fertilization is dependent on both water motion and temperature. Release of gametes typically occurs in F. vesiculosus at low turbulence and during daylight (Pearson and Serr~ao, 2006). Calm conditions are required to ensure fertilization success and facilitate recruitment of the non-dispersive zygotes within the adult habitat. Active photosynthesis is needed to detect turbulence (Serr~ao et al., 1996b); in the absence of turbulence, photosynthesis depletes dissolved inorganic carbon in the boundary layer covering the thallus and this depletion, in addition to a general semilunar periodicity, is used as one cue for synchronous gamete release (Pearson and Brawley, 1998; Pearson et al., 1998). Fertilization success typically approaches 100% (Berndt et al., 2002). Temporal patterns of reproduction may vary even at the local scale. For example, the reproductive period varies between 4 and 12 months among F. vesiculosus populations separated by 100 km or less along the German Baltic coast (K. Maczassek, personal communication). Along the Swedish South and East coast summer- and autumn-reproducing forms of F. vesiculosus are heterogeneously distributed with a tendency for a dominance of autumn breeders on the central mainland coast and a dominance of summer breeders along the coasts of islands (Oland and Gotland) (Berger et al., 2001). Autumn recruits are expected to suffer less from sedimentation and competing ephemeral green algae than spring recruits (Berger et al., 2004). Although such differentiation of reproductive phenology may be expected to create temporal isolation and promote genetic differentiation, no genetic differentiation (using five microsatellites) was observed between the summer and autumn reproducing forms (Tatarenkov et al., 2007). Mortality in fucoids is typically very high during the microscopic stages (Schiel and Foster, 2006). Estimates of survival from an egg to a benthic settler vary from 1.5 to 10%, and from a settler to a visible germling from 5 to 12% (Chapman, 1995). As thalli grow, mortality decreases but remains temporally variable (Wright et al., 2004). Egg production in the well-studied F. distichus is about 1 105– 2 106 eggs m2 mo1 (Ang, 1991). Of these, only a few hundred per m2 (i.e. less than 0.015%) succeed in the transition to a visible juvenile (Fig. 2.2). Fucus spp. typically reach an age of 5–10 years (Chapman, 1995). Due to high mortality of early life-history stages, there is often a paucity of juveniles in the mature stands, rendering populations vulnerable to catastrophic mortality (Dudgeon and Petraitis, 2005).

60

Martin Wahl et al.

[(Figure_2)TD$IG]

Figure 2.2 (a) Typical survivorship curve of Fucus species (expressed as surviving individuals N m2) through sequential developmental stages and (b) an indication of the relative sensitivities of the various stages to various stresses. The survivorship curve is an approximation based on data from F. distichus (Ang, 1991).

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

61

However, two recruitment strategies help counter the high early mortality and contribute to population replenishment. First, the microscopic stages may exhibit delayed growth of several months, which creates a propagule bank under the canopy (Schiel and Foster, 2006). A second strategy involves fragmentation (Tatarenkov et al., 2005) and regeneration (Schiel and Foster, 2006), which allows population re-growth after catastrophic mortality. Asexual reproduction by reattachment of fragments has been documented at high frequency only in F. radicans (Tatarenkov et al., 2005). Regeneration of fronds from attached adult holdfasts is common in F. vesiculosus (Kiirikki and Ruuskanen, 1996) and allows population recovery after mechanical damage, that is, by ice scour. Dispersal is highly variable. As distinct rocky intertidal habitats are often separated by soft bottom areas unsuitable for Fucus attachment, reproduction takes place mostly among the local individuals (Coyer et al., 2003). Direct estimates of average dispersal distances of fucoid zygotes are only a few meters, rarely exceeding 10 m (Dudgeon and Petraitis, 2001). Indirect estimates based on spatial auto-correlation and pairwise FST () analysis suggested a panmictic unit (area of random reproduction) of 0.5–2 km (see Section 5) (Coyer et al., 2003). Longdistance dispersal and gene flow are possible by rafting of intact individuals or detached pieces of receptacle-bearing thalli (Viejo and Arrontes, 1992; Thiel and Gutow, 2005).

3.3. Latitudinal and zonational shifts Fucus spp. have high photosynthetic rates, surpassing phytoplankton species on a per-unit, bottom-area basis, and provide settlement substratum, food and shelter for a large number of microbial, algal and animal species (e.g. Kautsky et al., 1992; Middelboe et al., 2006; Rohde et al., 2008). Consequently, population, as well as community dynamics of Fucus spp. will affect the well-being of numerous community members (Altieri et al., 2007). In the atidal Baltic Sea, F. vesiculosus has declined substantially in large areas over the past 50 years (e.g. Berger et al., 2004; e.g. Vogt and Schramm, 1991, and references therein). The frequently observed upward shift of the lower depth limit has been attributed to direct and indirect effects of eutrophication such as decreased light availability and increased competition and sedimentation (Kautsky et al., 1986; Eriksson et al., 1998; Berger et al., 2004; Korpinen et al., 2007b). However, changes in top-down regulation through a cascading effect of fish abundance on mesograzer abundance may contribute to local shifts by impacting competing filamentous algae (Eriksson et al., 1998) or the recruitment success of Fucus spp. (Korpinen et al., 2007a). Ongoing eutrophication in the Baltic changes the composition of F. vesiculosus associated invertebrate

62

Martin Wahl et al.

community (Korpinen et al., 2010) and the decline of F.vesiculosus populations is expected to entail even more substantial changes (Wikstr€ o m and Kautsky, 2007). Salinity sets the distributional edge for F. vesiculosus in the northern Baltic Sea, which is expected to shift southwards with the climate change associated increase in precipitation in the northern catchment area (The BACC Author Team, 2008). A more complex situation exists along the rocky intertidal shores of the North Atlantic, as at least 2–3 species of Fucus commonly co-exist along with the large fucoid Ascophyllumnodosum and small, high intertidal Pelvetiacanaliculata. Latitudinal range shifts in seaweeds associated with sea surface temperature increases have been documented along the Portuguese coast (Lima et al., 2007). There is also evidence that the southernmost populations of F. serratus are maladapted and more vulnerable to stresses than more northerly core populations (Pearson et al., 2009). Although declines in overall abundance of fucoids (mostly A. nodosum) have been reported from a number of European areas (Davies et al., 2007), the specific changes in coverage of Fucus spp. have not been quantified. Reasons for fucoid decline have ranged from increased limpet grazing to increased numbers/intensity of storms. Similarly, the relative abundances of F. vesiculosus, F. distichus and F. spiralis are changing in the Canadian Maritimes, where F. vesiculosus is gradually increasing its cover within A. nodosum beds independent of harvesting regimes (Ugarte et al., 2010). The effect of climate change on range shifts in Fucus spp. specifically and along the rocky intertidal more generally is unknown (Thompson et al., 2002). Nevertheless, growing evidence suggests that the distributional ranges of Fucus spp. are in a process of reorganization as a consequence of species introductions and stresses associated with climate change, eutrophication and, more indirectly, overfishing (e.g. Ugarte et al., 2010).

3.4. Genomic introductions and invasions Species introductions are also a major factor in shaping geographical distributions and community dynamics, including competitive displacement. For example, F. serratus was introduced to both Nova Scotia and Iceland from Europe within the last 100–200 years and in each area has expanded at a rate of 0.2–0.6 km yr1 (Coyer et al., 2006c; Brawley et al., 2009). In another example, Fucus evanescens (=F. distichus) was introduced to the Oslo fjord in the mid-1890s then expanded south into western Sweden (by 1933) and the southwestern Baltic (by 1992), where it experienced less herbivore pressure both compared to native Fucus species and to populations in the native range (Wikstr€ o m et al., 2006; Forslund et al., 2010). While the genus Fucus is considered a powerful invader itself (Williams and Smith, 2007), it may be impacted by invaders into its home

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

63

range. Thus, the introduced red alga Gracilaria vermiculophylla impacts the native Fucus vesiculosus doubly, by direct competition and by favouring Fucus grazers (Idotea baltica) to which Gracilaria itself is relatively insensitive (Weinberger et al., 2008). Another important aspect of species introductions is the notion of genomic invasions (e.g. Mallet, 2005), which can be inter-specific (i.e. congeneric contact between sibling species of Fucus) and/or intra-specific (i.e. conspecific contact among different biogeographic populations of a single species). In a natural example of genomic invasion, a process of ‘genetic surfing’ involving asymmetric introgression and exclusive northward spread of alien (F.vesiculosus) organellar genomes following postglacial range expansion in F. ceranoides was recently discovered (Neiva et al., 2010). In this case, the background nuclear genome of F. ceranoides was observed to retain remarkable integrity despite organellar introgression. Congeneric introductions of Fucus species create hybrid zones (as described in Section 3.1), thus complicating species identification and potentially resulting in long-term competitive displacement of parentals through evolution of new lineages via multiple generations of reproductive F1 hybrids, as well as by exchange of genes between parental species (introgression) via backcrossing with hybrids. In extreme cases, extensive backcrossing and interbreeding among hybrids could lead to complete homogenization of parental genomes or a hybrid swarm (extinction through hybridization) (see Coyer et al., 2007). Perhaps even more worrisome are conspecific introductions that go unnoticed. A new species of Fucus might be easily recognized as not part of the local marine flora but a foreign population of a resident Fucus species would not be. This is almost certainly the case in Nova Scotia (Brawley et al., 2009) where F. serratus was conclusively identified as an introduction from Europe and was of interest because it was not part of the Canadian Maritime flora. However, it is highly probable that European F. vesiculosus is also a part of the Canadian F. vesiculosus flora although this has not been confirmed. The significance of this from the stress perspective lies in the fact that conspecific hybridization potentially leads to outbreeding depression and lower fitness of an individual or population, as has been shown in intertidal copepods (Edmands et al., 2005). Thus, increased extrinsic stresses might have even greater effects on such populations. Alternatively, it is also possible that intra-populational hybridization might simultaneously release beneficial variation and faster development (Edmands, 2008). Doubtlessly, humanmediated transport will increasingly bring hitherto separated species and populations into contact, as will climate change. For example, as the Arctic Ocean opens on a more permanent basis, contact between Pacific and Atlantic biotas will accelerate (Vermeij and Roopnarine, 2008).

64

Martin Wahl et al.

4. THE STRESSFUL ENVIRONMENT While marginal habitats may be dominated by single stresses (e.g. low salinity in the eastern Baltic, low light with increasing depth), environmental change typically involves the simultaneous shift of many stresses (Harley et al., 2006; IPCC Climate Change, 2007; Darling and Cote, 2008; Przeslawski et al., 2008). It is therefore important to understand the nature and influence of single stresses as well as their many combinations.

4.1. Abiotic stress 4.1.1. Single stresses Single stresses along environmental gradients can influence the performance and distribution of Fucus spp. although acclimation and adaptation to marginal and stressful habitats is possible to some degree (Kawecki, 2008). At the largest scale, latitudinal gradients (thousands of kilometres) are governed primarily by sea surface temperatures and light regimes (L€ uning, 1990). At regional scales (tens of kilometres) there are transitions between fully marine and low salinity conditions in fjords and estuaries, as well as eutrophication gradients. Finally, the local scale (several metres) includes (1) the intertidal, where desiccation, high light and excessively high and low temperatures (air and water) create sharp and fluctuating gradients over a distance of a few metres and (2) the subtidal, where light and turbulence can be rapidly attenuated by depth, the former being further modulated by plankton blooms, turbidity or epibiosis. Below we provide an overview of the major abiotic stresses and how their effects may vary with Fucus life-history stage. 4.1.1.1. Irradiance Light is likely the most variable abiotic component of inter- and subtidal shores (Schubert et al., 2001) and is an obvious essential resource for all photo-autotrophic organisms. Nevertheless, both, an excess and deficit of light are significant stresses. Low-light stress occurs when irradiance is below the light compensation point of a photosynthetic species and compromises carbon accumulation (Lehvo et al., 2001; Middelboe et al., 2006). In the northern Baltic Sea, for instance, the lower distribution limits of F. vesiculosus correlate strongly with light penetration through the water; and in exposed areas (low sedimentation) F. vesiculosus individuals can survive down to 9–10 m depth (Kautsky et al., 1986; Eriksson and Bergstr€ o m, 2005). When F. vesiculosus of the western Baltic, where the lower distributional limit is about 3 m, is transplanted to greater depths, some acclimation to the resulting low-light stress occurred (Rohde et al., 2008). However, the observed increase in chlorophyll-a concentration (by a factor 1.4 following transplantation from 1 to 6 m depth) does not compensate for

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

65

the simultaneous decrease in irradiance (by a factor 7.3). With respect to seasonal irradiance regimes, F.vesiculosus is able to acclimate by shifting its compensation point between 35 mmol m2 s1 (summer) and 8 mmol m2 s1 (winter) (Middelboe et al., 2006). Similarly, in the northeastern Pacific intertidal F. distichus becomes saturated at 1000 mmol m2 s1 in summer and 100 mmol m2 s in winter (Dethier and Williams, 2009). In the western Baltic the daily dose of irradiation (24 h average) falls below the compensation point of F.vesiculosus between October and March at 1 m depth and between August and April at 3 m depth (Wahl, unpublished) but—following the law of reciprocity—a few hours of high light per day may suffice for an equilibrated energy budget. Fucus species, like many brown algae, are able to store surplus solar energy in the form of mannitol (Lehvo et al., 2001), which sustains individuals through extended periods of sub-compensation. However, the observation that F.vesiculosus can survive the dark winter period in the western Baltic at 1 m depth but rarely occurs at 3 m depth or deeper (Rohde et al., 2008) may indicate limitations in the energy storage. Though it is likely that low-light stress affects all developmental stages equally, smaller individuals are often subject to additional shading by larger neighbours as a result of their understory position. In the western Baltic Sea, the development of juvenile F.vesiculosus over a growing season was limited by shade from the adult canopy in a field experiment manipulating both light and canopy cover (Eriksson et al., 2006). Light exceeding the saturation level for photosynthesis may produce reversible photoinhibition as a protection mechanism or, at more extreme intensities, irreversible photodamage (Huppertz et al., 1990). Photodamage only occurs when the cellular mechanisms of photo-protection are exceeded. Particularly severe stress may be caused by highenergy short wavelengths (UVA and B), which have negligible photomorphogenic effects, but potentially large photo-destructive effects (Hanelt et al., 1997). Fucus species are often exposed to high levels of UV radiation, especially for intertidal populations during emersion. Repair mechanisms or shield compounds can accumulate in tissue to reduce UV stress, but may bear some metabolic cost and, thus, reduce physiological performance. While intertidal populations of Fucus exhibit regular photoinhibition, mostly in response to afternoon intensities, photodamage and reduction in physiological performance seem rare (Huppertz et al., 1990; Hanelt et al., 1997; Michler et al., 2002). The high mitotic activities during germination may be particularly susceptible to UV radiation (Wiencke et al., 2000), although some adaptation to increased UV in particular habitats (e.g. UV absorbing compounds) can be expected. For example, embryos and juveniles of F. distichus in the upper intertidal on Spitsbergen, and F. serratus in the mid-intertidal on Helgoland, exhibited high sensitivity to UVA and

66

Martin Wahl et al.

particularly UVB radiation at natural levels, while upper-intertidal F. spiralis from Helgoland were insensitive (Schoenwaelder et al., 2003). UV sensitivity in F. serratus was highest at the zygote stage and decreased non-linearly with age (Altamirano et al., 2003). Phenolic phloroglucinol (a naturally occurring monomer of brown algal phlorotannins) tends to be more concentrated in younger tissue and was found to provide UV protection for developing embryos (Schoenwaelder et al., 2003). Early life stages of F.spiralis are less sensitive to ultra-violet radiation (UVR) than those of F. serratus presumably due to the higher density of phlorotannincontaining physodes in the former (Schoenwaelder et al., 2003). 4.1.1.2. Temperature Temperature stress is typically caused by the long-wavelength range of solar radiation (720 nm–300 mm), which is photosynthetically inactive, but thermally effective. The severity of stress caused by warming depends not only on the absolute temperature but also on the duration of exposure to it in air (emersion) and water (submersion), and the developmental stage considered. In intertidal areas, both high- and low-temperature stresses are generally more extreme during emersion. While germination success of F. vesiculosus is reduced by 90% at 25 C compared to 15 C in Kiel Bight (Germany), adults during emersion can survive transitory heating of up to 45 C (Maczassek and Wahl, unpublished). Moderately warm temperatures, as in many other poikilotherm species, actually increased growth in A. nodosum (Keser et al., 2005). Severe high-temperature stress begins when the rate of protein denaturation cannot be neutralized by mechanisms such as increased production of chaperons (heat-shock proteins) or acceleration of protein biosynthesis (Csikasz-Nagy and Soyer, 2008). However, stress can also occur at sub-denaturating temperatures because impaired biochemical pathways may accumulate harmful intermediates (Davison and Pearson, 1996) and/or because repair mechanisms may be costly (Weidner and Ziemens, 1975). Elevated temperatures also negatively influence membrane properties that are crucial for gas exchange and nutrient uptake (Maheswari et al., 1999). Low-temperature stress reduces metabolic rates, but can be partially compensated by an increase in the concentration of key enzymes (reviewed in Middelboe et al., 2006). Nonetheless, growth may be severely reduced in winter (but see Lehvo et al., 2001; Dethier and Williams, 2009) and biomass losses to grazing, ice scouring or wave action cannot be regained before spring growth. As fucoid tissue can recover from extended freezing (Davison et al., 1989; Pearson and Davison, 1993; Pearson et al., 2000), the primary detriment of ice scouring is physical detachment of the holdfast. In some species, holdfasts remain intact following ice scour and may sprout new fronds in the subsequent growth season (see Section). In the northern Baltic Sea, subtidal F. vesiculosus

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

67

have two distinct strategies to avoid ice damage (Kiirikki and Ruuskanen, 1996). In exposed areas, pack ice can reach several meters depth and an effective regeneration from holdfasts enables fast recovery of the vegetation in the shallow zone. In sheltered areas, ice forms on the surface and here the large growing individuals decrease their buoyancy during winter, which causes them to lie flat on the bottom and thereby prevent them from freezing into the ice cover. 4.1.1.3. Desiccation Extended exposure to air, especially in summer, can lead to variable degrees of desiccation for intertidal species of Fucus. Severe desiccation and the associated osmotic stress, especially when combined with high temperatures and light, can increase mortality (Pearson et al., 2009). For example, increasing duration of daily airexposure for Fucus gardneri (F. distichus complex) had a negative effect on the growth rate of the adult thalli, as well as a cumulative negative effect on survival of all life-history stages (Wright et al., 2004). Juveniles are most susceptible to emersion and associated stresses (Schoenwaelder et al., 2003; Henry and Van Alstyne, 2004). For example, fucoid germlings lose their photosynthetic ability within a few hours of air exposure, although they can recover quickly after re-immersion (Lamote et al., 2007). High mortality may result from desiccation, high light and high temperatures (Dudgeon and Petraitis, 2001; Wright et al., 2004). The smaller life stages, however, may gain a refuge from emersion stress under a canopy of adults (Lamote et al., 2007). For example, germling survival under an intertidal canopy approaches 100%, whereas survival is near zero on adjacent bare rock or on exposed habitats (Brawley and Johnson, 1991). 4.1.1.4. Pollution Pollution is defined as the accumulation of toxic compounds and/or abnormally high levels of nutrients in a local environment and/or tissue. Toxic compounds are an important marine stress because of bioaccumulation (Volterra and Conti, 2000). Fucus spp. readily adsorb and accumulate heavy metals (e.g. Mata et al., 2008) with concomitant negative effects on germination and growth (Andersson and Kautsky, 1996; Brooks et al., 2008; Nygard and Dring, 2008). In addition, a biotic toxin (nodularin) produced by cyanobacteria is biosorbed by F. vesiculosus and induces oxidative stress (Pflugmacher et al., 2007). Increased nutrient loads mostly affect macroalgae indirectly through increased shading (pre-empting light) (e.g. Vogt and Schramm, 1991) and organic sedimentation (pre-empting space for recruitment; Eriksson and Johansson, 2003). Nutrient enrichment may enhance surface-fouling ranging from biofilms (Wahl et al., 2010) to ephemeral macroalgae (Korpinen et al., 2007b) and epifauna with diverse effects on the host (see Section 4.2). Nutrient enrichment also alters the selective environment favouring fast growing species (Berger et al., 2004) or by

68

Martin Wahl et al.

changing the palatabilty of the thallus to grazers (Hemmi and Jormalainen, 2002). Direct effects arise through high concentrations of nitrate, which delayed attachment and decreased germination success in F. vesiculosus zygotes (Bergstr€ o m et al., 2003). 4.1.1.5. Osmotic stress Salinity stress mostly affects populations in the middle and uppermost intertidal. Rainfall increases the water potential (C ) of the environment, whereas evaporation and desiccation obviously decreases the potential. Sensitivity to low-salinity stress varies enormously among Fucus species, populations and (presumably) life-history stages (e.g. Pearson et al., 2000). For example, F. vesiculosus and F. radicans have adapted to the low salinity of the Baltic Sea (Serr~ao et al., 1996a), but fertilization success in F. serratus decreases substantially with strongly reduced salinity: a fertilization success of 87% at 9 psu declines to 5% at 6 psu (Malm et al., 2001). In a western Baltic population of F. vesiculosus, however, fertilization success was not affected by salinity between 7 and 17 psu (Maczassek and Wahl, unpublished). Low salinity decreases swimming performance and fertilization ability of fucoid sperm (Serr~ao et al., 1996a) and increases the rate of polyspermy (Serr~ao et al., 1999b). The present range of F. vesiculosus in the Baltic Sea corresponds to the osmotic tolerance of its gametes (Serr~ao et al., 1996a), illustrating that environmental factors affecting performance of a particular life-history stage may be important determinants of distributional ranges and colonizing abilities. 4.1.1.6. Water motion Water motion is an important consideration for shallow water organisms; as with light, too much or too little is stressful to an individual. In the case of Fucus and other large macrophyte genera, increasing flow speeds and/or wave action increases mechanical stress through increased drag, which must be counterbalanced by structurally resistant thalli and holdfasts (Haring et al., 2002). A negative correlation exists between increasing water motion and thallus size, as evidenced by extensive piles of beach wrack following high wave action. In F. vesiculosus and F. spiralis, a flow speed of 7–8 m s1 completely dislodges individuals larger than 10 cm (Jonsson et al., 2006). Consequently, exposed sites have smaller-sized individuals than sheltered sites, either due to high mortality (especially of the larger individuals) or size reduction by thallus tattering of the larger individuals (Blanchette, 1997). The risk of dislodgement is more pronounced in areas where Fucus attaches to pebbles or mussel shells in lieu of bedrock. One strategy to deal with wave-induced forces is thallus toughening. In both the North Sea and the Baltic Sea, thalli from exposed F.vesiculosus were 30% more resistant to tear and breakage as compared with conspecifics from more sheltered sites (Nietsch, 2009). The formation of

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

69

bladders on F. vesiculosus seems to be a disadvantage under conditions of high turbulence and is negatively correlated with wave exposure (Burrows and Lodge, 1951). Fucoids enhance fertilization success by releasing gametes only under hydrodynamic conditions that optimize sperm–egg encounters (Pearson and Brawley, 1996; Serr~ao et al., 1996b; Pearson and Serr~ao, 2006). Water motion can interact with timing of gamete release during tidal cycles in species-specific ways, affecting gamete and zygote dispersal (Ladah et al., 2008). Low wave exposure is also critical to successful attachment and survival of zygotes (Vadas et al., 1990), and the rate of fucoid recruitment is negatively correlated with wind speed (Lamote and Johnson, 2008). Consequently, water motion strongly influences local distribution patterns of Fucus spp. (Ladah et al., 2003, 2008). Conversely, calm conditions leading to a thicker boundary layer result in a slower absorption of nutrients and CO2, and a faster accumulation of O2 and exudates at the thallus/water interface (Jørgensen and Revsbech, 1985). The effects of depletion/enrichment, however, may be compensated for by morphological adaptations, enhancing surface area and thallus rugosity that in turn reduces the boundary layer by enhanced microturbulence (Steen, 2003; Stewart and Embrey, 2003). Furthermore, calm conditions may facilitate sedimentation on algal thalli (Umar et al., 1998), resulting in shading and oxygen deficiency (if the sediment is rich in organic particles) (Kautsky et al., 1986; Duggins et al., 1990). High sedimentation rates on rocky substrata also impede attachment of Fucus embryos and decrease the survival and growth of juveniles through scour and burial (Eriksson et al., 1998; Chapman and Fletcher, 2002; Schiel et al., 2006). In field and laboratory bio-assays, increasing amounts of deposited matter have been shown to decrease germling survival of F. vesiculosus by >60% (Berger et al., 2003), while experimental removal of sediment in the field increased recruitment twofold (Eriksson and Johansson, 2003). Thus, sedimentation regimes partly define recruitment opportunities of fucoid propagules, and strongly influence distributional patterns at the regional and local scale (Eriksson et al., 1998; Berger et al., 2003; Eriksson and Johansson, 2005). 4.1.2. Simultaneous abiotic stresses Stresses vary strongly in space and time. Spatial variation is usually linked to habitat properties such as depth, exposure or geographical position. Temporal variations may be rhythmic (diurnal, seasonal, decadal) or stochastic (rainfall, storms). Likewise stress sensitivity of an individual will vary with genotype, life-history stage or physiological phase (Fig. 2.2), which will determine the impact of a given stress at various scales (see Section 3.2). Temporal variations tend to decrease with increasing depth (e.g. Wahl et al., 2010) and lower latitudes. When different stresses

70

Martin Wahl et al.

[(Figure_3)TD$IG]

Figure 2.3 Hypothetical effects of single environmental factors on performance. Condition sets A and B exemplify conditions in different habitats or seasons where the combined effects of stressors determine a species’ performance. Environmental shift typically realizes as changes in the levels of several stressors, that is, a change in stress regime. If single stressor effects alone are considered, the stressor level with a minimum performance within a set of conditions determines performance. With additive stressor effects, performance is determined by the sum of negative stressor effects. With interactive stressor effects, performance cannot be predicted without knowledge on the quality of the interaction.

fluctuate at different frequencies and/or amplitudes, the variability of their combined action may be complex (Fig. 2.3). Responses to combined abiotic stresses included antagonistic (5), synergistic (4) and additive (5) effects (Table 2.1). These studies collectively highlight the role of local conditions and local adaptation to stresses, not only in determining tolerance of single stresses but also in modulating the combined stress effects. For example, low nutrients and low salinity together decreased the performance of F.vesiculosus in an additive manner (Nygard and Dring, 2008). Antagonistic interactions were found in stresses including temperature, salinity, desiccation and UV, but the type of interaction was sometimes equivocal. In one case, low-salinity stress decreased the sensitivity of F.vesiculosus to UV in a population originating from the Atlantic (Nygard and Ekelund, 2006), but in a population from the Baltic Sea, the effects of salinity and UVR were additive. In another example, the combined effect of UV and temperature was either antagonistic or additive depending on the response variable (Hoffman et al., 2003). The microfouling–enhancing effect of warming in one study was buffered by light reduction (Wahl et al., 2010). Similarly, emersion and consequent partial desiccation antagonistically reduced the damage inflicted by high irradiation (Huppertz et al., 1990). Antagonistic interactions were also described for the combination of hyposalinity with unfavourably high or low temperature for various Fucus spp. (Munda

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

71

and Kremer, 1977), as well as of hypersalinity with stressful heat-shock for F. spiralis and F. vesiculosus (Li and Brawley, 2004). As an example of a synergistic stress effect, Maczassek (unpublished) found that experimental reductions of temperature (15–5 C) and salinity (17–10 psu) lowered germination rates by 15 and 46%, respectively, when acting in isolation; but when the two stresses were present simultaneously, the germination rate was lowered by 90% (the additive expectation being 61%). Other synergistic interactions were found between desiccation and wave action, between additions of different nutrients and between coldtemperature stress at parental stage and heat-shock at embryonic stage (Table 2.1). The last case provides an example of temporally separated synergistic stresses, where a stress in one ontogenetic stage (parent) amplified the stress response in another stage (embryo) (Li and Brawley, 2004). 4.1.3. Meta-analysis of abiotic stresses Results of the meta-analysis indicated that a single abiotic stress decreased algal performance of Fucus spp. by 40–50%, on average, as compared with the control (Fig. 2.4a and b). However, depending on the type of experiment, the average stress effect was significant only in experiments conducted in recruit/germling stages (lab experiments only, Fig. 2.4a) or only with adults (mainly field experiments, Fig. 2.4b). Averaged over all combinations, the effect of co-occurring abiotic stresses was highly variable but on average additive (Fig. 2.4a, Fig. S1a). The same was true with the combined effect of abiotic stress and nutrient enrichment (Fig. 2.4b, Fig. S1b). It is worth noting that nutrient enrichment alone had neither negative nor positive average effects (Fig. 2.4b). However, behind the average combined effects there was considerable variability of interactive effects within individual experiments (Fig. S1a and b) as noted earlier. This variability illustrates how a second abiotic stress may modulate the impact of a first abiotic stress and how much the outcome depends on the nature of stresses combined and the environmental setting.

4.2. Biotic stress 4.2.1. Single biotic stresses The biotic stresses of grazing, fouling and competition vary enormously over space and time. For example, the number of grazer and epibiont species decreases from the fully marine North Atlantic Ocean into the brackish Baltic Sea (e.g. Kautsky et al., 1986; Ojaveer et al., 2010) as does genetic diversity (Johannesson and Andre, 2006). Although the various biotic stresses interact in myriad ways in nature, understanding the effect of single biotic stresses is a necessary first step to understanding the higher order interactions.

72

Martin Wahl et al.

[(Figure_4)TD$IG] Figure 2.4 Effects of single and combined stresses based on a meta-analysis of 41 experiments extracted from articles in Table 2.1. Effect size (LRR) is plotted against a single stress or stress category (a–f) and their combined effects (see Table 2.2) following the method of Hedges et al. (1999). The number of experiments compared is given above each bar. The error bar depicts the 95% confidence interval of the mean effect ratio. When the confidence interval does not include zero, the stress is considered significant. Note that for different meta-analyses (a–f) different sets of experiments were used, which accounts for differences in the magnitude of a particular result. See Section 2 for details of the assumptions and calculations using the method of Hedges et al. (1999). See Figure S1 for results based on the method of Gruner et al. (2008).

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

73

[(Figure_4)TD$IG] Figure 2.4

Continued

4.2.1.1. Competition and epibiotism The strong intertidal zonation patterns typically exhibited by Fucus species (Fig. 2.1), together with other macroalgae and sessile animals, clearly reveals the importance of competition and competitive displacement among these species (reviewed in Karez and Chapman, 1998; e.g. Schonbeck and Norton, 1978; Schonbeck and Norton, 1980). Ultimately, the ability of Fucus to persist and successfully compete intra- or inter-specifically is determined by its growth rates (Airoldi, 1998; Johnson et al., 1998), canopy height (Dayton et al., 1984), reproductive output (Malm and Kautsky, 2003),

74

Martin Wahl et al.

production of allelopathic compounds (Gross, 2003) and tolerance towards biotic and abiotic stresses (e.g. Worm and Chapman, 1998; Pearson et al., 2009; Schiel, 2009). Competition for light and space occurs among Fucus individuals (conspecifics and congeners), as well as with epibionts attached to their thalli surfaces (Creed et al., 1996; Karez and Chapman, 1998; Karez, 2003a,b). In general, competition varies with ontogenetic stage. For example, periphyton and ephemeral algae may decrease germling survival (Lubchenco, 1983; Lotze et al., 2000), whereas microalgae and sessile invertebrates decrease growth, interfere with nutrient absorption and increase drag on the thallus (Brock et al., 2007; Jormalainen et al., 2008b; Rohde et al., 2008; reviewed in Wahl, 1996; see also Wahl and Mark, 1999; Wahl et al., 2010). Epibionts on Fucus thalli also include bacterial biofilms (microfoulers), a largely unknown interaction that may be important, particularly as a host-specific assemblage of microbes influences many interactions of the host with its environment (Dobretsov, 2008; Lachnit et al., 2009). Furthermore, microfouling pressure is a conditioning phase in the fouling process and varies substantially with temperature, light and depth. For example, in the western Baltic fouling pressure (substratum occupation per unit time) by macroscopic species is 30 times lower in February as compared to July, and the number of simultaneously settling species is 10 times lower in winter than in summer (Thomsen and Melzner, 2010; Wahl et al., 2010). By colonizing the functional interface between the host alga and the environment, dense epibiosis will clearly modulate most interactions of the host (Wahl, 2008a). 4.2.1.2. Herbivory and parasitism Herbivory on Fucus, primarily by isopods and, to a lesser extent, by amphipods and snails, ranges from mild to severe and may differentially impact Fucus from the germling stage through the adult stage (Pennings et al., 2000; Long et al., 2007; Dethier and Williams, 2009). High grazing rates by isopods (mainly the genus Idotea) can eliminate local Fucus stands (i.e. Engkvist et al., 2000), while grazing by herbivorous fishes is less common in the distributional range of Fucus (Connell and Slatyer, 1977; Floeter et al., 2005). In the species-poor northern Baltic Sea, grazing pressure by isopods varies seasonally (e.g. Kotta et al., 2006): low in winter (when isopods are inactive) and early summer (when isopod densities are low); high in late summer, which is characterized by high densities of juvenile isopods (Engkvist et al., 2000; Korpinen et al., 2010). Grazing pressure also varies spatio-temporally with exposure (wave action) (e.g. Jonsson et al., 2006) and the patchy distribution of consumers (e.g. Korpinen and Jormalainen, 2008b). Isopod grazing pressure may also vary with fishing pressure on isopod predators (e.g. Bostrom and Mattila, 2005; Jormalainen and

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

75

Ramsay, 2009). Simultaneous grazing by several consumer species may interact. Thus, defences developed by F. vesiculosus against one herbivorous species may reduce grazing by another species (Long et al., 2007; Yun et al., 2010). Very little is known about parasitism or infections in Fucus. Coles (1958) identified parasitic nematodes that caused galls on F. vesiculosus and F.serratus along the Cornwall coast and Zuccaro et al. (2008) recently detected a number of fungal species associated with F. serratus. Unlike A. nodosum, which harbours an obligate endophytic fungus (Xu et al., 2008, and references therein), few, if any mycobionts are associated with species of Fucus (but see Parker and Chapman, 1994 for impact by endophytes). However, the potential for increased biotic interactions involving invasive parasites or pathogens is on the rise in many marine systems (e.g. Torchin et al., 2002). 4.2.2. Simultaneous biotic stresses Because Fucus exists within a multi-species community, it will simultaneously or sequentially interact with a multitude of micro- and macrofoulers, competitors (annual or perennial macroalgae, sessile animals) and consumers (crustaceans, molluscs), as well as an unknown number of pathogens and parasites. Consequently, the co-occurrence of different biotic stresses is expected to be as common as the co-occurrence of abiotic stresses. However, only few studies have investigated the interactive effects of two or more biotic stresses (Table 2.1). Several studies illustrate the complexity of interactive biotic effects. The competitive impact of turf algae (Rhodophyta) on F. vesiculosus is alleviated by limpet grazing, but not the similarly negative effect of larger canopy algae (Jenkins et al., 1999). Similarly, limpet grazing and competition with turf algae had an additive effect on Fucus recruitment, but at the same time limpet grazing inhibited recruitment of A.nodosum that has the potential to suppress Fucus by canopy effects (Cervin et al., 2005). In another study, epibiotism and grazing had an additive effect on the growth of F. vesiculosus, but the effects were functionally interrelated in the sense that fouling attracted grazers (Jormalainen etal. 2008b). Finally, in F.distichus, grazing by littorinids and gammarids has been reported to reduce the rate of fatal endophyte infections (Parker and Chapman, 1994). Thus, if grazers feed on both the host and the epibiota, epibionts may attract grazers with consequent ‘shared doom’ for both, epibionts and host. On the other hand, if the epibionts are resistant to grazers, the host may gain associational resistance. 4.2.3. Results of the meta-analysis of biotic stresses Experiments with two biotic stresses revealed additive (5), synergistic (1) and antagonistic (2) interactions (Table 2.1). The meta-analysis of single

76

Martin Wahl et al.

biotic stresses (competition, herbivory and epibiotism) showed a mean 10–50% decrease in performance relative to controls (Fig. 2.4c and d) and no differences in biotic stress responses among life-history stages. With all life-history stages combined, both the single and combined effects of biotic stresses were statistically significant, which was not the case when the data were stratified by life-history stages (Fig. 2.4c). The meta-analysis indicated that the combined effect of two biotic stresses, in general, was additive (Fig. 2.4c, Fig. S1c). Still, the high variation in interaction effect sizes reflects the contingency of multi-stress responses in which interactive effects are more complex and depend on the host spectrum and feeding preferences of the grazers (Wahl and Hay, 1995; Karez et al., 2000).

4.3. Protection against abiotic and biotic stresses Fucus spp. have evolved several defence mechanisms for a variety of abiotic and biotic stresses. One example is development of an elastic, but tough thallus that resists mechanical damage (e.g. Nietsch, 2009), whereas wound healing and re-sprouting from holdfasts should favour fast population recovery when damage has occurred (e.g. Malm et al., 2001; Tatarenkov et al., 2005). High concentrations of phlorotannins may offer protection from UV radiation (Pavia et al., 1997; Swanson and Druehl, 2002) or deter herbivory (e.g. Hay and Steinberg, 1992; Jormalainen and Honkanen, 2008). Metabolic repair mechanisms of UV damage exist, but may be jeopardized under multiple stresses (e.g. Hoffman et al., 2003). Palatability of Fucus species to grazers is affected by a number of lipophilic and polar metabolites; in particular, it is augmented by high concentrations of mannitol and proteinic amino acids, as well as by high water content, and reduced by high concentrations of phlorotannins and high toughness of the thallus (Tuomi et al., 1989; Hemmi and Jormalainen, 2004; Nietsch, 2009; Jormalainen et al., 2011; Weinberger et al., 2011). Although the reduction of nutritive value (e.g. fewer storage compounds such as mannitol) may save energy and discourage grazing (Viejo and Arrontes, 1992; Worm et al., 1999; Engkvist et al., 2004), it may also jeopardize the chances to survive seasons of low energy supply (Lehvo et al., 2001). Chemical defences are present in many terrestrial and marine species, but their production, storage and ultimate detoxification may well impose fitness costs on the producer (reviewed in Stamp, 2003; Pohnert, 2004). Costs of chemical defence traits have been documented in macroalgae (Dworjanyn et al., 2006; Jormalainen and Ramsay, 2009), but because the relevance of costs varies with resource availability (Davison and Pearson, 1996; Dethier et al., 2005), and may vary with respect to the measured fitness component, costs have not been detected in all studies of chemically defended algal species (Rohde and Wahl, 2008; Pansch et al., 2009; Appelhans et al., 2010). In addition, net defence costs are only expected to

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

77

arise under conditions in which natural enemies do not impose significant performance costs and thus when defences are superfluous. Nevertheless, at least four Fucus species employ chemical defences against fouling and herbivory. For example, F. vesiculosus alters the composition of epibiotic fouling by producing a combination of pro- and antifouling compounds (e.g. Lachnit et al., 2010), and macrofoulers such as barnacles are deterred by secondary metabolites of F. vesiculosus (Brock et al., 2007; Jormalainen et al., 2008b; Rohde and Wahl, 2008) and F. evanescens (=F.distichus, Wikstr€ o m and Pavia, 2004). Seasonal fluctuations in anti-microfouling chemical activity have been reported in F.vesiculosus (Wahl et al., 2010), but whether this is resource-driven (availability of energy) or demand-driven (fouling pressure) and whether similar rhythms exist in anti-macrofouling defences has not yet been investigated. Induced chemical defences against herbivores have been detected in F.distichus (van Alstyne, 1989), F. vesiculosus (Rohde et al., 2004), F. serratus (Rohde and Wahl, 2008) and F. evanescens (Rohde and Wahl, 2008). While some chemical defences are produced or deployed at variable intervals after the onset of herbivory (‘induced’), they may also be reduced before herbivory is terminated (Weinberger et al., 2011).

4.4. Modulation of stresses When two negatively interacting species exhibit unequal sensitivities towards a given abiotic stress, the least sensitive species may indirectly benefit, as the more sensitive species is more strongly inhibited. Furthermore, when abiotic stress jeopardizes the production of defence chemicals, the overall stress impact will be magnified. Thus, when the costs of defence and of stress compensation mechanisms must be paid from the same limited resource, defences may decline, subsequently increasing biotic stress between two species. Accordingly, acute abiotic stress should most impact inducible defences that are produced denovo, rather than as a by-product of some primary metabolic pathway. The meta-analysis revealed that, on average, interactions between abiotic and biotic stresses were additive (Fig. 2.4d and e), although there was a wide scatter of variable effects among separate experiments, varying from synergistic to highly antagonistic ones (Fig. S1d and e). Below, we focus on studies examining how abiotic stress modulates various biotic interactions among Fucus species, their grazers, competitors and epibiota. Although several studies did not utilize a factorial design, they do provide indirect evidence about the possible type of stress interactions. 4.4.1. Irradiance effects on biotic interactions Sensitivity to high and low light stress contributes to the patterns of intertidal zonation exhibited by Fucus species (Karez and Chapman,

78

Martin Wahl et al.

1998). Although epibiont shading may help to protect Fucus thalli against excessive irradiation (as shown for other organisms by Przeslawski et al., 2008), the effect is often transient, as epibiota rapidly increase and thus exacerbate light limitation for the host (Oswald et al., 1984). Low light may weaken anti-microfouling defences in F.vesiculosus and uncontrolled bacterial fouling, in turn, can reduce available light by more than 90% within a few weeks (Wahl et al., 2010). Low light and fouling had additive negative effects on growth (Rohde et al., 2008) and both macro- and microepibionts may raise the light compensation depth by 2 m in the western Baltic (Rohde et al., 2008). Susceptibility to light stress (and competitive interactions) also is influenced by emersion time. For example, F.serratus is more susceptible to high light stress during emersion than F.vesiculosus (Malm and Kautsky, 2003) and this can alter local distribution patterns of the species. Low light stress also affects the susceptibility of Fucus spp. to herbivory, although there is no indication that irradiation affects the behaviour of herbivores directly (e.g. Eriksson et al., 2006). For example, light limitation can reduce the production of chemical defences in F. vesiculosus thus enhancing consumption by the isopod I. baltica (Weinberger et al., 2011). At the same time, reduced growth rates under light limitation (e.g. Rohde et al., 2008) increases the effect of herbivory, as grazed tissue is more slowly replaced; an effect that may be countered by decreased mannitol production in low light, which subsequently renders the alga less attractive to isopod grazers (Weinberger et al., 2011). A synergistic effect of grazing and low light stress has been demonstrated, as grazing losses in F.vesiculosus increase under low light, possibly by decreasing resistance to herbivory (fewer defence chemicals) (Jormalainen and Ramsay, 2009). Deleterious effects of UVR on the defence capacity have been shown in some macroalgae (Cronin and Hay, 1996; Pavia et al., 1997), but not in others (Macaya et al., 2005). While enhanced UVR exposure in various Fucus spp. increases phlorotannin concentration (Pavia and Toth, 2000; Schoenwaelder et al., 2003; Henry and Van Alstyne, 2004), it is unclear whether increased levels constitute an anti-herbivore defence or protection from UVR (reviewed in Amsler and Fairhead, 2006). Defence against epibionts is another consideration and in F. vesiculosus, is strongest at moderate irradiance (Wahl et al., 2010) or levels typical of its normal depth distribution (1–2 m) in the western Baltic Sea. 4.4.2. Temperature effects on biotic interactions Increasing temperatures (>15 C) enhance biotic stress in Baltic Sea Fucus by (1) increasing micro- and macrofouling rates (Wahl et al., 2010); (2) increasing grazing rates of I. baltica in summer (but not in the other seasons) (M. Zimmer, personal communication) and (3) decreasing levels of defence chemicals (under both high and fluctuating temperatures)

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

79

(Weinberger et al., 2011). In contrast, anti-fouling defence chemicals in F. vesiculosus were unaffected by a 2-week exposure to temperatures between 8 and 23 C (Wahl et al., 2010). Thus, temperature and grazing stress can generate synergistic interactions, whereas temperature and fouling stress are additive. Freezing temperatures can shift competitive interactions among Fucus spp. Laboratory experiments revealed that F. vesiculosus had a higher tolerance to freezing than its congener F.serratus: aerial exposure to 15 C for 1 h killed F. serratus, while F. vesiculosus survived (Malm and Kautsky, 2003). Thus, for this species pair freezing and competitive stress is likely to be antagonistic for F. vesiculosus (direct physiological stress partially compensated for by reduced competition) and negatively synergistic for F. serratus. 4.4.3. Salinity and emersion effects on biotic interactions Emersion may decrease salinity by removing seawater and/or exposing intertidal organisms to rain and/or freshwater runoff or increase salinity by evaporation. The combination of diverse factors associated with emersion will determine the strength or even the sign of stress during emersion. In F. vesiculosus, for example, emerged individuals experienced a transient refuge from aquatic herbivory and epibiont settlement, as well as an accumulation of anti-fouling defence chemicals at the thallus surfaces, which produced a more effective defence after re-immersion (Brock et al., 2007). Thus, emersion and fouling may act as antagonistic stresses. 4.4.4. Water motion effects on biotic interactions Canopy sweeping or whiplash removes or tatters early post-settlement stages (Vadas et al., 1992) and illustrates how wave action, which is not a strong stress for small individuals or recruits, may be lethally amplified by the presence of larger (con)specific individuals. Thus, whiplash represents a synergistic stress for juveniles and may shift competitive interactions between F.serratus and F.vesiculosus in favour of F.serratus because F.serratus juveniles are less sensitive to whiplash (Vadas et al., 1992; Malm and Kautsky, 2003). However, because F.vesiculosus can regenerate from holdfasts, the slight competitive advantage of F.serratus recruits ultimately may be negligible (Kiirikki and Ruuskanen, 1996; Malm and Kautsky, 2003; Malm and Isaeus, 2005). Whiplash also reduces the abundance of competing filamentous epiphytic and understory macroalgae (Kiirikki, 1996), as well as the effect of swimming grazers (either intertidally or subtidally) (Davis et al., 2003), thus creating an antagonistic interaction between wave action and both competition and herbivory. As isopods (I.baltica) experience more attachment difficulties on F. serratus than on F. vesiculosus due to differences in surface morphology, in high water motion grazing intensity is shifted to

80

Martin Wahl et al.

F.vesiculosus (Engkvist et al., 2004). Thus, the effects of water motion and grazing in a mixed stand of F. serratus and F. vesiculosus are antagonistic for F. serratus and synergistic for F. vesiculosus. Though not tested, it is conceivable that where the two species compete, F.serratus may benefit from a combined wave action and grazing stress, as long as F. vesiculosus density remains at or above some threshold level. In the more exposed mid- to high-intertidal zone, non-swimming grazers such as limpets (Patella vulgata) are dominant and their grazing preference for F. vesiculosus and F. spiralis over F.serratus further affects the competitive hierarchy by allowing F. serratus to move upshore (Jonsson et al., 2006). An additional consideration is that while the risk of thallus dislodgement is higher at waveexposed sites, water motion may indirectly lead to reduced grazing by inducing increased toughness of the thallus (Nietsch, 2009). 4.4.5. Eutrophication effects on biotic interactions Nutrient enrichment has been the most frequently studied abiotic modifier of biotic interactions, with individual responses being additive (2), antagonistic (3) or synergistic (9) (Table 2.1). With respect to nutrient enrichment–herbivory interaction, grazers counteract the negative effect of high nutrient availability on colonization success of F. vesiculosus by reducing competition for space (Lotze et al., 2000). In later stages, gastropod grazing counteracts nutrient stress by removing epibiota (Jormalainen et al., 2003; Ra berg and Kautsky, 2008). Field experiments on F. vesiculosus in the Baltic Sea (where abundant gastropod grazers regularly graze early recruits and nutrient enrichment severely restricts recruitment success) showed that negative effects of grazing and nutrient enrichment on recruitment success remained additive, but varied depending on the community structure of other grazers (Korpinen and Jormalainen, 2008a) and the presence of canopy species (Eriksson et al., 2007). On the other hand, synergistic effects of nutrient enrichment and herbivory may arise when nutrient enrichment improves the nutritional quality of Fucus thalli. Thus, high-quality Fucus tissue greatly benefits growth and reproduction of I. baltica (Hemmi and Jormalainen, 2002) and the amphipod, Gammarus locusta (Kraufvelin et al., 2006), which concomitantly leads to higher grazing pressure. Palatability of F. vesiculosus to littorinid grazers also increases as a result of increased nutritive value (increased N: Yates and Peckol, 1993; increased mannitol: F. Weinberger, personal communication). Finally, negative effects of grazing on F.vesiculosus may be stronger under increased nutrient conditions (Hemmi et al., 2005). With respect to nutrient enrichment and competition, interactions were mainly synergistic (Table 2.1), suggesting that nutrient enrichment benefits competitors/epibiota of Fucus more than Fucus itself (Jormalainen et al., 2003, 2008b). As expected, growing F. vesiculosus in

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

81

a nutrient enriched environment without epiphytes positively affected growth (Jormalainen et al., 2003). Thus, two effects of epibiosis have a synergistic impact on their Fucus host by shading and inhibiting nutrient uptake (through insulation or a ‘smothering’ effect). Furthermore, nutrient enrichment may completely reorganize competitive hierarchies as eutrophication typically favours ephemeral, fast growing algae (Berger et al., 2004) at the cost of slow growing perennials (Korpinen et al., 2010). The addition of an abiotic stress to two biotic stresses may render the biotic–biotic interactive effects more complex and non-additive. For example, under ambient levels of nutrients, grazers had a large positive and competitors a slightly negative effect on growth of F. vesiculosus recruits, with an additive or a minor synergistic interaction (Worm et al., 2001). Under nutrient enrichment, however, the combined effect turned highly antagonistic as the increased competition cancelled out the positive grazing effect. 4.4.6. Biotic modulation of abiotic stress Biotic interactions may, in turn, modulate the impact of abiotic stresses. Thus, as shown for non-Fucus spp., epibionts may protect their host from UVR radiation and desiccation stress (Penhale, 1977; Przeslawski et al., 2008). However, they may also render the fouled host more vulnerable to drag (Witman and Suchanek, 1984; Wahl, 1997). Furthermore, canopyforming algae, which might act as inter- or intra-specific competitors, may at the same time shelter small Fucus from abiotic stress during emersion (Brawley and Johnson, 1991; Lamote et al., 2007). Results of the meta-analysis showed that the average combined effect of herbivory or competition (or both) with nutrient enrichment did not differ from additivity (Fig. 2.4e and f). However, the nutrient enrichment– herbivory interaction was less consistent than the nutrient enrichment– competition interaction, which was also found in the individual studies.

5. GENETIC LEVELS OF STRESS RESPONSE On a temporal scale, responses to stress range from fast acclimation by phenotypic plasticity to slower adaptation by stress-driven selection (i.e. adaptive evolution or ecotypic differentiation). The efficacy of both relates to an individual’s or a population’s genetic composition. Traditionally, physiologists and ecologists have been more interested in immediate responses, while population geneticists have been more interested in changes over multiple generations (Allendorf and Luikart, 2007). However, it is now realized that the temporal differential between ecological and genetic changes can be small or negligible and most species

82

Martin Wahl et al.

are not driven to local extinction before genetic factors play a role (Spielman et al., 2004). In short, dynamic selective regimes in the environment can have a significant and nearly simultaneous impact on both ecological and evolutionary processes, which subsequently shape genetic structure and performance of populations. The ability to distinguish these effects is an emerging challenge.

5.1. Sensitivity versus genetic diversity of a population: stress from the evolutionary perspective The extrinsic component of stress (abiotic and biotic factors in the environment) shapes the phenotype through natural selection and leads to changes in allele frequencies and new gene complexes, collectively resulting in adaptation (i.e. the integration of the phenotype with the genotype). But it is crucial to also recognize the intrinsic component of stress tolerance (Bijlsma and Loeschcke, 2005), estimated as the standing genetic variation or diversity (evolutionary potential) of a population. Genetic diversity stems from the relationship between the effective population size (roughly the number of individuals that successfully contribute to the next generation) and the processes of gene flow, selection, genetic drift and mutation. Effective population size (Ne) is a crucial parameter for assessing the adaptive potential of a population. Large sexually reproducing populations with high Ne have more genetic diversity and collectively experience fewer negative ramifications from the stochastic effects of genetic drift (random gain and loss of alleles through time). Moreover, at least some individuals within large populations are likely to have genotypes that are already able to cope (preadapted) with a new selective pressure such as a higher temperature. In contrast, populations with low Ne—often arising from habitat fragmentation or isolation—tend to have less genetic diversity, are highly subject to the negative effects of genetic drift, and are collectively more likely to be affected by stress due to their low adaptive potential (Harley et al., 2006). As pre-adapted genotypes in these populations would be few or absent, it follows that they also are at higher risk of local extinction. Estimating Ne is extremely difficult. Direct estimates require extensive tagging studies over long time periods, whereas indirect estimates are based on temporal changes in allelic frequencies. Recent work on F. serratus using microsatellites suggested that despite large census population sizes Ne was very small with a concomitant loss of fitness (Coyer et al., 2008). If loss of genetic variation is combined with a deteriorating environment, then a negatively synergistic effect is likely to arise in which a population’s susceptibility and tolerance to extrinsic stress is magnified.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

83

5.2. Indirect detection of adaptation to stress on relevant spatial scales Neutral loci are not under selection and as they are affected only by gene flow and genetic drift, cannot be used to directly measure adaptation. Neutral loci do, however, provide necessary basic information for studies of adaptation by resolving genetic population structure, measuring gene flow and providing indirect evidence for adaptation when correlated with environmental parameters. Genetic population structure can identify spatial scales over which selection (e.g. adaptation to stress at a population level) may act and has been assessed at one or more spatial scales for F.serratus (Coyer et al., 2003; Hoarau et al., 2007), F. vesiculosus (Engel et al., 2005; Tatarenkov et al., 2007), F. distichus (Coyer et al., 2011c) and F. spiralis (Engel et al., 2005; Coyer et al., 2011a) using microsatellite (Coyer et al., 2002a,b,c, 2009; Engel et al., 2003, 2005) and mitochondrial loci (mt DNA) (Hoarau et al., 2007; Engel et al., 2008). Population structure may be homogenized by gene flow as high gene flow dampens local adaptation to a stress (Kawecki, 2008). However, estimates of gene flow for F.serratus based on population differentiation at the regional scale (1–100 s km) suggest that gene flow can be as little as 500 m to 2 km (Coyer et al., 2003), between 10 m and <1 km in F. vesiculosus from the Baltic Sea (Tatarenkov et al., 2007) and 150 m to 20 km in Brittany (estimated from Billard et al., 2005a). In the Kattegat Sea, gene flow distances for F.serratus increased to 70 km (Coyer et al., 2003). Taken together, the aforementioned results illustrate how oceanographic currents and local coastal topologies affect dispersal and variation (see below). Changes in putatively neutral allele frequencies along various stress gradients in replicated geographic locations provide strong correlative evidence for adaptation. This, however, is not conclusive as long as the actual genes involved in the stress response remain unknown. Conversely, strong changes in phenotypic traits may be observed across a gradient that are not mirrored by neutral allele frequencies. In Fucus spp., both of these situations have been encountered. For example, no correlation was found in the neutral allele frequency of F. vesiculosus along a gradient in salinity from the North Sea (35 psu), to the Kattegat, and the SW Baltic Sea (12 psu) using neutral markers (Tatarenkov et al., 2005; Johannesson and Andre, 2006), despite strong phenotypic differences in emersion stress tolerances (F. vesiculosus is submerged in the atidal Baltic Sea) (Pearson et al., 2000) and fertilization success (Serr~ao et al., 1996a,b). Deeper within the Baltic Sea (from Bornholm and then extending north into the Bothnian Gulf), however, a strong shift in allele frequencies is correlated with decreasing salinity and a transition to clonal reproduction of F. radicans (Tatarenkov et al., 2005). The salinity shift is even considered a strong

84

Martin Wahl et al.

enough selective force to contribute to the recent and rapid formation of the newly described endemic species F.radicans within the past 400–2000 years (Pereyra et al., 2009). In F.serratus, there is no change in neutral allele frequencies over salinity gradients either within or between the Kattegat and Baltic Seas (Coyer et al., 2003) or along steep salinity gradients in a Norwegian fjord (Coyer et al., 2011b). However, using a genome scan approach these authors found evidence for selection in six candidate loci (three derived from expressed sequence tag (EST) libraries and three from anonymous libraries; see Section 5.3) of which at least one was related to osmotic stress (Coyer et al., 2011b). Thus, a lack of correlation using neutral markers does not imply a lack of selection. At the local scale of a single patch or stand, populations may display variation in quantitative genetic traits as has been shown in F. vesiculosus from the Baltic Sea regarding the resistance to both epiphytes and grazers (Jormalainen and Honkanen, 2008; Jormalainen et al., 2008b; Koivikko et al., 2008; Jormalainen and Ramsay, 2009), as well as to concentrations of phlorotannins (Jormalainen and Honkanen, 2004). Similarly, strength and/or inducibility of anti-herbivory and anti-fouling defences may vary enormously among conspecifics of F.vesiculosus, F.serratus and F.evanescens (Rohde et al., 2004; Wahl et al., 2010; F. Symanowski, unpublished). This variation indicates the potential for local adaptation to natural enemy regimes.

5.3. Direct detection of adaptation to abiotic and biotic stress The interplay between environment and genes is the focus of the new field of EEG (Ouborg and Vriezen, 2007; Tautz et al., 2010). Until full genome sequences are available for the various Fucus spp. (whose genomes range in size from 800 to 1000 Mb, Kapraun, 2005), a more rapid and economical approach is to develop EST libraries, which represent the expressed genes of the individual under a given stress condition (compared to a recovery condition). EST libraries provide a rich source for gene discovery and the development of gene-linked markers that can be tested for evidence of selection using genome scans (Storz, 2005). Comparative gene expression studies based on EST libraries provide a snapshot of organismal physiology and a fine-scale picture of plastic and constitutive changes in cellular metabolism associated with acclimation and genetic adaptation to the stress under investigation. Thus, EST-linked markers and genomic scans can distinguish phenotypic plasticity (acclimation capacity) from ecotypic differentiation directly rather than correlatively. Salinity clines and heat-shock in Fucus spp. have recently been investigated in an EEG framework. Species distributions from the North, Kattegat and Baltic Seas are governed by both salinity and (emersion) temperature gradients. Replicated clinal variation along such parallel

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

85

environmental gradients provides good evidence for natural selection in the wild. For example, four populations of F. serratus along a 12-km long salinity gradient (2.6–33.0 psu) in a northern Norwegian fjord were investigated with a genome scan approach using both anonymously and EST-linked microsatellite loci (Coyer et al., 2011b). Six genes revealed putative signatures of selection, of which one was annotated to a putative mannitol transporter (Coyer et al., 2011b). As mannitol is regulated in highly saline environments to control cell turgor (Iwamoto and Shiraiwa, 2005, and references therein), significant selection has occurred for at least some part of the mannitol pathway over the 12-km scale. Disentangling the dual roles of mannitol in osmoregulation and energy storage will require manipulative mesocosm experiments, which have not yet been performed. Intertidal gradients present several physiological stresses and it is along these gradients that classical transplant experiments have been conducted with several species of Fucus (see Section 4.1 and Table 2.1). Using an EEG approach, Pearson et al. (2009) compared emersion stress at the distributional centre versus the distributional edges in two Fucus species. Southern edge populations of F. serratus (northern Portugal) were less resilient to desiccation and heat-shock than central (UK) populations and expression of heat-shock genes was higher at the same temperatures, suggesting greater cellular stress (Pearson et al., 2009). In contrast, F. vesiculosus showed no such divergence in heat-shock response and little variation in gene expression. These results suggest that for F.serratus, edge populations are maladapted, less genetically diverse and display lower fitness. Thus, changing climate conditions may threaten small, fragmented and/or marginal populations because of inherently reduced fitness and lower adaptive capacity. Using comparative gene expression studies of several heat-shock proteins in naturally occurring, sympatric populations of Baltic Sea F.radicans and closely related F. vesiculosus, Lago-Leston et al. (2010) were able to show that F. radicans was more sensitive to mild heat-shock than F. vesiculosus. Also from earlier experiments, the tolerance of F. vesiculosus did not vary substantially in contrast to its physiological variability in desiccation and freezing tolerance (Pearson et al., 2000). At present, the interpretation of these results remains general, but suggests that changes in transcriptional regulation, combined with local ecological dynamics, may significantly impact functional traits in marginal habitats, coincident with genome-wide reductions in genetic diversity due to genetic drift (Pearson et al., 2009). At the level of biotic stress (i.e. community genomics), comparative transcriptomics is still in its infancy with the exception of host–parasite and plant–herbivore studies conducted mainly on model systems (e.g. Walley and Dehesh, 2010).

86

Martin Wahl et al.

In order to explore the genetic basis of inducible chemical defence against herbivores in F.vesiculosus, a cDNA microarray has been developed from libraries based on grazed and non-grazed F. vesiculosus (Weinberger et al., unpublished). F.vesiculosus is capable of inducing a chemical defence when exposed to grazers (Rohde et al., 2004). Both chemical elicitators (oligoguluronate, methyl-jasmonate) and direct feeding by isopod grazers altered gene expression. While Fucus has not yet entered the post-genome era, the rapidity of advances in next generation sequencing technologies (Metzker, 2010) is paving the way for the types of multi-level experiments essential to understanding complex ecological interactions involving stress (Gilad et al., 2007). Importantly, the demands for multi-factorial experimental designs and sufficient replication are already accessible and the costs are coming within reach of individual investigator budgets.

6. CONCLUSIONS The simultaneous effects of different stresses and the dynamics of positive and/or negative feed-backs form the selective environment on the genetic potential to determine whether a given species can (1) tolerate a transient stress regime (plasticity, acclimation); (2) adapt to a permanent regime (ecotypic differentiation via selection) or (3) become locally extinct (unable to maintain positive population growth). As the combination of stresses is variable over space and time, the challenge for an individual is to cope with the spectrum of stresses occurring during its lifetime; the challenge for the population is to adapt to shifting stress regimes within a few generations. Finally, in the context of climate change, abiotic stress-driven effects on biotic interactions are expected to shift stress impact from the population level to the community level by altering competitive hierarchies and feedbacks among trophic and other interactions. At this point, we have a reasonable understanding of the effects of individual stresses on Fucus spp. at the population level, but our knowledge of their higher order interactions remains rudimentary. Consequently, our ability to predict interactions and their effects on community maintenance/assembly declines precipitously as the number of stresses increases. The model in Fig. 2.5 is a hypothesis of the network of single stressor effects and their interdependencies that may generate various interactive effects as they are currently understood. However, rigorous testing of this network of system-level hypotheses will require a different approach that takes into account non-linear and stochastic dynamics (Section 7). Results from the meta-analysis (Fig. 2.4) show that although all combined effects were, on average, additive this was not necessarily the case for

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

87

[(Figure_5)TD$IG]

Figure 2.5 Example of an interaction network of abiotic and biotic stresses on F. vesiculosus (based on Wahl et al., 2010; Weinberger et al., 2011, Table 2.1). Black circles used as arrow heads indicate negative interactions, black diamonds indicate positive interactions. Dark grey shading indicates biotic variables and light grey shading indicates abiotic variables. Examples: shading weakens energy reserves (a) leading to weakened anti-fouling (AF) and anti-grazing (AG) defences, which enhances fouling (2) and grazing (20 ) pressure. Both reduce (3, 30 ) the photosynthetic area of the thallus (c), which amplifies (4) the energy shortage under low light conditions. Low light (10 ) and temperature–stress (‘warming’, 5) reduce growth (b), which jeopardizes the alga’s ability to compensate for tissue lost to grazers, the activity of which is enhanced by temperature stress (50 ). Emersion reduces fouling and strengthens anti-fouling defences (70 ). Eutrophication, in contrast, enhances fouling (80 ) and competition (8) by favouring filamentous algae relative to Fucus. 1-2-3-4 is one of several examples for a positive feedback loop, which may be amplified or dampened by other abiotic and biotic stresses.

individual case studies. There, the interaction between two stresses was often synergistic or antagonistic contingent upon the environmental context. The statistical test of interaction effects (Fig. S1) suggested a similar result although a slight trend towards synergistic interactions was stronger and more evident given the range of variation in individual studies. We emphasize that the results from the meta-analysis must be interpreted with caution, as the number of published studies fulfilling all requirements (factorial design, adequate replication, controlled treatments) was small.

7. PROSPECTS The many species of Fucus span 45 of latitude in the northern hemisphere with dominance in the North Atlantic. Collectively they

88

Martin Wahl et al.

Table 2.3 Distribution of Fucus species (left) and regions with habitats (right) used in the meta-analysis

F. distichus F. evanescens F. gardneri F. serratus F. spiralis F. vesiculosus

1 2 3 9 2 26

Baltic Sea (subtidal) Eastern Atlantic (intertidal) Eastern Pacific (intertidal) Western Atlantic (intertidal)

16 15 1 1

inhabit a broad suite of habitats: exposed outer coast to sheltered bays/ fjords; fully marine to nearly freshwater; rocky substrata to saltmarsh; high intertidal to shallow subtidal. Despite this diversity, however, experimental work on stress is highly skewed with >60% of the experiments having been conducted on F. vesiculosus in the Baltic Sea or eastern North Atlantic (Table 2.3) and >95% of all studies having been conducted in the mid-range of the biogeographic distribution of the genus. Clearly, a fuller understanding of how members of the genus have colonized and continue to successfully exploit northern hemisphere shores, requires greater emphasis on the less studied species, as well as greater emphasis on their performance across clines and along distributional edges. Indeed, studies at distributional edges are likely to offer insights on how Fucus (and other species) will respond to global climate change (Pearson et al., 2009).

7.1. Experiments and modelling Our call for research on all species of Fucus and over their respective clinal and distributional ranges also acknowledges that our growing capacity to integrate physiology, genetics and experimental ecology provides numerous avenues to understanding the role of stress. In methodological terms, this will require (1) the design of manipulative experiments in a factorial manner to address multivariate interactions, (2) the up-scaling of experiments by including several interacting groups and by allowing enough time for effects to spread from the individual to the community level, (3) comparison of stress impact on marginal versus core populations, (4) attention to genotypic–phenotypic links and (5) the use of multivariate modelling techniques to explore and analyse the network system. Factorial ecological experiments with appropriate replication for >3 stress factors, however, are logistically difficult (if not impossible) in the field and are only slightly less daunting to establish and maintain in the laboratory (mesocosms). Thus, future studies should strategically acquire

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

89

data that are appropriate for complex analyses as it is precisely the multivariate effects that need to be understood. Structural equation modelling (SEM) provides a framework for estimating causal effects through the study of path relations (reviewed in Grace, 2006; Grace et al., 2010). Because the focus of SEMs is on understanding direct and indirect pathways, the approach is well suited for both building and testing hypotheses of multiple stress processes (e.g. Tonsor and Scheiner, 2007, Arabidopsis). In a marine setting, SEM has been used to model indirect facilitation of the benthic communities of kelp forests (Arkema et al., 2009). Its application to the hypothetical interaction network proposed in Fig. 2.5 has not yet been attempted but would greatly contribute to our understanding of community stress.

7.2. Next generation molecular ecology Ecologists as a group tend to shun molecular methods in their research programs (Johnson et al., 2009). Nevertheless, the capacity to examine interactions among stress reactions—almost all of which are complex, multi-gene-regulated traits—will open fundamentally new avenues in community genetics–genomics. At this stage, we still know very little about the genetic basis of osmoregulation, thermal tolerance, chemical defence and the boundaries between plastic and selective responses for any of these traits. As distinguishing phenotypic plasticity from ecotypic differentiation can be approached directly by examining gene expression/regulation via differential transcription and structural changes in the DNA sequences of key genes, it is increasingly possible to integrate our understanding of ecological stress with complex genetic responses (Tautz et al., 2010). Thus, integrating the genetic perspective into field studies of stress ecology will enable us to (1) more finely identify plasticity in stress responses and expression of stress-related genes; (2) evaluate the adaptive potential of populations and (3) investigate how selection driven by one stress affects sensitivity towards other stresses. For example, differential mortality in response to one stress will lead to the survival of a non-random (selected) sub-sample of the original genotypic diversity, which in turn will harbour a sub-sample of traits ancillary to tolerance for the selective stress (e.g. tolerance to a subsequent stress, productivity, light harvesting, etc.). Traits of the genotypic sub-sample may be non-random if the traits are linked to the tolerance of the first stress, or random if they are not.

7.3. How will climate change affect fucus? As a foundational taxon, the genus Fucus has a large influence on the associated community as a whole; likewise, the associated community

90

Martin Wahl et al.

influences Fucus. This duality highlights the importance of gaining a more integrative understanding of stress responses in species of Fucus and the communities they support. Species with wide ecological niches are more easily able to cope with change; likewise, the ongoing and rapid diversification of the genus creates multiple overlapping niches. This suggests that Fucus spp. from stressful and fluctuating habitats may be better equipped to tolerate (via pre-adaptation) the superimposition of increased fluctuations (e.g. Schneider, 2008) than species from habitats with more subtle fluctuations (e.g. the deep subtidal, or low latitude habitats). As the predicted changes in the North Atlantic region over the next one or two centuries (3–6 C increase in temperature, a 0.5 units decline in pH, IPCC Climate Change, 2007) are small relative to the daily environmental fluctuations characterizing any temperate northern hemisphere intertidal habitat, the direct impact of rapid climate changes on Fucus may be small. In contrast, the indirect impacts could be large if Fucus spp. were to be competitively displaced (as, e.g. by de novo interactions with introduced competitors, consumers and/or parasites and pathogens). However, the niche breadth of several sympatrically occurring Fucus spp. taken together, provides further insurance in terms of holding space and by ensuring its own ‘invasive’ potential (Peterson, 2003). As more than a third of Fucus spp. have been characterized as successful invaders (Williams and Smith, 2007), the genus has high expansion potential, be it by anthropogenic or natural means. Invasiveness, that is, the ability of a species to establish in new habitats, might be a proxy for its capacity to tolerate climate change. Reproductive success in a new environment is favoured by phenotypic plasticity and/or rapid ecotypic selection. Even if the effects of climate change are disproportionally strong on marginal habitats (Melzner et al., 2010; Thomsen and Melzner, 2010), Fucus spp. certainly represent promising candidates to colonize areas abandoned by more sensitive macroalgae. The scale and importance of indirect effects of stresses, however, are only beginning to be understood. As a complicated yet realistic hypothetical example, moderate warming may not affect Fucus directly, but could have a large indirect impact via a synergistic interaction involving (i) a slight increase in the metabolic rate of consumers (=increased grazing pressure); (ii) a slight decrease in the defence strength of Fucus leading to; (iii) slightly higher fouling pressure and, in turn leading to; (iv) a slight decrease in available solar energy, further reducing; (v) defence strength and (vi) overall fitness (Fig. 2.5). The key realization is that accumulation of small effects acting in a non-linear manner could suddenly move towards a tipping point and major regime shift (Scheffer and van Nes, 2004; Christensen et al., 2006; Harley et al., 2006).

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

91

7.4. Open questions Stress ecology has gained a new dimension of relevance and urgency in our contemporary world. At the same time we are in a position to integrate disciplines and exploit new types of data and multivariate analyses to address the role of climate change at the species and community level. With these considerations in mind, the following questions are now tractable.

Are there consistent combinations of stress interactions that may provide general predictions about community responses to climate change? What suites of genes are associated with particular stress interaction networks affecting, for example, salinity or temperature tolerance, chemical defence and susceptibility to predation? What is the potential of biotic interactions to modulate the effects of abiotic stress and community resources; and how does this feedback to the long-term resilience of Fucus communities? Will marginal and core populations differ in their response to global change? What specific insights about climate change effects can be gained from understanding the responses of Fucus-based communities as compared with other foundational species and their associated communities (e.g. seagrasses, mussels)?

Appendix A Supplementary Materials Supplementary data associated with this chapter can be found, in the online version, at doi:10.1016/B978-0-12-385536-7.00002-9.

REFERENCES Airoldi, L. (1998). Roles of disturbance, sediment stress, and substratum retention on spatial dominance in algal turf. Ecology 79, 2759–2770. Allison, G. (2004). The influence of species diversity and stress intensity on community resistance and resilience. Ecol. Monogr. 74(1), 117–134. Allendorf, F. W., and Luikart, G. (2007). Conservation and the genetics of populations. Blackwell Publishing, Malden, MA. Altamirano, M., Flores-Moya, A., Kuhlenkamp, R., and Figueroa, F. L. (2003). Stagedependent sensitivity to ultraviolet radiation in zygotes of the brown alga Fucus serratus. Zygote 11, 101–106. Altieri, A. H., Silliman, B. R., and Bertness, M. D. (2007). Hierarchical organization via a facilitation cascade in intertidal cordgrass bed communities. Am. Nat. 169, 195–206. Amsler, C. D., and Fairhead, V. A. (2006). Defensive and sensory chemical ecology of brown algae. Adv. Bot. Res. 43, 1–91.

92

Martin Wahl et al.

Andersson, S., and Kautsky, L. (1996). Copper effects on reproductive stages of Baltic Sea Fucus vesiculosus. Mar. Biol. 125, 171–176. Ang, P. O. (1991). Natural dynamics of a Fucus distichus (Phaeophyceae, Fucales) population—reproduction and recruitment. Mar. Ecol. Prog. Ser. 78, 71–85. Appelhans, Y. S., Lenz, M., Medeiros, H. E., da Gama, B. A. P., Pereira, R. C., and Wahl, M. (2010). Stressed, but not defenceless: no obvious influence of irradiation levels on antifeeding and antifouling defences of tropical macroalgae. Mar.Biol. 157, 1151–1159. Arkema, K. K., Reed, D. C., and Schroeter, S. C. (2009). Direct and indirect effects of giant kelp determine benthic community structure and dynamics. Ecology 90, 3126–3137. Bartsch, I., Wiencke, C., Bischof, K., Buchholz, C. M., Buck, B. H., Eggert, A., Feuerpfeil, P., Hanelt, D., Jacobsen, S., Karez, R., Karsten, U., Molis, M., Roleda, M. Y., Schubert, H., Schumann, R., Valentin, K., Weinberger, F., and Wiese, J. (2008). The genus Laminaria sensulato: recent insights and developments. Eur. J. Phycol. 43, 1–86. Benedetti-Cecchi, L. (2000). Variance in ecological consumer-resource interactions. Nature 407, 370–374. Berger, R., Bergstr€ o m, L., Graneli, E., and Kautsky, L. (2004). How does eutrophication affect different life stages of Fucus vesiculosus in the Baltic Sea? A conceptual model. Hydrobiologia 514, 243–248. Berger, R., Henriksson, E., Kautsky, L., and Malm, T. (2003). Effects of filamentous algae and deposited matter on the survival of Fucus vesiculosus L. germlings in the Baltic Sea. Aquat. Ecol. 37, 1–11. Berger, R., Malm, T., and Kautsky, L. (2001). Two reproductive strategies in Baltic Fucus vesiculosus (Phaeophyceae). Eur. J. Phycol. 36, 265–273. Bergstr€ o m, L., Berger, R., and Kautsky, L. (2003). Negative direct effects of nutrient enrichment on the establishment of Fucusvesiculosus in the Baltic Sea. Eur.J.Phycol. 38, 41–46. Bergstr€ o m, L., Tatarenkov, A., Johannesson, K., J€ onsson, R. B., and Kautsky, L. (2005). Genetic and morphological identification of Fucus radicans sp. Nov. (Fucales, Phaeophyceae) in the brackish Baltic Sea. J. Phycol. 41, 1025–1038. Berndt, M. L., Callow, J. A., and Brawley, S. H. (2002). Gamete concentrations and timing and success of fertilization in a rocky shore seaweed. Mar.Ecol.Prog.Ser. 226, 273–285. Bijlsma, R., and Loeschcke, V. (2005). Environmental stress, adaptation and evolution: an overview. J. Evol. Biol. 18, 744–749. Billard, E., Daguin, C., Pearson, G. A., Serr~ao, E. A., Engel, C. R., and Valero, M. (2005a). Genetic isolation between three closely related taxa: Fucusvesiculosus, F.spiralis and F. ceranoides. J. Phycol. 41, 900–905. Billard, E., Serrao, E., Pearson, G., Destombe, C., and Valero, M. (2010). Fucus vesiculosus and spiralis species complex: a nested model of local adaptation at the shore level. Mar. Ecol. Prog. Ser. 405, 163–174. Billard, E., Serr~ao, E. A., Pearson, G. A., Engel, C. R., Destombe, C., and Valero, M. (2005b). Analysis of sexual phenotype and prezygotic fertility in natural populations of F. spiralis, F. vesiculosus (Fucaceae, Phaeophyceae) and their putative hybrids. Eur. J. Phycol. 40, 397–407. Bischof, K., Krabs, G., Wiencke, C., and Hanelt, D. (2002). Solar ultraviolet radiation affects the activity of ribulose-1,5-bisphosphate carboxylase-oxygenase and the composition of photosynthetic and xanthophyll cycle pigments in the intertidal green alga Ulva lactuca L. Planta 215, 502–509. Blanchette, C. A. (1997). Size and survival of intertidal plants in response to wave action: a case study with Fucus gardneri. Ecology 78, 1563–1578. Bostrom, C., and Mattila, J. (2005). Effects of isopod grazing: an experimental comparison in temperate (Idotea balthica, Baltic Sea, Finland) and subtropical (Erichsonella attenuata, of Mexico, USA) ecosystems. Crustaceana 78, 185–200.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

93

Brawley, S. H., Coyer, J. A., Blakeslee, A. M. H., Hoarau, G., Johnson, L. E., Byers, J. E., Stam, W. T., and Olsen, J. L. (2009). Historical invasions of the intertidal zone of Atlantic North America associated with distinctive patterns of trade and emigration. Proc. Natl. Acad. Sci. U.S.A. 106, 8239–8244. Brawley, S. H., and Johnson, L. E. (1991). Survival of fucoid embryos in the intertidal zone depends upon developmental stage and microhabitat. J. Phycol. 27, 179–186. Brock, E., Nylund, G., and Pavia, H. (2007). Chemical inhibition of barnacle larval settlement by the brown alga Fucus vesiculosus. Mar. Ecol. Prog. Ser. 337, 165–174. Brooks, S. J., Bolam, T., Tolhurst, L., Bassett, J., La Roche, J., Waldock, M., Barry, J., and Thomas, K. V. (2008). Dissolved organic carbon reduces the toxicity of copper to germlings of the macroalgae, Fucus vesiculosus. Ecotoxicol. Environ. Saf. 70, 88–98. Burrows, E. M., and Lodge, S. M. (1951). Autecology and the species problem in Fucus. J. Mar. Biol. Assoc. U.K. 30, 161–176. Cervin, G., A berg, P., and Jenkins, S. R. (2005). Small-scale disturbance in a stable canopy dominated community: implications for macroalgal recruitment and growth. Mar. Ecol. Prog. Ser. 305, 31–40. Chapman, A. R. O. (1995). Functional ecology of fucoid algae: twenty-three years of progress. Phycologia 34, 1–32. Chapman, A. S., and Fletcher, R. L. (2002). Differential effects of sediments on survival and growth of Fucus serratus embryos (Fucales, Phaeophyceae). J. Phycol. 38, 894–903. Christensen, M. R., Graham, M. D., Vinebrooke, R. D., Findlay, D. L., Paterson, M. J., and Turner, A. M. (2006). Multiple anthropogenic stressors cause ecological surprises in boreal lakes. Global Change Biol. 12, 2316–2322. Coleman, M. A., and Brawley, S. H. (2005). Are life history characteristics good predictors of genetic diversity and structure? A case study of the intertidal alga Fucus spiralis (Heterokontophyta; Phaeophyceae). J. Phycol. 41, 753–762. Coles, J. W. (1958). Nematodes parasitic on sea weeds of the genera Ascophyllum and Fucus. J. Mar. Biol. Assoc. U.K. 37, 145–155. Connell, J. H. (1972). Community interactions on marine rocky intertidal shores. Ann. Rev. Ecol. Syst. 3, 169–192. Connell, J. H., and Slatyer, R. (1977). Mechanisms of succession in natural communities and their role in community stability and organization. Am. Nat. 111, 1119–1144. Coyer, J. A., Hoarau, G., Beszteri, B., Pearson, G., and Olsen, J. L. (2009). Expressed sequence tag-derived polymorphic SSR markers for Fucus serratus and amplification in other species of Fucus. Mol. Ecol. Res. 9, 168–170. Coyer, J. A., Hoarau, G., Costa, J. F., Hogerdijk, B., Serr~ao, E. A., Billard, E., Valero, M., Pearson, G. A., and Olsen, J. L. (2011a). Evolution and diversification within the intertidal brown macroalgae Fucus spiralis/F. vesiculosus species complex in the North Atlantic. Mol. Phylogenet. Evol., 58:283–296. Coyer, J. A., Hoarau, G., Oudot-Le Secq, M. P., Stam, W. T., and Olsen, J. L. (2006a). AmtDNA-based phylogeny of the brown algal genus Fucus (Heterokontophyta; Phaeophyta). Mol. Phylogenet. Evol. 39, 209–222. Coyer, J. A., Hoarau, G., Pearson, G., Mota, C., J€ uterbock, A., Alpermann, T., John, U., and Olsen, J. L. (2011b). Genomic scans detect signatures of selection along a salinity gradient in populations of the intertidal seaweed Fucus serratus on a 12 km scale. Mar. Genom., 4:41-49. Coyer, J. A., Hoarau, G., Pearson, G. A., Serr~ao, E. A., Stam, W. T., and Olsen, J. L. (2006b). Convergent adaptation to a marginal habitat by homoploid hybrids and polyploidecads in the seaweed genus Fucus. Biol. Lett. 2, 405–408. Coyer, J. A., Hoarau, G., Schaik, J. V., Luijckx, P., and Olsen, J. L. (2011c). Trans-Pacific and trans-Arctic pathways of the intertidal macroalgal Fucus distichus reveal multiple

94

Martin Wahl et al.

glacial refugia and colonisations from the North Pacific to the North Atlantic. J. Biogeogr., 38:756–771. Coyer, J. A., Hoarau, G., Sjøtun, K., and Olsen, J. L. (2008). Being abundant is not enough: a decrease in effective population size over eight generations in a Norwegian population of the seaweed, Fucus serratus. Biol. Lett. 4, 755–757. Coyer, J. A., Hoarau, G., Skage, M., Stam, W. T., and Olsen, J. L. (2006c). Origin of Fucus serratus (Heterokonotophyta; Fucaceae) populations on Iceland and The Faroes: a microsatellite-based assessment. Eur. J. Phycol. 41, 235–246. Coyer, J. A., Hoarau, G., Stam, W. T., and Olsen, J. L. (2007). Hybridization and introgression in a mixed population of the intertidal seaweeds Fucus distichus and F. serratus. J. Evol. Biol. 16, 3606–3616. Coyer, J. A., Peters, A. F., Hoarau, G., Stam, W. T., and Olsen, J. L. (2002a). Hybridisation of the marine seaweeds, Fucus serratus and F. evanescens (Heterokontophyta; Phaeophyceae) in a century-old zone of secondary contact. Proc. R. Soc. Lond. B Biol. Sci. 269, 1829–1834. Coyer, J. A., Peters, A. F., Hoarau, G., Stam, W. T., and Olsen, J. L. (2002b). Inheritance patterns of ITS1, chloroplasts, and mitochondria in artificial hybrids of the marine rockweeds, Fucus serratus and F. evanescens (Heterokontophyta; Fucaceae). Eur. J. Phycol. 37, 173–178. Coyer, J. A., Peters, A. F., Stam, W. T., and Olsen, J. L. (2003). Post-ice age recolonization and differentiation of Fucus serratus L. (Fucaceae: Phaeophyta) populations in Northern Europe. Mol. Ecol. 12, 1817–1829. Coyer, J. A., Veldsink, J. H., Stam, W. T., and Olsen, J. L. (2002c). Characterization of microsatellite loci in the marine rockweeds, Fucus serratus and F. evanescens (Heterokontophyta; Fucaceae). Mol. Ecol. Notes 2, 35–37. Crain, C. M., Kroeker, K., and Halpern, B. S. (2008). Interactive and cumulative effects of multiple human stressors in marine systems. Ecol. Lett. 11, 1304–1315. Creed, J. C., Norton, T. A., and Kain (Jones), J. M. (1996). Are neighbours harmful or helpful in Fucus vesiculosus populations? Mar. Ecol. Prog. Ser. 133, 191–201. Cronin, G., and Hay, M. E. (1996). Effects of light and nutrient availability on the growth, secondary chemistry, and resistance to herbivory of two brown seaweeds. Oikos 77, 93–106. Csikasz-Nagy, A., and Soyer, O. S. J. (2008). Adaptive dynamics with a single two-state protein. J. R. Soc. Interface 5, S41–S47. Darling, E. S., and Cote, I. M. (2008). Quantifying the evidence for ecological synergies. Ecol. Lett. 11, 1278–1286. Davies, A. J., Johnson, M. P., and Maggs, C. A. (2007). Limpet grazing and loss of Ascophyllum nodosum canopies on decadal time scales. Mar. Ecol. Prog. Ser. 339, 131–141. Davis, J. A., McGuire, M., Halse, S. A., Hamilton, D., Horwitz, P., McComb, A. J., Froend, R. H., Lyons, M., and Sim, L. (2003). What happens when you add salt: predicting impacts of secondary salinisation on shallow aquatic ecosystems by using an alternative-states model. Aust. J. Bot. 51, 715–724. Davis, M. B., Shaw, R. G., and Etterson, J. R. (2005). Evolutionary responses to changing climate. Ecology 86, 1704–1714. Davison, I. R., Dudgeon, S. R., and Hang-Ming, R. (1989). Effect of freezing on seaweed photosynthesis. Mar. Ecol. Prog. Ser. 58, 123–131. Davison, I. R., and Pearson, G. A. (1996). Stress tolerance in intertidal seaweeds. J.Phycol. 32, 197–211. Dayton, P. K., Currie, V., Gerrodette, T., Keller, B. D., Rosenthal, R., and Tresca, D. V. (1984). Patch dynamics and stability of some California kelp communities. Ecol. Monogr. 54, 253–289.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

95

Dethier, M. N., and Williams, S. L. (2009). Seasonal stresses shift optimal intertidal algal habitats. Mar. Biol. 156, 555–567. Dethier, M. N., Williams, S. L., and Freeman, A. (2005). Seaweeds under stress: manipulated stress and herbivory affect critical life-history functions. Ecol. Monogr. 75, 403–418. Dobretsov, S. (2008). Inhibition and induction of marine biofouling by biofilms. R. Venkatesan., P. S. Murthy., K. Cooksey, and H. -C. Flemming, eds.), Volume 4, pp. 293–314. Springer, Heidelberg. Dudgeon, S., and Petraitis, P. S. (2001). Scale-dependent recruitment and divergence of intertidal communities. Ecology 82, 991–1006. Dudgeon, S., and Petraitis, P. S. (2005). First year demography of the foundation species, Ascophyllum nodosum, and its community implications. Oikos 109, 405–415. Duggins, D. O., Eckman, J. E., and Sewell, A. T. (1990). Ecology of understory kelp environments. II. Effects of kelp on recruitment of benthic invertebrates. J.Exp.Mar. Biol. Ecol. 143, 27–45. Dworjanyn, S., Wright, J., Paul, N., de Nys, R., and Steinberg, P. (2006). Cost of chemical defence in the red alga Delisea pulchra. Oikos 113, 13–22. Edmands, S. (2008). Recombination in interpopulation hybrids of the copepod Tigriopus californicus: release of beneficial variation despite hybrid breakdown. J. Hered. 99, 316–318. Edmands, S., Feaman, H. V., Harrison, J. S., and Timmerman, C. C. (2005). Genetic consequences of many generations of hybridization between divergent copepod populations. J. Hered. 96, 114–123. Elser, J. J., Bracken, M. E. S., Cleland, E. E., Gruner, D. S., Harpole, W. S., Hillebrand, H., Ngai, J. T., Seabloom, E. W., Shurin, J. B., and Smith, J. E. (2007). Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol. Lett. 10, 1135–1142. Engel, C. R., Billard, E., Voisin, M., and Viard, F. (2008). Conservation and polymorphism of mitochondrial intergenic sequences in brown algae (Phaeophyceae). Eur. J. Phycol., 43. Engel, C. R., Brawley, S., Edwards, K. J., and Serr~ao, E. A. (2003). Isolation and crossspecies amplification of microsatellite loci from the fucoid seaweeds Fucus vesiculosus, F.serratus, and Ascophyllum nodosum (Heterokontophyta, Fucaceae). Mol.Ecol.Notes 3, 180–182. Engel, C. R., Daguin, C., and Serr~ao, E. A. (2005). Genetic entities and mating system in hermaphroditic Fucus spiralis and its close dioecious relative F. vesiculosus (Fucaceae, Phaeophyceae). Mol. Ecol. 14, 2033–2046. Engkvist, R., Malm, T., and Nilsson, J. (2004). Interaction between isopod grazing and wave action: a structuring force in macroalgal communities in the southern Baltic Sea. Aquat. Ecol. 38, 403–413. Engkvist, R., Malm, T., and Tobiasson, S. (2000). Density dependent grazing effects of the isopod Idotea baltica Pallas on Fucus vesiculosus L in the Baltic Sea. Aquat. Ecol. 34, 253–260. Eriksson, B. K., and Bergstr€ o m, L. (2005). Local distribution patterns of macroalgae in relation to environmental variables in the northern Baltic Proper. Estuar. Coast. Shelf Sci. 62, 109–117. Eriksson, B. K., and Johansson, G. (2003). Sedimentation reduces recruitment success of Fucus vesiculosus (Phaeophyceae) in the Baltic Sea. Eur. J. Phycol. 38, 217–222. Eriksson, B. K., and Johansson, G. (2005). Effects of sedimentation on macroalgae: species-specific responses are related to reproductive traits. Oecologia 143, 438–448.

96

Martin Wahl et al.

Eriksson, B. K., Johansson, G., and Snoeijs, P. (1998). Long-term changes in the sublittoral zonation of brown algae in the southern Bothnian Sea. Eur. J. Phycol. 33, 241–249. Eriksson, B. K., Rubach, A., and Hillebrand, H. (2006). Community dominance by a canopy species controls the relationship between macroalgal production and species richness. Limnol. Oceanogr. 51, 1813–1818. Eriksson, B. K., Rubach, A., and Hillebrand, H. (2007). Dominance by a canopy forming seaweed modifies resource and consumer control of bloom-forming macroalgae. Oikos 116, 1211–1219. Floeter, S. R., Behrens, M. D., Ferreira, C. E. L., Paddack, M. J., and Horn, M. H. (2005). Geographical gradients of marine herbivorous fishes: patterns and processes. Mar.Biol. 147, 1435–1447. Forslund, H., Wikstr€ o m, S. A., and Pavia, H. (2010). Higher resistance to herbivory in introduced compared to native populations of a seaweed. Oecologia 164, 833–840. Gamfeldt, L., and Kallstrom, B. (2007). Increasing intraspecific diversity increases predictability in population survival in the face of perturbations. Oikos 116, 700–705. Gilad, E., Shachak, M., and Meron, E. (2007). Dynamics and spatial organization of plant communities in water-limited systems. Theor. Popul. Biol. 72, 214–230. Grace, J. B. (2006). Structural equation modeling and natural systems. Cambridge University Press, Cambridge. Grace, J. B., Anderson, T. M., Olff, H., and Scheiner, S. M. (2010). On the specification of structural equation models for ecological systems. Ecol. Monogr. 80, 67–87. Green, D. G., and Sadedin, S. (2005). Interactions matter—complexity in landscapes and ecosystems. Ecol. Complex. 2, 117–130. Grime, J. P. (1989). The stress debate—symptom of impending synthesis. Biol.J.Linn.Soc. 37, 3–17. Gross, E. M. (2003). Allelopathy of aquatic autotrophs. Crit. Rev. Plant Sci 22, 313–339. Gruner, D. S., Smith, J. E., Seabloom, E. W., Sandin, S. A., Ngai, J. T., Hillebrand, H., Harpole, W. S., Elser, J. J., Cleland, E. E., Bracken, M. E. S., Borer, E. T., and Bolker, B. M. (2008). A cross-system synthesis of consumer and nutrient resource control on producer biomass. Ecol. Lett. 11, 740–755. Guiry, M. D., and Guiry, G. M. (2010). Algaebase version 01/10 World-wide electronic publication. National University of Ireland, Galway. http://www.algaebase.org/; searched on 10 March 2010. Hanelt, D., Wiencke, C., and Nultsch, W. (1997). Influence of UV radiation on the photosynthesis of arctic macroalgae in the field. J. Photochem. Photobiol. B 38, 40–47. Haring, N. R., Dethier, M. N., and Williams, S. L. (2002). Desiccation facilitates wave-induced mortality of the intertidal alga Fucus gardneri. Mar. Ecol. Prog. Ser. 232, 75–82. Harley, C. D. G., Hughes, A. R., Hultgren, K. M., Miner, B. G., Sorte, C. J. B., Thornber, C. S., Rodriguez, L. F., Tomanek, L., and Williams, S. L. (2006). The impacts of climate change in coastal marine systems. Ecol. Lett. 9, 228–241. Hay, M. E., and Steinberg, P. D. (1992). The chemical ecology of plant-herbivore interactions in marine versus terrestrial communities. G. A. Rosenthal, and M. R. Berenbaum, eds.), Volume II, pp. 371–413. Academic Press, San Diego. Hedges, L. V., Gurevitch, J., and Curtis, P. S. (1999). The meta-analysis of response ratios in experimental ecology. Ecology 80, 1150–1156. Helmuth, B., Harley, C. D. G., Halpin, P. M., O’Donnell, M., Hofmann, G. E., and Blanchette, C. A. (2002). Climate change and latitudinal patterns of intertidal thermal stress. Science 298, 1015–1017.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

97

Helmuth, B., Mieszkowska, N., Moore, P., and Hawkins, S. J. (2006). Living on the edge of two changing worlds: Forecasting the responses of rocky intertidal ecosystems to climate change. Annu. Rev. Ecol. Evol. Syst. 37, 373–404. Hemmi, A., and Jormalainen, V. (2002). Nutrient enhancement increases performance of a marine herbivore via quality of its food alga. Ecology 83, 1052–1064. Hemmi, A., and Jormalainen, V. (2004). Geographic covariation of chemical quality of the host alga Fucus vesiculosus with fitness of the herbivorous isopod Idotea baltica. Mar. Biol. 145, 759–768. Hemmi, A., Makinen, A., Jormalainen, V., and Honkanen, T. (2005). Responses of growth and phlorotannins in Fucus vesiculosus to nutrient enrichment and herbivory. Aquat. Ecol. 39, 201–211. Henry, B. E., and Van Alstyne, K. L. (2004). Effects of UV radiation on growth and phlorotannins in Fucus gardneri (Phaeophyceae) juveniles and embryos. J. Phycol. 40, 527–533. Hillebrand, H., Soininen, J., and Snoeijs, P. (2010). Warming leads to higher species turnover in a coastal ecosystem. Global Change Biol. 16, 1181–1193. Hoarau, G., Coyer, J. A., Veldsink, J. H., Stam, W. T., and Olsen, J. L. (2007). Glacial refugia and recolonization patterns in the brown seaweed Fucus serratus. Mol.Ecol. 16, 3606–3616. Hoffman, J. R., Hansen, L. J., and Klinger, T. (2003). Interactions between UV radiation and temperature limit inferences from single-factor experiments. J.Phycol. 39, 268–272. Hughes, A. R., Inouye, B. D., Johnson, M. T. J., Underwood, N., and Vellend, M. (2008). Ecological consequences of genetic diversity. Ecol. Lett. 11, 609–623. Huppertz, K., Hanelt, D., and Nultsch, W. (1990). Photoinhibition of photosynthesis in the marine brown alga Fucus serratus as studied in field experiments. Mar. Ecol. Prog. Ser. 66, 175–180. IPCC Climate Change. (2007). Synthesis Report. IPCC, Geneva, Switzerland. Iwamoto, K., and Shiraiwa, Y. (2005). Salt-regulated mannitol metabolism in algae. Mar. Biotechnol. 7, 407–415. Jenkins, S. R., Norton, T. A., and Hawkins, S. J. (1999). Interactions between canopy forming algae in the eulittoral zone of sheltered rocky shores on the Isle of Man. J. Mar. Biol. Assoc. U.K. 79, 341–349. Johannesson, K., and Andre, C. (2006). Life on the margin—genetic isolation and loss of variation in a peripheral marine ecosystem. Mol. Ecol. 15, 2013–2030. Johnson, M. P., Hawkins, S. J., Hartnoll, R. G., and Norton, T. A. (1998). The establishment of fucoid zonation on algal-dominated rocky shores: hypotheses derived from a simulation model. Funct. Ecol. 12, 259–269. Johnson, J. B., Peat, S. M., and Adams, B. J. (2009). Where’s the ecology in molecular ecology? Oikos 118, 1601–1609. Jonsson, P. R., Granhag, L., Moschella, P. S., A berg, P., Hawkins, S. J., and Thompson, R. C. (2006). Interactions between wave action and grazing control the distribution of intertidal macroalgae. Ecology 87, 1169–1178. Jørgensen, B. B., and Revsbech, N. P. (1985). Diffusive boundary layers and the oxygen uptake of sediments and detritus. Limnol. Oceanogr. 30, 111–122. Jormalainen, V., and Honkanen, T. (2004). Variation in natural selection for growth and phlorotannins in the brown alga Fucus vesiculosus. J. Evol. Biol. 17, 807–820. Jormalainen, V., and Honkanen, T. (2008). Macroalgal chemical defenses and their roles in structuring temperate marine communities. In ‘‘Algal Chemical Ecology’’ (C. D. Amsler ed.), pp. 57–89. Springer, Berlin. Jormalainen, V., Honkanen, T., Koivikko, R., and Eranen, J. (2003). Induction of phlorotannin production in a brown alga: defense or resource dynamics? Oikos 103, 640–650.

98

Martin Wahl et al.

Jormalainen, V., Honkanen, T., and Vesakoski, O. (2008a). Geographical divergence in host use ability of a marine herbivore in alga-grazer interaction. Evol. Ecol. 22, 545–559. Jormalainen, V., Koivikko, R., Ossipov, V., and Lindqvist, M. (2011). Quantifying variation and chemical correlates of bladderwrack quality-herbivore population makes a difference. Functional Ecology DOI: 10.1111/j.1365–2435.2011.01841.x. Jormalainen, V., and Ramsay, T. (2009). Resistance of the brown alga Fucus vesiculosus to herbivory. Oikos 118, 713–722. Jormalainen, V., Wikstrom, S. A., and Honkanen, T. (2008b). Fouling mediates grazing: intertwining of resistances to multiple enemies in the brown alga Fucus vesiculosus. Oecologia 155, 559–569. Kapraun, D. F. (2005). Nuclear DNA content estimates in multicellular green, red, and brown algae: phylogenetic considerations. Ann. Bot. (Lond.) 95, 7–44. Karez, R. (2003a). Do monospecific stands of three Fucus species (Phaeophyta) comply with the ‘self-thinning rule’? Eur. J. Phycol. 38, 171–180. Karez, R. (2003b). Competitive ranks of three Fucus spp. (Phaeophyta) in laboratory experiments—testing of Keddy’s competitive hierarchy model. Helgol. Mar. Res. 57, 83–90. Karez, R., and Chapman, A. R. O. (1998). A competitive hierarchy model integrating roles of physiological competence and competitive ability does not provide a mechanistic explanation for the zonation of three intertidal Fucus species in Europe. Oikos 81, 471–494. Karez, R., Engelbert, S., and Sommer, U. (2000). ‘Co-consumption’ and ‘protective coating’: two new proposed effects of epiphytes on their macroalgal hosts in mesograzer-epiphyte-host interactions. Mar. Ecol. Prog. Ser. 205, 85–93. Kautsky, H., Kautsky, L., Kautsky, N., Kautsky, U., and Lindblad, C. (1992). Studies on the Fucus vesiculosus community in the Baltic Sea. Acta Phytogeogr. Suec. 78, 33–48. Kautsky, N., Kautsky, H., Kautsky, U., and Waern, M. (1986). Decreased depth penetration of Fucusvesiculosus (L.) since the 1940s indicates eutrophication of the Baltic Sea. Mar. Ecol. Prog. Ser. 28, 1–8. Kawecki, T. J. (2008). Adaptation to marginal habitats. Annu. Rev. Ecol. Evol. Syst. 39, 321–342. Keser, M., Swenarton, J. T., and Foertch, J. F. (2005). Effects of thermal input and climate change on growth of Ascophyllum nodosum (Fucales, Phaeophyceae) in eastern Long Island Sound (USA). J. Sea Res. 54, 211–220. Kiirikki, M. (1996). Experimental evidence that Fucus vesiculosus (Phaeophyta) controls filamentous algae by means of the whiplash effect. Eur. J. Phycol. 31, 61–66. Kiirikki, M., and Ruuskanen, A. (1996). How does Fucus vesiculosus survive ice scraping? Bot. Mar. 39, 133–139. Koivikko, R., Eranen, J. K., Loponen, J., and Jormalainen, V. (2008). Variation of phlorotannins among three populations of Fucus vesiculosus as revealed by HPLC and colorimetric quantification. J. Chem. Ecol. 34, 57–64. Korpinen, S., Honkanen, T., Vesakoski, O., Hemmi, A., Koivikko, R., Loponen, J., and Jormalainen, V. (2007a). Macroalgal communities face the challenge of changing biotic interactions: review with focus on the Baltic Sea. Ambio 36, 203–211. Korpinen, S., and Jormalainen, V. (2008a). Grazing and nutrients reduce recruitment success of Fucus vesiculosus L. (Fucales: Phaeophyceae). Estuar. Coast. Shelf Sci. 78, 437–444. Korpinen, S., and Jormalainen, V. (2008b). Grazing effects in macroalgal communities depend on timing of patch colonization. J. Exp. Mar. Biol. Ecol. 360, 39–46. Korpinen, S., Jormalainen, V., and Honkanen, T. (2007b). Effects of nutrients, herbivory, and depth on the macroalgal community in the rocky sublittoral. Ecology 88, 839–852.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

99

Korpinen, S., Jormalainen, V., and Pettay, E. (2010). Nutrient availability modifies species abundance and community structure of Fucus-associated littoral benthic fauna. Mar. Environ. Res. 70, 283–292. Kotta, J., Orav-Kotta, H., Paalme, T., Kotta, I., and Kukk, H. (2006). Seasonal changes in situ grazing of the mesoherbivores Idoteabaltica and Gammarus oceanicus on the brown algae Fucus vesiculosus and Pylaiella littoralis in the central Gulf of Finland, Baltic Sea. Hydrobiologia 554, 117–125. Kraufvelin, P., Salovius, S., Christie, H., Moy, F. E., and Karez, R. (2006). Eutrophicationinduced changes in benthic algae affect the behavior and fitness of the marine amphipod Gammarus locusta. Aquat. Bot. 84, 199–209. Lachnit, T., Bl€ umel, M., Imhoff, J. F., and Wahl, M. (2009). Specific epibacterial communities on macroalgae: phylogeny matters more than habitat. Aquat. Biol. 5, 181–186. Lachnit, T., Wahl, M., and Harder, T. (2010). Isolated thallus-associated compounds from the macroalga Fucus vesiculosus mediate bacterial surface colonization in the field similar to that on the natural alga. Biofouling 26, 247–255. Ladah, L., Bermudez, R., Pearson, G., and Serrao, E. (2003). Fertilization success and recruitment of dioecious and hermaphroditic fucoid seaweeds with contrasting distributions near their southern limit. Mar. Ecol. Prog. Ser. 262, 173–183. Ladah, L. B., Feddersen, F., Pearson, G. A., and Serrao, E. A. (2008). Egg release and settlement patterns of dioecious and hermaphroditic fucoid algae during the tidal cycle. Mar. Biol. 155, 583–591. Lago-Leston, A., Mota, C., Kautsky, L., and Pearson, G. A. (2010). Functional divergence in heat shock response following rapid speciation of Fucus spp. in the Baltic Sea Mar. Biology 157, 683–688. Lamote, M., and Johnson, L. E. (2008). Temporal and spatial variation in the early recruitment of fucoid algae: the role of microhabitats and temporal scales. Mar. Ecol. Prog. Ser. 368, 93–102. Lamote, M., Johnson, L. E., and Lemoine, Y. (2007). Interspecific differences in the response of juvenile stages to physical stress: fluorometric responses of fucoid embryos to variation in meteorological conditions. J. Phycol. 43, 1164–1176. Lehvo, A., Back, S., and Kiirikki, M. (2001). Growth of Fucus vesiculosus L. (Phaeophyta) in the northern Baltic proper: energy and nitrogen storage in seasonal environment. Bot. Mar. 44, 345–350. Li, R., and Brawley, S. H. (2004). Improved survival under heat stress in intertidal embryos (Fucus spp.) simultaneously exposed to hypersalinity and the effect of parental thermal history. Mar. Biol. 144, 205–213. Lima, F. P., Ribeiro, P. A., Queiroz, N., Hawkins, S. J., and Santos, A. M. (2007). Do distributional shifts of northern and southern species of algae match the warming pattern? Global Change Biol. 13, 2592–2604. Long, J. D., Hamilton, R. S., and Mitchell, J. L. (2007). Asymmetric competition via induced resistance: specialist herbivores indirectly suppress generalist preference and populations. Ecology 88, 1232–1240. Lotze, H., Worm, B., and Sommer, U. (2000). Propagule banks, herbivory and nutrient supply control population development and dominance patterns in macroalgal blooms. Oikos 89, 46–58. Lubchenco, J. (1983). Littorina and Fucus: effects of herbivores, substratum heterogeneity and plant escapes during succession. Ecology 64, 1116–1123. L€ uning, K. (1990). Seaweeds: Their Environment, Biogeography and Ecophysiology. John Wiley & Sons, New York. Macaya, E. C., Roth€ausler, E., Thiel, M., Molis, M., and Wahl, M. (2005). Induction of defenses and within-alga variation of palatability in two brown algae from the

100

Martin Wahl et al.

northern-central coast of Chile: effects of mesograzers and UV radiation. J.Exp.Mar. Biol. Ecol. 325, 214–227. Maczassek, K., and Wahl, M. (in prep.). Sensitivity of Fucus vesiculosus germlings to interactive effects of temperature and salinity. Maheswari, M., Joshi, D. K., Saha, R., Nagarajan, S., and Gambhir, P. N. (1999). Transverse relaxation time of leaf water protons and membrane injury in wheat (Triticum aestivum L.) in response to high temperature. Ann. Bot. 84, 741–745. Mallet, J. (2005). Hybridization as an invasion of the genome. Trends Ecol. Evol. 20, 229–237. Malm, T., and Isaeus, M. (2005). Distribution of macroalgal communities in the central Baltic Sea. Ann. Bot. Fenn. 42, 257–266. Malm, T., and Kautsky, L. (2003). Differences in life-history characteristics are consistent with the vertical distribution pattern of Fucus serratus and Fucus vesiculosus (Fucales, Phaeophyceae) in the central Baltic Sea. J. Phycol. 39, 880–887. Malm, T., Kautsky, L., and Engkvist, R. (2001). Reproduction, recruitment and geographical distribution of Fucus serratus L. in the Baltic Sea. Bot. Mar. 44, 101–108. Mata, Y. N., Blazquez, M. L., Ballester, A., Gonzalez, F., and Munoz, J. A. (2008). Characterization of the biosorption of cadmium, lead and copper with the brown alga Fucus vesiculosus. J. Hazard. Mater. 158, 316–323. Melzner, F., K€ ortzinger, A., Koeve, W., Oschlies, A., Gutowska, M., Bange, H., and Hansen, H. P. (2010). Future ocean acidification will be amplified by hypoxia in coastal habitats. Global Change Biol., submitted. Menge, B. A. (1995). Indirect effects in marine rocky intertidal interaction webs: patterns and importance. Ecol. Monogr. 65, 21–74. Metzker, M. L. (2010). Sequencing technologies—the next generation. Nat. Rev. Genet. 11, 31–46. Michler, T., Aguilera, J., Hanelt, T., Bischof, K., and Wiencke, C. (2002). Long term effects of ultraviolet radiation on growth and photosynthetic performance of polar and cold-temperate macroalgae. Mar. Biol. 140, 1117–1127. Middelboe, A. L., Sand-Jensen, K., and Binzer, T. (2006). Highly predictable photosynthetic production in natural macroalgal communities from incoming and absorbed light. Oecologia 150, 464–476. Moore, P., Hawkins, S. J., and Thompson, R. C. (2007). Role of biological habitat amelioration in altering the relative responses of congeneric species to climate change. Mar. Ecol. Progr. Ser. 334, 11–19. Muhlin, J. F., and Brawley, S. H. (2009). Recent versus relic: discerning the genetic signature of Fucus vesiculosus (Heterokontophyta; Phaeophyceae) in the northwestern Atlantic. J. Phycol. 45, 828–837. Munda, I. M., and Kremer, B. P. (1977). Chemical composition and physiological properties of fucoids under conditions of reduced salinity. Mar. Biol. 42, 9–15. Neiva, J., Pearson, G. A., Valero, M., and Serrao, E. A. (2010). Surfing the wave on a borrowed board: range expansion and spread of introgressed organellar genomes in the seaweed Fucus ceranoides L. Mol. Ecol. 19, 4812–4822. Nietsch, B. (2009). Messung der Z€ahigkeit der Makroalge Fucus vesiculosus an verschiedenen Standorten und Untersuchung der Auswirkungen auf die trophische Interaktion. University of Kiel, Kiel, Diploma thesis. Nygard, C. A., and Dring, M. J. (2008). Influence of salinity, temperature, dissolved inorganic carbon and nutrient concentration on the photosynthesis and growth of Fucus vesiculosus from the Baltic and Irish Seas. Eur. J. Phycol. 43, 253–262. Nygard, C. A., and Ekelund, N. G. A. (2006). Photosynthesis and UV-B tolerance of the marine alga Fucus vesiculosus at different sea water salinities. J.Appl.Phycol. 18, 461–467.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

101

Ojaveer, H., Jaanus, A., MacKenzie, B. R., Martin, G., Olenin, S., Radziejewska, T., Telesh, I., Zettler, M. L., and Zaiko, A. (2010). Status of biodiversity in the Baltic Sea. PLoS One 5, e12467. Oswald, R. C., Telford, N., Seed, R., and Happey-Wood, C. M. (1984). The effect of encrusting bryozoans on the photosynthetic activity of Fucus serratus L. Estuar. Coast. Shelf Sci. 19, 697–702. Ouborg, N. J., and Vriezen, W. H. (2007). An ecologist’s guide to ecogenomics. J. Ecol. 95, 8–16. Pansch, C., Cerda, O., Lenz, M., Wahl, M., and Thiel, M. (2009). Consequences of light reduction for anti-herbivore defense and bioactivity against mussels in four seaweed species from northern-central Chile. Mar. Ecol. Prog. Ser. 381, 83–97. Parker, T., and Chapman, A. R. O. (1994). Separating the grazing effects of periwinkles and amphiphods on a seaweed community dominated by Fucus distichus. Ophelia 39, 75–91. Parmesan, C. (2006). Ecological and evolutionary responses to recent climate change. Annu. Rev. Ecol. Evol. Syst. 37, 637–669. Pavia, H., Cervin, G., Lindgren, A., and A berg, P. (1997). Effects of UV-B radiation and simulated herbivory on phlorotannins in the brown alga Ascophyllum nodosum. Mar. Ecol. Prog. Ser. 157, 139–146. Pavia, H., and Toth, G. B. (2000). Influence of light and nitrogen on the phlorotannin content of the brown seaweeds Ascophyllum nodosum and Fucus vesiculosus. Hydrobiologia 440, 299–305. Pearson, G. A., and Brawley, S. H. (1996). Reproductive ecology of Fucus distichus (Phaeophyceae): an intertidal alga with successful external fertilization. Mar. Ecol. Prog. Ser. 143, 211–223. Pearson, G. A., and Brawley, S. H. (1998). A model for signal transduction during gamete release in the fucoid alga Pelvetia compressa. Plant Physiol. 118, 305–313. Pearson, G. A., and Davison, I. R. (1993). Freezing rate and duration determine the physiological response of intertidal fucoids to freezing. Mar. Biol. 115, 353–362. Pearson, G., Kautsky, L., and Serr~ao, E. A. (2000). Recent evolution in Baltic Fucus vesiculosus: reduced tolerance to emersion stresses compared to intertidal (North Sea) populations. Mar. Ecol. Prog. Ser. 202, 67–79. Pearson, G. A., Lago-Leston, A., and Mota, C. (2009). Frayed at the edges: selective pressure and adaptive response to abiotic stressors are mismatched in low diversity edge populations. J. Ecol. 97, 450–462. Pearson, G. A., and Serr~ao, E. A. (2006). Revisiting synchronous gamete release by fucoid algae in the intertidal zone: fertilization success and beyond? Integr. Comp. Biol. 46, 587–597. Pearson, G. A., Serrao, E. A., and Brawley, S. H. (1998). Control of gamete release in fucoid algae: sensing hydrodynamic conditions via carbon acquisition. Ecology 79, 1725–1739. Penhale, P. A. (1977). Macrophyte-epiphyte biomass and productivity in an eelgrass (Zostera marina L.) community. J. Exp. Mar. Biol. Ecol. 26, 211–224. Pennings, S. C., Carefoot, T. H., Zimmer, M., Danko, J. P., and Ziegler, A. (2000). Feeding preferences of supralittoral isopods and amphipods. Can. J. Zool. 78, 1918–1929. Pereyra, R. T., Bergstr€ o m, L., Kautsky, L., and Johannesson, K. (2009). Rapid speciation in a newly opened postglacial marine environment, the Baltic Sea. BMCEvol.Biol. 9, . doi: 10.1186/1471-2148-1189-1170. Perrin, C., Daguin, C., Van De Vliet, M., Engel, C. R., Pearson, G. A., and Serrao, E. A. (2007). Implications of mating system for genetic diversity of sister algal species: Fucus spiralis and Fucus vesiculosus (Heterokontophyta, Phaeophyceae). Eur. J. Phycol. 42, 219–230.

102

Martin Wahl et al.

Peterson, A. T. (2003). Predicting the geography of species’ invasions via ecological niche modeling. Q. Rev. Biol. 78, 419–433. Pflugmacher, S., Olin, M., and Kankaanpaa, H. (2007). Nodularin induces oxidative stress in the Baltic Sea brown alga Fucusvesiculosus (Phaeophyceae). Mar.Environ.Res. 64, 149–159. Pohnert, G. (2004). Chemical defense strategies of marine organisms. In ‘‘Chemistry of Pheromones and Other Semiochemicals I’’ (G. Pohnert ed.), pp. 179–219. Springer, Berlin. Przeslawski, R., Ahyong, S., Byrne, M., Worheide, G., and Hutchings, P. (2008). Beyond corals and fish: the effects of climate change on noncoral benthic invertebrates of tropical reefs. Global Change Biol. 14, 2773–2795. Ra berg, S., and Kautsky, L. (2008). Grazer identity is crucial for facilitating growth of the perennial brown alga Fucus vesiculosus. Mar. Ecol. Prog. Ser. 361, 111–118. Rohde, S., Hiebenthal, C., Wahl, M., Karez, R., and Bischof, K. (2008). Decreased depth distribution of Fucus vesiculosus (Phaeophyceae) in the Western Baltic: effects of light deficiency and epibionts on growth and photosynthesis. Eur. J. Phycol. 43, 143–150. Rohde, S., Molis, M., and Wahl, M. (2004). Regulation of anti-herbivore defence by Fucus vesiculosus in response to various cues. J. Ecol. 92, 1011–1018. Rohde, S., and Wahl, M. (2008). Antifeeding defense in baltic macroalgae: induction by direct grazing versus waterborne cues. J. Phycol. 44, 85–90. Scheffer, M., and van Nes, E. H. (2004). Mechanisms for marine regime shifts: can we use lakes as microcosms for oceans? Prog. Oceanogr. 60, 303–319. Schiel, D. R. (2009). Multiple stressors and disturbances: when change is not in the nature of things. In ‘‘Marine Hard Bottom Communities: Patterns, Dynamics, Diversity, and Change’’ (M. Wahl ed.), pp. 281–294. Springer, Stuttgart. Schiel, D. R., and Foster, M. S. (2006). The population biology of large brown seaweeds: ecological consequences of multiphase life histories in dynamic coastal environments. Ann. Rev. Ecol. Evol. Syst. 37, 343–372. Schiel, D. R., Steinbeck, J. R., and Foster, M. S. (2004). Ten years of induced ocean warming causes comprehensive changes in marine benthic communities. Ecology 85, 1833–1839. Schiel, D. R., Wood, S. A., Dunmore, R. A., and Taylor, D. I. (2006). Sediment on rocky intertidal reefs: effects on early post-settlement stages of habitat-forming seaweeds. J. Exp. Mar. Biol. Ecol. 331, 158–172. Schmidt, P. S., Serr~ao, E. A., Pearson, G. A., Riginos, C., Rawson, P. D., Hilbish, T. J., Brawley, S. H., Trussell, G. C., Carrington, E., Wethey, D. S., Grahame, J. W., Bonhomme, F., and Rand, D. M. (2008). Ecological genetics in the North Atlantic: environmental gradients and adaptation at specific loci. Ecology 89, S91–S107. Schneider, K. R. (2008). Heat stress in the intertidal: comparing survival and growth of an invasive and native mussel under a variety of thermal conditions. Biol. Bull. (Woods Hole) 215, 253–264. Schoenwaelder, M. E. A., Wiencke, C., Clayton, M. N., and Glombitza, K. W. (2003). The effect of elevated UV radiation on Fucus spp. (Fucales, Phaeophyta) zygote and embryo development. Plant Biol. 5, 366–377. Schonbeck, M., and Norton, T. (1978). Factors controlling the upper limits of fucoid algae on the shore. J. Exp. Mar. Biol. Ecol. 31, 303–313. Schonbeck, M., and Norton, T. A. (1980). Factors controlling the lower limits of fucoid algae on the shore. J. Exp. Mar. Biol. Ecol. 43, 131–150. Schubert, H., Sagert, S., and Forster, R. M. (2001). Evaluation of the different levels of variability in the underwater light field of a shallow estuary. Helgol.Mar.Res. 55, 12–22. Serr~ao, E. A., Alice, L. A., and Brawley, S. H. (1999a). Evolution of the Fucaceae (Phaeophyceae) inferred from nrDNA-ITS. J. Phycol. 35, 382–394. Serr~ao, E. A., Brawley, S. H., Hedman, J., Kautsky, L., and Samuelsson, G. (1999b). Reproductive success of Fucus vesiculosus (Phaeophyceae) in the Baltic Sea. J. Phycol. 35, 254–269.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

103

Serr~ao, E. A., Kautsky, L., and Brawley, S. H. (1996a). Distributional success of the marine seaweed Fucusvesiculosus L in the brackish Baltic Sea correlates with osmotic capabilities of Baltic gametes. Oecologia 107, 1–12. Serr~ao, E. A., Kautsky, L., Lifvergren, T., and Brawley, S. (1997). Gamete dispersal and pre-recruitment mortality in Baltic Fucus vesiculosus. Phycol. Suppl. 36, 101–102. Serr~ao, E. A., Pearson, G., Kautsky, L., and Brawley, S. H. (1996b). Successful external fertilization in turbulent environments. Proc. Natl. Acad. Sci. U.S.A. 93, 5286–5290. Spielman, D., Brook, B. W., and Frankham, R. (2004). Most species are not driven to extinction before genetic factors impact them. Proc. Natl. Acad. Sci. U.S.A. 101, 15261–15264. Stamp, N. (2003). Out of the quagmire of plant defense hypotheses. Q.Rev.Biol. 78, 23–55. Steen, H. (2003). Apical hair formation and growth of Fucus evanescens and F. serratus (Phaeophyceae) germlings under various nutrient and temperature regimes. Phycologia 42, 26–30. Steen, H., and Scrosati, R. (2004). Intraspecific competition in Fucus serratus and F. evanescens (Phaeophyceae: Fucales) germlings: effects of settlement density, nutrient concentration, and temperature. Mar. Biol. 144(1), 61–70. Steen, H. (2004). Interspecific competition between Enteromorpha (Ulvales: Chlorophyceae) and Fucus (Fucales: Phaeophyceae) germlings: effects of nutrient concentration, temperature, and settlement density. Mar.Ecol.Progr.Ser. 278, 89–101. Stewart, T. W., and Embrey, T. R. (2003). Demonstrating that habitat structure facilitates coexistence of prey & predator—a laboratory investigation using goldfish & invertebrates. Am. Biol.Teach. 65, 292–297. Storz, J. F. (2005). Nonrandom dispersal and local adaptation. Heredity 95, 3–4. Swanson, A. K., and Druehl, L. D. (2002). Induction, exudation and the UV protective role of kelp phlorotannins. Aquat. Bot. 73, 241–253. Tatarenkov, A., Bergstr€ o m, L., J€ onsson, R. B., Serr~ao, E. A., Kautsky, L., and Johannesson, K. (2005). Intriguing asexual life in marginal populations of the brown seaweed Fucus vesiculosus. Mol. Ecol. 14, 647–651. Tatarenkov, A., J€ onsson, R. B., Kautsky, L., and Johannesson, K. (2007). Genetic structure in populations of Fucus vesiculosus (Phaeophyceae) over spatial scales from 10 m to 800 km. J. Phycol. 43, 675–685. Tautz, D., Ellegren, H., and Weigel, D. (2010). Next generation molecular ecology. Mol. Ecol. Notes 19, 1–3. Team, T. B. A. (2008). Assessment of Climate Change for the Baltic Sea Basin. SpringerVerlag, Berlin. Thiel, M., and Gutow, L. (2005). The ecology of rafting in the marine environment. II. The rafting organisms and community. Oceanogr. Mar. Biol. 43, 279–418. Thompson, R. C., Crowe, T. P., and Hawkins, S. J. (2002). Rocky intertidal communities: past environmental changes, present status and predictions for the next 25 years. Environ. Conserv. 29, 168–191. Thomsen, J., and Melzner, F. (2010). Moderate seawater acidification does not elicit longterm metabolic depression in the blue mussel Mytilusedulis. Mar.Biol. 157, 2667–2676. Tomanek, L., and Helmuth, B. (2002). Physiological ecology of rocky intertidal organisms: a synergy of concepts. Integr. Comp. Biol. 42, 771–775. Tonsor, S. J., and Scheiner, S. M. (2007). Plastic trait integration across a CO2 gradient in Arabidopsis thaliana. Am. Nat. 169, E119–E140. Torchin, M. E., Lafferty, K. D., and Kuris, A. M. (2002). Parasites and marine invasions. Parasitol. Int. 124, 137–151. Tuomi, J., Ilvessalo, H., Niemele, P., Siren, S., and Jormalainen, V. (1989). Within-plant variation in phenolic content and toughness of the brown alga Fucusvesiculosus L. Bot. Mar. 32, 505–509.

104

Martin Wahl et al.

Ugarte, R., Craigie, J., and Critchley, A. (2010). Fucoid flora of the rocky intertidal of the Canadian Maritimes: implications for the future with rapid climate change. In ‘‘Seaweeds and Their Role in Globally Changing Environments’’ (A. Israel., R. Einav, and J. Seckbach, eds.), Springer, Dordrecht, The Netherlands. Umar, M. J., McCook, L. J., and Price, I. R. (1998). Effects of sediment deposition on the seaweed Sargassum on a fringing coral reef. Coral Reefs 17, 169–177. Vadas, R. L., Johnson, S., and Norton, T. A. (1992). Recruitment and mortality of early postsettlement stages of benthic algae. Br. Phycol. J. 27, 331–351. Vadas, R. L., Wright, W. A., and Miller, S. L. (1990). Recruitment of Ascophyllum nodosum: wave action as a source of mortality. Mar. Ecol. Prog. Ser. 61, 263–272. van Alstyne, K. L. (1989). Adventitious branching as a herbivore-induced defense in the intertidal brown alga Fucus distichus. Mar. Ecol. Prog. Ser. 56, 169–176. van den Hoek, C., Mann, D., and Jahns, H. (1995). Algae, An Introduction to Phycology. Cambridge University Press, Cambridge. Vermeij, G. J., and Roopnarine, P. D. (2008). The coming Arctic invasion. Science 321, 780–781. Viejo, R. M., and Arrontes, J. (1992). Interactions between mesograzers inhabiting Fucus vesiculosus in Northern Spain. J. Exp. Mar. Biol. Ecol. 162, 97–111. Vinebrooke, R. D., Cottingham, K. L., Norberg, J., Scheffer, M., Dodson, S. I., Maberly, S. C., and Sommer, U. (2004). Impacts of multiple stressors on biodiversity and ecosystem functioning: the role of species co-tolerance. Oikos 104, 451–457. Vogt, H., and Schramm, W. (1991). Conspicuous decline of Fucus in Kiel Bay (Western Baltic)—what are the causes. Mar. Ecol. Prog. Ser. 69, 189–194. Volterra, L., and Conti, M. E. (2000). Algae as biomarkers, bioaccumulators and toxin producers. Int. J. Environ. Pollut. 13, 92–125. Wahl, M. (1996). Living attached: fouling, epibiosis. In ‘‘Fouling Organisms of the Indian Ocean: Biology and Control Technology’’ (R. Nagabhushanam, and M. F. Thompson, eds.), pp. 31–83. Oxford & IBH Publishers, New Delhi, India. Wahl, M. (1997). Increased drag reduces growth of snails: comparison of flume and in situ experiments. Mar. Ecol. Prog. Ser. 151, 291–293. Wahl, M. (2008a). Ecological lever and interface ecology: epibiosis modulates the interactions between host and environment. Biofouling 24, 427–438. Wahl, M. (2008b). Ecological modulation of environmental stress: interactions between ultraviolet radiation, epibiotic snail embryos, plants and herbivores. J.Anim.Ecol. 77, 549–557. Wahl, M., and Hay, M. E. (1995). Associational resistance and shared doom—effects of epibiosis on herbivory. Oecologia 102, 329–340. Wahl, M., and Mark, O. (1999). Epibiotic associations. Mar. Ecol. Prog. Ser., http://www. int-res.com/journals/meps/supplementary-material/#2002. Wahl, M., Shahnaz, L., Dobretsov, S., Saha, M., Symanowski, F., David, K., Lachnit, T., Vasel, M., and Weinberger, F. (2010). Ecology of antifouling resistance in the bladder wrack Fucus vesiculosus: patterns of microfouling and antimicrobial protection. Mar. Ecol. Prog. Ser. 411, 33–48. Wallace, A. L., Klein, A. S., and Mathieson, A. C. (2004). Determining the affinities of salt marsh fucoids using microsatellite markers: evidence of hybridization and introgression between two species of Fucus (Phaeophyta) in a Maine estuary. J.Phycol. 40, 1013–1027. Walley, J. W., and Dehesh, K. (2010). Molecular mechanisms regulating rapid stress signaling networks in Arabidopsis. J. Integr. Plant Biol. 52, 354–359. Weidner, W., and Ziemens, C. (1975). Preadaptation of protein synthesis in wheat seedlings to high temperature. Plant Physiol. 56, 590–594. Weinberger, F., Buchholz, B., Karez, R., and Wahl, M. (2008). The invasive red alga Gracilaria vermiculophylla in the Baltic Sea: adaptation to brackish water may compensate for light limitation. Aquat. Biol. 3, 251–264.

Stress Ecology in Fucus: Abiotic, Biotic and Genetic Interactions

105

Weinberger, F., Rohde, S., Oschmann, Y., Shahnaz, L., Dobretsov, S., and Wahl, M. (2011). Effects of limitation stress (light) and disruptive stress (temperature shift) on induced antigrazing defense in the bladder wrack Fucusvesiculosus. Mar.Ecol.Prog.Ser., 10.3354/meps09044. Wiencke, C., Gomez, I., Pakker, H., Flores-Moya, A., Altamirano, M., Hanelt, D., Bischof, K., and Figueroa, F. L. (2000). Impact of UV-radiation on viability, photosynthetic characteristics and DNA of brown algal zoospores: implications for depth zonation. Mar. Ecol. Prog. Ser. 197, 217–229. Wiencke, C., Gorham, J., Tomos, D., and Davenport, J. (1992). Incomplete turgor adjustment in Cladophora-Rupestris under fluctuating salinity regimes. Estuar. Coast. Shelf Sci. 34, 413–427. Wiencke, C., Roleda, M. Y., Gruber, A., Clayton, M. N., and Bischof, K. (2006). Susceptibility of zoospores to UV radiation determines upper depth distribution limit of Arctic kelps: evidence through field experiments. J. Ecol. 94, 455–463. Wikstr€ o m, S. A., and Kautsky, L. (2007). Structure and diversity of invertebrate communities in the presence and absence of canopy-forming Fucus vesiculosus in the Baltic Sea. Estuar. Coast. Shelf Sci. 72, 168–176. Wikstr€ o m, S. A., and Pavia, H. (2004). Chemical settlement inhibition versus postsettlement mortality as an explanation for differential fouling of two congeneric seaweeds. Oecologia 138, 223–230. Wikstr€ o m, S. A., Steinarsdottir, M. B., Kautsky, L., and Pavia, H. (2006). Increased chemical resistance explains low herbivore colonization of introduced seaweed. Oecologia 148, 593–601. Williams, S. L., and Smith, J. E. (2007). A global review of the distribution, taxonomy, and impacts of introduced seaweeds. Annu. Rev. Ecol. Evol. Syst. 38, 327–359. Witman, J. D., and Suchanek, T. H. (1984). Mussels in flow: drag and dislodgement by epizoans. Mar. Ecol. Prog. Ser. 16, 259–268. Worm, B., and Chapman, A. R. O. (1998). Relative effects of elevated grazing pressure and competition from a red algal turf on two post-settlement stages of Fucus evanescens C. Ag. J. Exp. Mar. Biol. Ecol. 220, 247–268. Worm, B., Lotze, H. K., Bostr€ o m, C., Engkvist, R., Labanauskas, V., and Sommer, U. (1999). Marine diversity shift linked to interactions among grazers, nutrients and propagule banks. Mar. Ecol. Prog. Ser. 185, 309–314. Worm, B., Lotze, H. K., and Sommer, U. (2001). Algal propagule banks modify competition, consumer and resource control on Baltic rocky shores. Oecologia 128, 281–293. Wright, J. T., Williams, S. L., and Dethier, M. N. (2004). No zone is always greener: variation in the performance of Fucus gardneri embryos, juveniles and adults across tidal zone and season. Mar. Biol. 145, 1061–1073. Xu, H., Deckert, R. J., and Garbary, D. J. (2008). Ascophyllum and its symbionts. X. Ultrastructure of the interaction between A. nodosum (Phaeophyceae) and Mycophycias ascophylli (Ascomycetes). Botany-Botanique 86, 185–193. Yates, J. L., and Peckol, P. (1993). Effects of nutrient availability and herbivory on polyphenolics in the seaweed Fucus vesiculosus. Ecology 74, 1757–1766. Yun, H. Y., Rohde, S., Linnane, K., Wahl, M., and Molis, M. (2010). Geographic difference in trait-mediated indirect interactions between a seaweed and two species of meso-herbivores. Mar. Ecol. Prog. Ser. 408, 47–53. Zardi, G., Nicastro, K., Canovas, F., Costa, J., Serr~ao, E., and Pearson, G. (submitted). Adaptive traits are maintained on steep selective gradients despite gene flow and hybridization in the intertidal zone. Zuccaro, A., Schoch, C. L., Spatafora, J. W., Kohlmeyer, J., Draeger, S., and Mitchel, J. I. (2008). Detection and identification of fungi intimately associated with the brown seaweed Fucus serratus. Appl. Environ. Microbiol. 74, 931–941.

C H A P T E R T H R E E

Hydrozoans and the Shape of Things to Come €bler S.R. Dudgeon and J.E. Ku

Contents 1. Introduction 108 1.1 Hydrozoans as models for studying physiological regulation of 109 colony development 2. Experimental Studies of Hydrozoan Colony Form 111 2.1 Genetic basis of colony form 111 2.2 Colony integration by means of the gastrovascular system 115 2.3 Gastrovascular physiology and colony form 119 2.4 Vascular architecture and constraints on plasticity 124 3. Colony Development as a Dynamical System 131 4. Future Directions and Conclusions 134 4.1 The heritability of colony form 134 4.2 The heritability of morphological plasticity 134 4.3 Architectural versus genotypic effects 135 4.4 Physiological mechanisms underlying morphological plasticity 135 4.5 Empirical tests of nutrient-driven polyp behaviour 136 4.6 Colonial hydrozoans as models of vascular development 136 4.7 Conclusions 138 Acknowledgements 138 References 139

Department of Biology, California State University, Northridge, CA, USA

Corresponding author. Corresponding author.

Advances in Marine Biology, Volume 59 ISSN 0065-2881, DOI 10.1016/B978-0-12-385536-7.00003-0

# 2011 Elsevier LTD. All rights reserved.

107

108

¨bler Dudgeon and Ku

Abstract The physiological mechanisms that regulate adaptive plasticity of clonal organisms are key to their success in changing environments. Here, we review the mechanisms that regulate morphological plasticity of colonial hydrozoans. There is a heritable, genetic basis to colony form, but environmentally-induced plasticity and self-reinforcing developmental physiology explain much of total phenotypic variance. Morphological development of colonial hydrozoans emerges from interactions among (1) behaviors which drive gastrovascular transport, (2) architecture of the gastrovascular system that determines hydrodynamic characteristics of vascular flow, and, (3) gene products that vary in response to physiological signals provided by gastrovascular transport. Several morphogenetic signaling mechanisms have been identified, including, reactive oxygen species and nutrient concentrations in the hydroplasm, and hydromechanical forces associated with gastrovascular transport. We present a conceptual model of the interacting forces that drive hydrozoan morphological development. Several avenues for future research are suggested by the synthesis of information from prior studies of hydrozoans. Elucidating the morphogenetic signaling pathways responsive to metabolites or hydromechanical forces and the epigenetic effect of vascular architecture on colony form may give new insight into the self-maintenance of indeterminately growing and continuously developing vascular systems.

1. INTRODUCTION Clonal organisms grow and propagate by self-replication of genetically identical units (i.e. ramets) that acquire resources, provide structure or reproduce and can survive on their own if separated from one another (Jackson, 1985). Multicellular clonal (or colonial) organisms occur in many lineages of the Eukarya including several invertebrate phyla, fungi, benthic macroalgae and plants. Ecologically, they cover much of the biosphere and are particularly common in marine benthic communities (Boardman etal., 1973; Jackson, 1979, 1985; Buss and Blackstone, 1991). Clonal organisms may occur as solitary individuals or be physically connected and physiologically integrated, often by means of a shared vascular system (Jackson, 1985; Harvell and Grosberg, 1988; Buss and Blackstone, 1991; Blackstone and Bridge, 2005). Here, we examine the scope for alternative patterning of those connections. Besides the phylogenetic diversity and ecological success of clonal lineages of eukaryotes, there are two other compelling reasons to study the morphological patterning of colonial organisms. First, longevity, size and shape of a clone can vary enormously such that a developmental

The shape of things to come

109

programme cannot pre-determine the timing and location of every morphogenetic event during the life span of the organism. This contrasts with the model systems of development (unitary, [aclonal] organisms; e.g. fruit flies, mice, worms) in which gene expression early in life results in form that thereafter grows. Second, different forms show different ecological traits and patterns of selection on whole colony morphology (Buss and Grosberg, 1990; Yund, 1991). Despite the lack of developmental genetic programmes to specify a finished form, coherent and repeatable colony morphologies are nevertheless generated from the physiological state of the organism interacting with the environment to regulate gene expression. We review what is known about the development and maintenance of morphology in Hydrozoa (Phylum Cnidaria) with respect to heritability of colony form and the extent of phenotypic plasticity. We discuss existing evidence and present data supporting hypothesized physiological mechanisms of plasticity and constraint on plasticity imposed by colony architecture. Synthesizing the above, we present a conceptual model describing colony growth and development as a context-dependent dynamical system of physiological responses to environmental cues. The outcome of colony development from these responses is contingent upon the existing colony architecture as well as genotype. In this way, feedback mechanisms and environmental signals operating during development enable a selfmaintaining system leading to one of multiple potential morphological states for a given genotype. Future directions for research are offered based on (1) unresolved questions and (2) the opportunity to use indeterminately growing species as models of internal vascular systems that develop continuously in an organism’s internal environment.

1.1. Hydrozoans as models for studying physiological regulation of colony development Clonal organisms differ markedly from aclonal (or unitary) organisms with respect to their morphological construction, ecological traits and patterns of growth (Harper, 1977; Jackson, 1979, 1985; Buss, 1987; Harvell and Grosberg, 1988; Caswell, 2001; Winston, 2010). These fundamental differences in the morphology and ecology of clonal and aclonal organisms derive primarily from differences in their respective patterns of development. In contrast to unitary organisms in which development of the adult form occurs during a relatively narrow window of time early in ontogeny, development of clonal organisms continues with growth by means of active somatic stem cell lineages (Buss, 1987; Blackstone and Bridge, 2005). Variation in the relative growth rates of different modules determines their spacing and arrangement (i.e. form). Growth may be indeterminate and, consequently, morphogenetic

110

¨bler Dudgeon and Ku

patterning occurs over a wider span of the lifetime. Continuous development can be adaptive in organisms with a sessile habit and great longevity because they likely face environmental challenges they cannot escape during their life span. A developmental programme that responds to environmental cues (i.e. environmental signalling, sensu Blackstone and Bridge, 2005) produces morphologically plastic phenotypes that ameliorate unfavourable conditions (Bradshaw, 1965; Thompson, 1991; Stearns, 1992; Blackstone, 2003). Morphological plasticity is widespread among clonal organisms and its ecological significance clearly established (Foster, 1979, 1980; Palumbi, 1984; Scheiner and Goodnight, 1984; Harvell, 1986; Stearns, 1992; Scheiner, 1993a; Schmitt, 1993). In the past two decades, research on colonial animals has emphasized physiological processes that regulate morphogenesis as colonies develop and features that limit the extent of morphological plasticity. Colonial hydrozoans (Phylum Cnidaria), especially those in the Family Hydractiniidae, are useful models of the physiological regulation of development. Hydrozoan colonies have relatively simple morphology, consisting of polyps (modules for feeding, transporting gastrovascular fluid, defence and reproduction) and stolons (network of vascular tubes; Fig. 3.1). The principal colony-wide physiological system is the gastrovascular system, fluid-filled canals that run throughout the stolonal network and join the gastric cavities of polyps (Berrill, 1949; Crowell, 1957; Mackie, 1973, 1986). The gastrovascular system performs most of the extracellular physiological functions of the colony including digestion, transport of food and metabolites and waste removal (Lenhoff, 1968; Rees et al., 1970; Murdock, 1978; Gladfelter, 1983; Blackstone, 1999, 2001, 2003). Hydrozoans are useful in experimental manipulation

[(Figure_1)TD$IG]

Figure 3.1 Hydrozoan colonies growing on glass slides showing principal elements of a colony. (A) H. symbiolongicarpus characterized by feeding polyps (gastrozooids), the ectodermally fused stolonal mat and peripheral stolons extending beyond the mat. (B) P. carnea, which lacks a stolon mat, displays polyps atop sparsely branched stolons. White scale bars at the bottom of each photo are 1 mm.

The shape of things to come

111

of colony form, being effectively an exposed vascular system unencumbered by organs and tissues. Variation in spacing and arrangement of polyps and stolons is partially heritable and representative of the range of morphologies observed among diverse clonal taxa (Buss and Blackstone, 1991; Dudgeon and Buss, 1996). Morphological variants have been shown to correlate with ecological traits (Buss, 1979; Jackson, 1979; Yund, 1987, 1991; Blackstone and Yund, 1989; Buss and Grosberg, 1990). In addition to heritable variation of morphology (McFadden et al., 1984; Blackstone and Buss, 1991), colonies can display remarkable plasticity in growth and form (Crowell, 1957; Buss and Blackstone, 1991; Blackstone and Buss, 1992, 1993; Dudgeon and Buss, 1996). Knowledge of the environmental signalling that underlies morphological plasticity may provide new insight into development not afforded by study of the gross morphology of unitary model organisms but directly analogous to the indeterminate development of vascular systems in both colonial and unitary organisms. Finally, including colonial organisms as model systems for studying development broadens perspective of the conceptual framework of development as a dynamical system of interaction between gene expression, the modular features of organisms and the environment. Such a perspective may complement emerging views of the modularity of development of all multicellular organisms (Schlosser and Wagner, 2004).

2. EXPERIMENTAL STUDIES OF HYDROZOAN COLONY FORM 2.1. Genetic basis of colony form The spacing and arrangement of polyps and stolons varies widely both among and within cnidarian species. At one end of the spectrum of morphological variation, colonies (called ‘runners’) show widely spaced polyps along long, sparsely branched stolonal connections. At the other end, colonies (called ‘sheets’) show closely packed polyps along short, densely branched and anastomosed stolons (Buss and Blackstone, 1991). A genetic basis to colony form is suggested by the fact that colony form is routinely used to distinguish among ecologically distinct morphotypes within species or, identify cnidarian species in situ (e.g. Knowlton et al., 1992) and a clone-specific proportion between stolon length and polyp number (Braverman, 1963), such that replicate explants from the same colony produce similar outcomes of morphology (McFadden et al., 1984). This is remarkable given the nature of indeterminate growth and variable environments that can influence growth rates of colony parts as well as the size, shape or longevity of the colony. To what extent is cnidarian colony form under direct genetic control?

112

¨bler Dudgeon and Ku

For most colonial invertebrates, the respective contributions of heritable genetic variation and environmentally determined variation to phenotypes are unknown. Morphological plasticity has been observed in laboratory and field studies of cnidarian species (Crowell, 1957; Dustan, 1975; Foster, 1979, 1980; Graus and McIntyre, 1982; Brown et al., 1985; Willis, 1985; Sebens and Done, 1992; Bruno and Edmunds, 1997) and so, environmental variance (VE) (see Table 3.1 for a summary of all abbreviations) is expected to represent a large proportion of total phenotypic variance of colony form. Attempts to partition phenotypic variance of colony form into genetic and environmental components have been made for a few species of hydrozoans. The approach used most frequently is growing clonal replicates of several genotypes in a common garden and measuring clonal repeatability, r. This approach does not technically distinguish genetic and environmental components because shared environmental history among clonal replicates may overestimate the genetic component (Falconer, 1986; Lynch and Walsh, 1998). Nevertheless, clonal repeatability should represent an upper estimate of broad-sense heritability, h2B (the sum of additive and non-additive genetic variance). Values of Table 3.1

Abbreviations for genetic effects and their descriptions

Component

description

VP VG

Total phenotypic variance of a trait; VP = VG + VE Measures the amount of phenotypic variation among averages of different genotypes; VG = VA + VNA Component of phenotypic variance attributable to environmental variation measured among individuals with the same genotype Additive genetic variance; the linear component of phenotypic change associated with allelic substitution Non-additive genetic variance; the non-linear component of phenotypic change associated with allelic substitution caused by dominance and/or epistasis Clonal repeatability; the ratio of the between-individual component to the total phenotypic variance, which includes both genetic and environmental components, the latter due to non-localized, permanent or historical effects; r = (VG + VEg)/VP Broad-sense heritability; the fraction of phenotypic variance attributable to differences in genotype; h2B ¼ V G =V P Narrow-sense heritability; the fraction of phenotypic variance attributable to additive genetic variance; h2N ¼ V A =V P

VE VA VNA r

h2B h2N

The shape of things to come

113

heritability range from 0 to 1. Those approaching 1 indicate a large genetic component, and those near 0 indicate a large environmental effect on morphology. Common-garden studies of clonal repeatability have been used to distinguish colony form among species and genotypes within species of Podocoryne carnea and four species of Hydractinia. In some cases, only statistics establishing differences in colony form among species or genotypes are given implying genetic sources of variation, but not estimates of repeatability (McFadden et al., 1984; Blackstone and Buss, 1991). For studies that present such estimates, h2B from measures of clonal repeatability of different Hydractinia species are typically quite small for overall colony morphology (Table 3.2). Values of repeatability range from 0.17 to 0.31 in Hydractinia polyclina, 0.21 in Hydractinia symbiolongicarpus and 0.24 in Hydractinia [GM] (an unnamed Hydractinia species in the Gulf of Mexico; D. Ferrell, personal communication). These low values of repeatability, representing an upper estimate of h2B , imply that most variation in colony form is environmentally determined. Yund (1991) observed values of clonal repeatability in H. polyclina of 0.48 and 0.58 for stolonal mat and peripheral stolon production, respectively, somewhat greater than those measured for overall colony form. Blackstone and Buss (1991) provided an important addition to the studies that report values of clonal repeatability. In addition to commongarden experiments (although repeatability was not estimated), it is the only breeding study of Cnidaria that has attempted to quantify the heritable genetic components of colony form. They mated two males to each of five females to analyse co-variation among both full- and half-siblings (average number of offspring per family = 24 after 50 days). In a nested analysis of variance, the among-male component measures h2B , whereas the among-female component, which estimates covariance among halfsiblings, measures additive genetic variance in order to estimate narrowsense heritability (h2N ). All of their analyses indicated a strong effect of the male parent and a weak effect of the female parent, which correspond to a large non-additive genetic variance component and a small additive genetic variance component. Because of the small number of families and offspring in the study they focused on qualitative interpretations of the analysis, rather than exact calculations of heritability. Blackstone and Buss (1991) inferred from these results that genetic control of colony form was manifested principally from dominance and epistasis. For comparisons with other estimates of h2B from measures of clonal repeatability, we estimated broad- and narrow-sense heritabilities from the data presented in Blackstone and Buss (1991) (we assumed common environmental variance = residual environmental variance, as is supported by the lack of position effects in their analyses). From their data, we estimated h2B ¼ 0:84, which was comprised largely of non-additive

114

Table 3.2

Measures of clonal repeatability as upper estimates of broad-sense heritability in Hydractinia spp.

Species

Analysis

Variable

r

H. polyclina H. polyclina H. symbiolongicarpus H. [GM] H. symbiolongicarpus

Repeatability Repeatability Repeatability Repeatability Half-sibling breeding

Peripheral/mat Stolon tips Stolon tips Stolon tips Perimeter/area1/2

0.31 0.17 0.21 0.24

h2B

Study

0.84

Yund (1991) D. Ferrell (personal communication) D. Ferrell (personal communication) D. Ferrell (personal communication) Blackstone and Buss (1991)

Note: Other similar studies did not report values for r, or h2B , and they could not be inferred from the data presented.

¨bler Dudgeon and Ku

The shape of things to come

115

genetic variance (dominance and epistasis, VNA = 0.69; VA = 0.15), and environmental variance comprised only 16% of phenotypic variance. The breeding study by Blackstone and Buss (1991) suggests a much greater genetic component underlying colony form of hydractiniid hydrozoans than do other studies measuring clonal repeatability. Unfortunately, clonal repeatability for neither species can be calculated from the data provided in Blackstone and Buss (1991) for direct comparison. The different interpretation of the importance of genetic versus environmental control of colony form has at least two potential explanations. Estimates of clonal repeatability were made on colonies freshly collected from the field, whereas colonies used in breeding experiments were cultured in the laboratory until reproductively mature (2 months for Hydractinia; Blackstone and Buss, 1991). The difference may also reflect different morphometric variables used in analyses among studies by different investigators. Morphological variation in clonal organisms is often described qualitatively in terms of the suites of traits characteristic of different forms (Buss, 1979; Jackson, 1979; Lovett-Doust, 1981; Harper, 1985; Buss and Blackstone, 1991). Consequently, there are several potential descriptor variables that may effectively represent morphology to varying degrees, but may differ in the strengths of the genetic bases underlying the respective traits. There is also the possibility that the colonies used in various experiments sample different amounts of the genetic scope for plasticity in the population at large. Reviewing multiple studies, we can only conclude that there is strong evidence for interrelated genetic and environmental determinants of clonal morphology of hydrozoans. The correlation between ecological traits of interest (e.g. competitive ability, demographic parameters) and colony morphology in hydractiniid hydrozoans, specifically, and clonal organisms in general, emphasizes the genetic basis of form (Buss and Grosberg, 1990; Yund, 1991; Bruno and Edmunds, 1997). In particular, traits like competitive ability, for which there appears to be an underlying morphogenetic basis can only be efficaciously acted upon by selection if much of the genetic basis to colony form is in the form of additive genetic variance. The present evidence is inconclusive as to the extent to which colony form is heritable, but available data suggest that little of the variation in morphology is attributable to additive genetic variance.

2.2. Colony integration by means of the gastrovascular system In the simply constructed cnidarians, the physiological system regulating morphological development is the gastrovascular system, which carries out much of extracellular physiological function of the colony. The hypothesized role of the gastrovascular system in colony-level patterning means that we need to understand the dynamics of gastrovascular transport.

116

¨bler Dudgeon and Ku

The gastrovascular system integrates polyps of a colony with respect to sharing of resources, coordinating behaviours and defensive responses. Several early studies recognized the importance of gastrovascular transport to colony growth (Crowell, 1957, 1974; Hale, 1960; Wyttenbach, 1968, 1973; Karlson and Marferin, 1984; Marferin, 1985; Beloussov et al., 1989). It is not certain whether polyps remain locally autonomous, or whether they serve as organizing centres initiating behaviours (Mackie, 1986). The role of polyps may vary with taxon. Parrin etal. (2010) summarize the behavioural control of cnidarian gastrovascular dynamics. Briefly, muscular contractions of polyps outweigh ciliary action in driving gastrovascular flow in hydrozoans and the derived anthozoan group, the pennatulacean octocorals (Parker, 1920; Berrill, 1949; Crowell, 1957; Hale, 1960; Fulton, 1963; Wyttenbach, 1968; Brafield, 1969; Schierwater et al., 1992; Blackstone, 1996; Wagner et al., 1998; Dudgeon et al., 1999; Anctil et al., 2005; Parrin et al., 2010; but see Roosen-Runge, 1967 for an exception among Hydrozoa). Studies of other octocorals and hexacorals indicate that gastrovascular transport is achieved by cilia-driven flow, which may be the ancestral state for the cnidarian gastrovascular system (Murdock, 1978; Gladfelter, 1983; Schlichter, 1991; Gaten˜o etal., 1998; Parrin et al., 2010). Behaviours of polyps and stolons driving the dynamics of gastrovascular flow have been quantified in vivo in hydrozoans. Polyp behaviour can be characterized by the differing input–output relationships between the polyp and either the external (via the mouth) or the internal (via the polyp–stolon junction) environment, and delineated with respect to the timing of ingestion of food (Wagner et al., 1998; Dudgeon, 2001a). Polyps prior to feeding, or long after regurgitation show infrequent contractions, which occur without periodicity, and the volume of fluid exchanged with the stolon is extremely small (Dudgeon et al., 1999; Dudgeon, 2001b). Fed polyps initiate the first phase of oscillatory behaviour (expansion– contraction cycles of polyps with respect to their length and width, hence volume) within 5–15 min of ingestion. In the second phase of oscillatory behaviour, polyps behave semi-autonomously and display constant amplitude oscillations. Digestion occurs in the gastric cavity of the polyp during this stage, but small volumes may be exported to the common gastrovascular system. The final, third phase is initiated by the export of dense streams of particulates from the polyp into the stolon and terminated by regurgitation of undigested food, which triggers a return to pre-feeding behaviour. This phase of maximal transport rates occurs between 2 and 8 h after feeding (Schierwater et al., 1992), but tends to occur later and for longer durations in single polyp systems, or very small colonies (Dudgeon etal., 1999). Direct evidence of cause and effect for polyps moving gastrovascular fluid through stolons, first in one direction, then the other, would be

The shape of things to come

117

alternating oscillations of polyps and stolon lumens. In a simple colony with a single polyp and known length of stolon, time series of changes in polyp volume correspond with changes in lumen diameter of the stolon (Fig. 3.2A). Stolon diameter is at a minimum when polyp volume is maximal and stolon diameter increases as polyp volume declines, the former reaching its maximum diameter when polyp volume is minimal. Since fluid moves in both directions in a stolon (to and from a polyp), it must go from being at rest to a maximal velocity in one direction and then decline to 0 velocity as it switches to moving in the other direction. Flowspeed is greatest immediately upon expansion of the stolon lumen as polyp volume decreases from maximum to minimum volume (Fig. 3.2B, see also Van Winkle and Blackstone, 1997). Flowspeeds in the stolon approach 0 when polyp volume is at, both, maxima and minima. Flowspeeds in the stolon are greater when moving away from the polyp (during export by polyps) than when moving towards the polyp (during import by polyps). The volumetric rate of fluid transport in the stolon follows the dynamics of flowspeed (Fig. 3.2C). Volumetric rate is maximal as the stolon lumen rapidly opens and expands during export by polyps. As the lumen approaches its maximum diameter, volumetric rate drops quickly and approaches 0 as the stolon is maximally expanded. Volumetric rate again increases as it moves towards the polyp upon closing of the stolon lumen, but the rate of transport back towards polyps is less and the duration slightly longer than during export. The dynamics of polyp behaviour and gastrovascular transport change dramatically following ingestion and continue to change through regurgitation. Wagner et al. (1998) identified alternative hypotheses to explain the dynamics observed and developed an ordinary differential equation model to test whether the hypothesis of nutrient generation (from digestion) and metabolism could produce model polyp behaviours in silico qualitatively similar to observed polyp behaviours following ingestion of food and during digestion. Modelled polyp behaviour showed a start-up of oscillatory behaviour, increased polyp length and oscillation amplitude during digestion and a steady state of oscillations until nutrients were depleted (Wagner et al., 1998). The model equations (differentiated with respect to time) represented (1) the quantity of food remaining in the polyp gastric cavity (a monotonically declining function until the next ingestion period), (2) the nutrients available to the polyp, which are generated by the digestion of food and depleted by metabolism and export from the polyp and (3) a function in which two state variables of the polyp, representing an underlying non-linear oscillator, are sensitive to the quantity of nutrients in the gastric cavity. Oscillations in values of these state variables are reflected in oscillatory behaviour by polyps. The alternative hypothesis that mechanical strain on polyp tissue caused by ingestion of food elicits polyp oscillations is compromised by observations that

118

¨bler Dudgeon and Ku

[(Figure_2)TD$IG]

Figure 3.2 Gastrovascular dynamics in P. carnea 3–5 h after feeding. Data represent behaviour of a single polyp connected to a stolon network with a volume capacity in excess of 5 nl. Dynamics over a 630 s interval showing oscillations of polyp volume (solid circles and line) and lumen diameter (dashed line) of stolons (A), polyp volume (solid circles and line) and flowspeed (dashed line) of gastrovascular fluid through stolons (B) and lumen diameter of stolon (dashed line) and volumetric rate (solid circles and line) of gastrovascular fluid transport (C). Volume estimates of polyps were sampled every 8 s. Stolon dynamics (lumen diameter, flowspeed and volumetric rate) were sampled at 30 frames s1. Polyp contractions export fluid into stolons which open rapidly to near their maximum diameter as the fluid accelerates to maximum speed and then declines to 0 velocity before switching direction as the polyp begins to expand.

The shape of things to come

119

oscillations may not begin for up to 15 min after strain is initiated upon ingestion and lacks a mechanism to induce regurgitation by polyps. The hypothesis that nutrients emanating from food substrates elicit oscillatory behaviour by polyps in silico, including a rise in oscillation amplitude to a steady state as nutrient concentration plateaus and the eventual return to a quiescent state when nutrients drop below levels that elicit polyp oscillations, is consistent with observed in vivo polyp behaviours. As expected, quantitative features of oscillating polyps were influenced by environmental conditions. Hydrozoan polyps oscillated with greater frequency with increasing temperature. At temperatures of 34 and 35 C, near the upper thermal limit of H. symbiolongicarpus, polyp behaviours underwent a transition in their behaviour from stable limit cycles to intermittent and erratic changes in oscillation frequency, to a chaotic regime of oscillations in which oscillation frequencies varied widely for indefinite periods of time (Buss and Vaisnys, 1993). Chaotic polyp behaviours were reversible by lowering temperature upon which stable limit cycle behaviours resumed. Quantitative models that characterize the dynamics of gastrovascular transport have time-varying behaviour of oscillations in polyp length or volume as output, not a morphogenetic pattern. Pumping behaviour of polyps is still not mechanistically linked to the expression of colony morphology. Conceptual models to explain colony-level morphogenesis and its plasticity (described below) invoke patterns and rates of gastrovascular transport, or substances transported within it, during the phase of maximal gastrovascular activity.

2.3. Gastrovascular physiology and colony form Patterns of fluid transport through the gastrovascular system emerge from the collective behaviour of polyps interacting with one another and the environment. Thus, physiological states of the gastrovascular system can represent environmental sensors from which adaptive morphological plasticity develops. For instance, Blackstone (2003) pointed out that developmental pathways that are sensitive to environmental conditions and link those cues to timing the action of morphogenetic mechanisms (i.e. initiation of polyps and stolon branches relative to stolon elongation) will be favoured by selection. Among modular organisms displaying continuous development, genetic information relevant to colony-level patterning has been hypothesized to represent a series of rules iteratively applied in response to the dynamics of the colony’s physiological state (Ulam, 1962; Braverman and Schrandt, 1966; Buss, 2001; Kaandorp and K€ ubler, 2001). Essentially the same idea of colony-level patterning was called environmental signalling by Blackstone and Bridge (2005), which they defined as developmental pathways influenced by cues that initiate

120

¨bler Dudgeon and Ku

outside the organism and underlie phenotypic plasticity. Both views share the idea that physiological mechanisms that can sense and respond to environmental cues are inextricably linked to morphological development of colonies. All evidence from hydractiniid hydrozoans indicates that gastrovascular transport mediates morphological development of colonies, with, potentially multiple signalling mechanisms at work. Colonies that exhibit higher volumetric rates of flow (via larger diameter stolons and/or faster flowspeeds) exhibit more rapid elongation of stolons and lower rates of stolon branching and polyp bud formation (Blackstone, 1996, 1998; Dudgeon and Buss, 1996). For instance, colonies of H. symbiolongicarpus that display a more reticulate vasculature compared to P.carnea show lower rates of flow than P. carnea at all developmental stages except at, and prior to, mat formation (very early in development; Blackstone, 1996). Colonies of P.carnea derived from inbred lines and selected for morphologies with long, sparsely branched stolons show greater volumetric rates of vascular transport than those with shorter branches and anastomosed stolons (Blackstone, 1998). Given the correlation between gastrovascular flow rate and growth rates of modules, simple principles of fluid mechanics, therefore, predict that the relative sizes and arrangement of stolons (i.e. vascular architecture) should influence subsequent colony development. The runner-to-sheet morphological continuum spans a wide range of colony form in Hydractinia spp. The vascular architecture of runner colonies of Hydractinia spp. is characterized by a highly variable size– frequency distribution of stolon diameters (Dudgeon and Buss, 1996). In contrast, the size–frequency distribution of diameters in the stolonal network of sheet colonies shows little variation. Stolon diameter is significant because volume flux and other hydrodynamic characteristics of transport vary with the dimensions of the stolon through which the fluid flows. For instance, in accord with the Law of LaPlace (Vogel, 1988) wider stolons disproportionately increase their diameter compared to narrower stolons when pressurized. Moreover, at the low Reynolds numbers relevant here (102–103; Dudgeon and Buss, 1996), wide stolons transport a much greater volume of fluid than narrow stolons for a given pressure drop and stolon length (Vogel, 1994). Consequently, runner colonies have an unequal distribution of flow to the growing periphery, whereas sheet colonies have equivalent volumes of flow delivered to the growing tips. These flow patterns reinforce their respective growth trajectories, as in a self-maintaining system. In addition to observing co-variation between rates of transport and colony form, experimental evidence shows that manipulating gastrovascular transport causes changes in colony form. Diminished flow rates in

The shape of things to come

121

stolons, either by treatments that shift mitochondrial redox state towards oxidation or excess feeding typically causes elevated rates of stolon branch and polyp bud formation resulting in sheet morph colonies (Blackstone and Buss, 1992, 1993). Moreover, in P.carnea a dose–response relationship between gastrovascular flow to peripheral stolon tips and colony development was observed; the greater the perturbation to gastrovascular transport, the more colony patterning was altered (Blackstone, 1997). There are two alternative, but not mutually exclusive, mechanisms by which gastrovascular transport may signal morphogenetic events. Endodermal cells lining the lumen of the stolon vasculature may respond to chemical substances (e.g. metabolites, diffusing morphogens, nutrients, etc.) transported in the flow, forces directly caused by gastrovascular flow or both. Multiple, independent lines of evidence from P. carnea indicate that reactive oxygen species (ROS) signal morphogenetic events. In fact, an increased oxidative state alone within colonies of P.carnea was sufficient to stimulate polyp bud and stolon tip formation in the absence of diminished flow (Blackstone and Buss, 1993; Blackstone, 1999, 2001, 2003). Treatment with pharmacological oxidizing agents, like 2,4-dinitrophenol (DNP) partially uncoupled oxidative phosphorylation diminishing ATP production and vascular transport while shifting colony redox state towards oxidation (Blackstone and Buss, 1992, 1993; Blackstone, 2009). Relative oxidation of colony physiological state (via DNP or other oxidizing agents with different associated effects, like carbonyl cyanide mchlorophenylhydrazone [CCCP]) increased rates of stolon branching and polyp formation (i.e. sheet growth; Blackstone, 1999, 2001, 2003). Treatment with pharmacological reducing agents, like sodium azide, blocked the electron transport chain, preventing O2 uptake, thereby diminishing ATP production and vascular transport and shifted colony redox state towards reduction (Blackstone, 2001, 2003, 2009). A shift to a more reduced physiological state correlated with lower rates of polyp bud and stolon tip formation (i.e. runner growth) compared to controls (Blackstone, 2001, 2003, 2009). ROS may participate in multiple signalling pathways from elongating stolons and inhibiting branching to cell death (Blackstone et al., 2005). Not only do stolons extend and sprout branches to varying degrees but they can regress. Treatment of hydroid colonies (P. carnea and Eirene viridula) with nitric oxide donors caused stolon regression (Cherry Vogt et al., 2008) as did treatment of peripheral stolon tips with the chemical antioxidant, vitamin C (Blackstone et al., 2005). Perturbations to the redox state of a colony generate signatures of ROS that emanate most strongly from the epitheliomuscular cells (EMCs) near the base of polyps (Blackstone, 2003; Blackstone etal., 2005). This observation is consistent with polyp oscillations driving the transport of

122

¨bler Dudgeon and Ku

gastrovascular fluid (Wagner et al., 1998; Dudgeon et al., 1999; Dudgeon, 2001a,b, Bumann and Buss 2008) and provides an ecological explanation linking adaptive morphological plasticity to environmentally heterogeneous food supplies via ROS signalling associated with polyp behaviour (Blackstone, 2001, 2003; Blackstone and Bridge, 2005). Polyps actively pumping following ingestion become relatively oxidized and ROS emanating from mitochondrion-rich EMCs and dispersed in gastrovascular fluid appear to signal increased rates of stolon branching and polyp formation to exploit locally abundant food supplies (Blackstone, 2001). Environmental signalling may operate through pathways in addition to ROS signalling in some hydrozoans. This is suggested by tests of different hypotheses as well as results from dinitrophenol experiments using H. symbiolongicarpus (Blackstone and Buss, 1993). DNP treatment shifted redox state towards oxidation but colonies showed reduced rates of bud and tip formation (i.e. runner-like growth). Developmental plasticity is sensitive to colony nutritional status, diffusing morphogens and vascular architecture, which determines volume flux and hydromechanical forces associated with gastrovascular transport (Turing, 1952; Crowell, 1957; Wyttenbach, 1974; Meinhardt, 1976; M€ uller and Plickert, 1982; Plickert et al., 1987; Lange and M€ uller, 1991; Blackstone and Buss, 1993; Dudgeon and Buss, 1996; Blackstone, 2001; Dudgeon, 2001c). With respect to nutritional status, Bumann and Buss (2008) elaborated upon earlier works by Crowell (1957) and Braverman (1974) and found that food supply and the distribution of fed and unfed polyps markedly affected colony morphology. They suggested that ROS may not be the cause of morphogenetic patterning, but may simply reflect epitheliomuscular activity of large, fed polyps generating local circulation patterns among themselves that biases the distribution of nutrients and, hence, zones of growth in a colony. The different vascular architectures of runner and sheet colonies of H. symbiolongicarpus were surgically manipulated to alter hydrodynamic characteristics of gastrovascular transport and assay subsequent trajectories of colony form (Dudgeon and Buss, 1996). Reduced variation in flow rate among stolons of runner colonies resulted in subsequent sheet growth. Conversely, increased variation in flow among stolons within sheet colonies resulted in subsequent runner growth. Even without disrupting the colony-wide volume flow rate through metabolic perturbations, colony development was malleable in response to changed gastrovascular flow patterns. Based on these results, Dudgeon and Buss (1996) suggested that hydromechanical forces of gastrovascular fluid flowing through colonies act as morphogenetic signals, as occurs in vertebrate vascular systems (Davies et al., 1986; Nollert et al., 1990; Ziegelstein et al., 1992; Chen et al., 1999; Yeh et al., 1999; Osawa et al., 2002; Shay-Salit et al., 2002; Urbich et al., 2002; Resnick et al., 2003; Milkiewicz et al., 2007).

The shape of things to come

123

Buss (2001) examined whether hydromechanical forces were plausible mechanisms regulating polyp formation and stolon branching based on the results of Dudgeon and Buss (1996) and observations by Crowell (1957) and Braverman (1963). Crowell (1957) noted that interpolyp distances were greater in Campanularia£exuosa colonies with wider stolon radii and Braverman (1963) observed that polyp number to stolon length was a constant in a clone of P. carnea. Buss (2001) established the relationship of spacing between a polyp and bud along a stolon of a given radius to polyp size in experimental colonies. This relationship was compared to a derived relationship that balanced opposing forces of the pressure of fluid over a cross-sectional area imparted by a polyp with the shear forces of gastrovascular fluid moving past itself and along the lumen wall of a stolon. The predicted relationship between polyp size and spacing between polyps normalized to stolon radius was observed and, therefore, is consistent with the hypothesis that shear stress signals polyp morphogenesis. Dudgeon and Buss established experimental replicates consisting of single polyps of P.carnea in the middle of a linear stolon of defined length (Fig. 3.3A) and grew them in six different seawater treatments in which viscosity was manipulated by the addition of Dextran over the range of 1.0 cp (unmanipulated control seawater) to 2.35 cp. Shear stress equals the product of dynamic viscosity and the velocity gradient across the stolon radius, so changing viscosity indirectly manipulates shear stress during gastrovascular transport. Growth rates of polyps and stolons were assayed in each treatment after 3 days and significant differences in morphological patterning were evident (Fig. 3.3B and C). An increase in seawater viscosity increased the rate of production of polyps per unit stolon length relative to controls (Fig. 3.4). In contrast, no relationship was evident between seawater viscosity and the number of new stolon tips (Fig. 3.5). These results suggest that shear forces along the lumen wall of stolons are signals transduced by endodermal cells that regulate polyp morphogenesis. Shear stress appears not to be involved in signalling stolon branch formation. Buss (2001) suggested that circumferential wall stress may act as a mechanosensor signalling stolon branch formation. The limits of morphological plasticity are potentially broad such that runner phenotypes may be transformed into sheet phenotypes and vice versa. Developmental plasticity of colony morphology is regulated by multiple signalling mechanisms including ROS and nutrient concentrations associated with gastrovascular transport and the hydrodynamic forces of that transport. Physiological regulation of development by multiple pathways also occurs in well-studied vascular systems of vertebrates and there are strong similarities between them. Both hydrozoan and vertebrate vascular systems are regulated by ROS and hydro-(hemo-) dynamic factors suggesting common mechanisms underlying plasticity.

124

¨bler Dudgeon and Ku

[(Figure_3)TD$IG]

Figure 3.3 Single polyp/single stolon systems of P. carnea used in viscosity manipulation experiments. (A) A representative replicate prior to manipulation— polyp positioned near the centre of a 2.7 mm length of stolon. (B) A control replicate after 72 h, note single new polyp bud emerging at lower right. (C) A replicate in 1.93fold viscosity of seawater following 72 h treatment, note three new polyps formed close together to the left of the original polyp.

2.4. Vascular architecture and constraints on plasticity The range of morphological plasticity of a colony may exceed the differences among genotypes, or even among species, but that range itself is likely to vary among colonies. For instance, in H. symbiolongicarpus, replicate colonies of a runner genotype were transformed more easily into a sheet phenotype than replicates of a sheet genotype were transformed into

The shape of things to come

125

[(Figure_4)TD$IG]

Figure 3.4 Production of new polyps relative to stolon growth in colonies grown under different seawater viscosities. Values represent mean s.e. of N = 5 replicates per treatment. The allocation to polyp growth increases relative to stolon growth with increased seawater viscosity up to 2.2 cp.

[(Figure_5)TD$IG]

Figure 3.5 Production of new branch tips of stolons grown under different seawater viscosities. Values represent mean s.e. of N = 5 replicates per treatment. New stolon tips branch independently of the dynamic viscosity of seawater.

126

¨bler Dudgeon and Ku

a runner phenotype (Dudgeon and Buss, 1996). There are two possible explanations for this asymmetry. Genetic differences between the clones used may have differed in their respective reaction norms to surgical perturbations of stolons. Alternatively, differences in their existing gastrovascular architectures may have influenced their respective ranges of plasticity. The greater extent of laterally branching anastomosed stolons of sheet colonies should facilitate sharing of resources and coordination of behaviours within the colony (i.e. enhanced colony integration). At the low Reynold’s numbers that occur in the gastrovascular system ‘slug flow’ characterizes transport of fluid, so ‘mixing’ of nutrient-laden fluid with nutrient-poor fluid occurs only by insertion of these respective parcels at intersections of stolon branches (M. LaBarbera, personal communication). Enhanced colony integration may reduce disparities in local environmental signals at different points in the colony and buffer against different morphogenetic responses. We tested the hypothesis that a runner morphology displays greater plasticity in (1) the polyp/stolon investment and (2) growth among stolons within a colony in response to food limitation than does a sheet morphology. A single ‘sheet morph’ genotype of P. carnea was used to unconfound genotypic and morphological sources of plasticity by surgically constructing experimental morphologies. Plasticities of different experimental morphologies were assessed by comparing patterns of growth of colonies before and after a transition from a ‘fed to repletion’ environment to a ‘food-limiting’ environment. In addition to the sheet morph treatment (unmanipulated controls), two other morphology treatments were established. The experimental treatment consisted of removing lateral stolons that connected radially oriented stolons. A ‘shammanipulated’ sheet served as a control for severing stolon connections, but lateral stolons were not removed enabling stolons to fuse. Colonies remained in a food replete environment for 4 days after initiating treatments and then were switched to a food-limiting environment in which only the same selected polyps received a single brine shrimp nauplius larva on feeding days for the remainder of the experiment. In the food replete environment, all replicates showed similar patterns of gastrovascular transport (Fig. 3.6) and growth among stolons within a colony (Fig. 3.7). In the food-limiting environment, stolons in sheet colonies continued to grow and produce polyps at similar rates, but runner colonies did not. In these colonies, stolons with unfed polyps grew more slowly (Fig. 3.7), but increased production of polyps (relative to stolons) compared to stolons with fed polyps (Fig. 3.8). Moreover, differences in gastrovascular behaviour were consistent with those observed with respect to polyp and stolon growth. In sheet colonies, both stolons with fed polyps and those with starved polyps showed similar maximum relative diameters when oscillating (Fig. 3.9). In contrast, stolons of runner colonies with

The shape of things to come

127

[(Figure_6)TD$IG]

Figure 3.6 Relative diameters (diameter of the stolon lumen divided by the total [external] diameter of the stolon) near stolon tips during a period of maximal gastrovascular transport after feeding. Measures were taken prior to beginning the experiment. Data points in the time series represent the mean (s.e.) of four stolon tips in each of five colonies for each treatment (N = 20). Gastrovascular behaviour shows no differences among morphology treatments prior to limiting colony food supply.

starved polyps exhibited lower maximum relative diameters, hence, diminished gastrovascular transport compared to stolons in the same colony with fed polyps (see also Bumann and Buss (2008) for the distribution of labelled food in colonies). These results indicate that colonies that vary in form, yet derive from the same clone, show different morphological plasticities in response to food supply. Moreover, the differences in plasticity resulted from differences in the transport of gastrovascular fluid imposed by the architecture of the gastrovascular system. Blackstone (2001) and Bumann and Buss (2008) have also conducted experiments with different objectives using P. carnea in which only selected polyps were fed with slightly different outcomes than reported above. Both prior studies observed that fed areas of colonies grew polyps and stolon tips more rapidly than unfed areas of colonies that continued linear extensions of stolons. Selected polyps in the study by Blackstone were fed to repletion but colonies in some treatments in the study by Bumann and Buss (2008) were food-limited. Like the latter study, in our study the selectively fed polyps were food-limited and some surgically manipulated to an experimental morphology. In all three studies, environmentally induced plasticity established patterns of vascular architecture and subsequent expressions of plasticity. Vascular architecture is important in colony development in at least two ways. Vascular architecture defines the characteristics of gastrovascular flow, including hydrodynamic forces on endodermal cells lining the

128

¨bler Dudgeon and Ku

[(Figure_7)TD$IG]

Figure 3.7 Specific growth rate of stolons having fed (open circles) or unfed (solid circles) polyps in (a) sheet colonies, (b) sham-manipulated colonies and (c) runner colonies over a 19-day time course after surgically manipulating colony form. Arrows on day 4 indicate the transition to food limitation. Data points represent means (s.e.) of N 3 stolons of each feeding level per colony and M = 5 colonies per treatment. On day 19, data points that have the same letters are not significantly different. No differences in growth rate between stolons with fed versus unfed polyps among morphology treatments prior to onset of food limitation (arrow). After 15 days of food limitation, growth differs between stolons with fed versus unfed polyps only on runner colonies.

The shape of things to come

129

[(Figure_8)TD$IG]

Figure 3.8 Ratio of polyp to stolon growth in runner, sham-manipulated control and sheet colony treatments from days 2–19 of the experiment. Dark grey bars represent data for fed stolons and light grey bars represent data for unfed stolons. Columns represent means (s.e.) of N 3 stolons of each feeding level per colony and M = 5 colonies in the sheet and runner treatments, but M = 3 in the shammanipulated control treatment. Columns sharing the same letters are not significantly different. Polyp production differed between stolons with fed versus unfed polyps on runner colonies, but not on other morphs.

vascular lumen. Vascular architecture indirectly determines the rate of delivery of morphogenetic substances distributed to endodermal cells of the lumen wall. Architectural effects are known to affect floral development in the modular, vascular plants (Diggle, 1995, 2003). We continue to investigate the relative strengths of architectural, genetic and environmental factors in morphogenesis of colonial invertebrates. Waddington (1942a) coined the term ‘epigenetics’ to describe inherited changes in phenotype caused by mechanisms other than changes in the underlying DNA sequence. This definition remains the broadest conceptualization, but typically epigenetics implies DNA methylation, histone modification, RNA actions or other regulatory mechanisms that transmit a non-genetic cellular memory to daughter cells, and in some cases transgenerationally, to specify a phenotype (Bonasio et al., 2010; Riddinhough and Zahn, 2010). Those are specific epigenetic mechanisms, but the essence of the basic definition includes mechanisms at other levels of development. As colonial invertebrates continue to develop as they grow, opportunities for variation to be transmitted to differentiating cells also occur at larger scales. Existing structures influence the development of new structures. The structural state of vascular architecture acts as an epigenetic factor influencing the range of morphological plasticity of colonies. Lateral and circumferential vascular connections buffer against disparities in gastrovascular transport in different parts of a colony, dampening morphological plasticity of colonies. In response to changes in food

130

¨bler Dudgeon and Ku

[(Figure_9)TD$IG]

Figure 3.9 Relative diameters (lumen diameter of the stolon divided by the total [external] diameter of the stolon) near tips of stolons having fed (open circles) or unfed (solid circles) polyps in (A) sheet colonies, (B) sham-manipulated colonies and (C) runner colonies, during a period of maximal gastrovascular transport after feeding. Measures were taken on day 19 of the experiment. Data points in the time series represent the mean (s.e.) of N 3 stolon tips of each feeding level in each of M = 5 colonies per treatment. Treatments sharing the same letters indicate that their relative maximum diameters are not significantly different. Gastrovascular behaviour in stolons with unfed polyps differed from those with fed polyps, but only among experimental runner morphs.

The shape of things to come

131

supply, the reaction norm of runner morphs is broader than that of sheet morphs, and the epigenetic factor controlling the norm of reaction is the dynamic pattern of vascular connectivity in colonies.

3. COLONY DEVELOPMENT AS A DYNAMICAL SYSTEM Buss and Blackstone (1991) applied Waddington’s (1942b) view of development as a dynamical system to colonial organisms. Their experiments, with a single clone of Hydractinia echinata, provided evidence that colonies regulate rates of polyp and stolon production, but morphology was not canalized by selection. They suggested that correlations between form and function were largely the outcome of a self-organizing process (sensuBonabeau et al., 1997) of dynamical behaviours of a developing colony. Genetic programmes of colonial cnidarians do not specify a stereotypical morphology, nevertheless repeatable and identifiable morphs are formed. Morphogenetic patterning occurs in response to physiological signals established by the interactions between collective polyp behaviour and the existing vascular architecture (Blackstone and Buss, 1992; Dudgeon and Buss, 1996; Wagner et al., 1998; Blackstone, 1999, 2001, 2003; Dudgeon et al., 1999, Bumann and Buss 2008; data presented above). The collective behaviour of a colony emerges from simple behaviours of polyps interacting with one another and their environment (Wagner et al., 1998; Dudgeon etal., 1999; Dudgeon, 2001a,b). With each new polyp or stolon branch added to the existing vasculature the physiological state may change. Colonies develop, therefore, by genotype- and context-dependent spatiotemporal patterns of gene expression dynamically responding to the physiological state (environment) and architecture of the gastrovascular system (Fig. 3.10). A population of cnidarian (or other invertebrate) colonies may include a variety of genotypic classes that vary in phenotypic expression in response to ontogenetic stage and environment. Some genotypes may be canalized early, some late, some not at all. There may be latitude in phenotypic values early in ontogeny that is canalized to a narrow outcome later in ontogeny (Fig. 3.10A), but Buss and Blackstone did not observe such a pattern in the genotype they studied. Plastic responses of colonies may vary from linear to threshold responses. Fig. 3.10B, C and E represent genotypes in which plasticity declines during ontogeny. Genotypes represented by Fig. 3.10B show a non-linear, but monotonic change in phenotype across environments early in ontogeny and a less responsive, linear change of phenotype across environments late in ontogeny. Genotypes represented by Fig. 3.10C show a strong threshold response of phenotype to environmental change early, but a less plastic, linear response late in ontogeny. Possibly, genotypes may have threshold plastic responses to environmental change throughout ontogeny (Fig. 3.10D). If

132

¨bler Dudgeon and Ku

[(Figure_0)TD$IG]

Figure 3.10 Phenotypic outcomes for different genotypic classes with respect to environmental conditions and ontogenetic stage (i.e. architectural inertia). Surfaces represent genotypes and show a range of hypothetical interactive effects with environment and existing architecture on phenotypes. (A) Canalized phenotype, (B) monotonically plastic phenotype with greater sensitivity to environmental variation at early stages, (C) threshold response of plasticity in early, but not late ontogeny, (D) threshold response of plasticity throughout ontogeny, (E) multiple stable phenotypic points late in ontogeny, but a threshold response early. These five patterns represent a sample of genotypic classes of effect that may exist in populations.

effects of vascular architecture increase later in ontogeny, then a plausible phenotypic response late in ontogeny may be one of discrete, alternative stable phenotypes of a genotype over some range of environmental conditions (Fig. 3.10E, see also Zeeman, 1976). Colonies may be very plastic early in ontogeny, but late in ontogeny they are not. Instead, colonies

The shape of things to come

133

become canalized to one of a few discrete, stable outcomes. Which of the multiple possible phenotypes prevails is contingent upon interactions between the existing phenotype and environment as the colony grows. The existing vascular architecture for a given ontogenetic stage is as important as the environmental conditions in determining plasticity, because architecture creates conditions for self-maintenance. We predict that canalization of a phenotype is much greater at later ontogenetic stages given the increasing inertia of architectural effects at large colony sizes. Modular organisms offer an illuminating contrast to unitary model systems (e.g. fruit flies, worms, mice) for study of development (Blackstone and Bridge, 2005). Both show tightly regulated spatiotemporal patterns of gene expression in the formation of their respective structures or modules. Body plans of modular organisms may vary greatly in the number, spacing and arrangement of modules relative to one another. At the level of patterning modules, environmental signalling plays an expanded role in morphogenesis (Blackstone and Bridge, 2005). Developmental genetic programmes of unitary and modular organisms can be arrayed along a spectrum of variation in the extent to which environmental signalling influences morphological pattern. At one extreme, gene expression in unitary organisms in response to the environment is largely the internal environment created by prior gene expression in the target and neighbouring cells with less opportunity for external environmental stimuli to influence morphological patterning. Phenotypic effects are more constrained to variation in size or shape of parts, with only rare variation in the number of parts, or body axes (Blackstone and Bridge, 2005). Toward the other extreme, gene expression in clonal organisms is highly responsive to external environmental signals. One can interpret the environmental signalling axis as representing the extent to which genes and their products directly drive morphological development. With increased environmental signalling, the role of genes changes to one of establishing a physiological state that is thereafter maintained by epigenetic feedbacks (or ‘local rules’; Ulam, 1962; Braverman and Schrandt, 1966; Buss, 2001; Kaandorp and K€ ubler, 2001) and modified in response to environmental signalling. Such dynamic regulation of development and resulting adaptive plasticity may enable maintenance of greater genetic variation in natural populations of clonal organisms. In Hydractinia spp., distributions of morphological phenotypes in natural populations remain variable in the face of intraspecific competition in which a clear dominance hierarchy of runners to sheets is known (Buss and Grosberg, 1990; Yund, 1991). A number of other processes, especially environmental variability, may act to maintain this variation through their effect on the dynamics of development. Selection against competitively inferior genotypes may be weakened, or masked, if competitive ability co-varies with morphological plasticity.

134

¨bler Dudgeon and Ku

4. FUTURE DIRECTIONS AND CONCLUSIONS Much has been learned in the past two decades about the physiological regulation of developmental plasticity in hydrozoans, which may serve as a model for many clonal organisms. These studies have reinvigorated old questions and stimulated new ones. We identify six avenues of research to enhance understanding of relationships between the development and ecology of colonial cnidarians, specifically, and clonal organisms, generally.

4.1. The heritability of colony form Evolutionary ecology studies of colonial cnidarians clearly documented a genetic basis of colony form (Foster, 1979; McFadden et al. 1984; Buss and Grosberg, 1990; Blackstone and Buss, 1991; Yund, 1991; Knowlton et al., 1992; Bruno and Edmunds, 1997). In light of data emphasizing morphological plasticity, disparities among estimates from different studies of broad-sense heritability and the lack of sufficient data to estimate narrow-sense heritability, this question needs greater resolution. It would be useful to have good estimates of the components of phenotypic variance for the different morphometric characters that capture the qualitative descriptions of colony form, in the context of competitive relationships among morphs. Estimates of additive genetic variance (VA) in populations could test the hypothesis that selection from competition strongly influences morphological phenotypes. Narrow-sense heritability and the response to selection from competition can be measured only from estimates of VA. Additional estimates of contributions of nonadditive genetic, and environmental, variances to phenotypic variance, from breeding studies, would resolve disparities among studies estimating broad-sense heritability and the scope for plasticity.

4.2. The heritability of morphological plasticity We ask whether morphological plasticity observed among colonial invertebrates is, itself, heritable. In other words, is there positive covariation between the plastic responses of parents and their offspring? Genotype by environment interactions (G*E interactions) contribute a significant fraction to phenotypic variance within populations of colonial cnidarians (Bruno and Edmunds, 1997), but it has yet to be quantified relative to other components of variance. The extent to which G*E interactions represent heritable variation would contribute to understanding quantitative genetics, as well as the developmental plasticity of clonal organisms. Whether plasticity is a heritable trait was hotly debated by theorists during the 1990s (Via and Lande, 1985; Schlichting, 1986; Stearns, 1989; Scheiner, 1993b; Schlichting and Pigliucci, 1993; Via, 1993; Via et al.,

The shape of things to come

135

1995; Pigliucci 1996) and colonial invertebrates appear to be ideal systems for empirical studies addressing the question because clonal variation in plasticity is known (Bruno and Edmunds, 1997) and cnidarians are amenable to the requisite experiments.

4.3. Architectural versus genotypic effects A related issue is the relative contribution of architectural versus genotypic variation in setting the limits of phenotypic plasticity. Experiments using a single clone suggested that vascular architecture influenced plastic responses of phenotypes. Experiments that cross different vascular geometries with multiple genotypes are needed to assess the importance of architectural effects more generally. The likely existence of genetic variation for phenotypic plasticity (whether or not it is heritable) raises two further questions. Are effects of vascular architecture consistent across genotypes? What are the relative effect sizes of plasticity associated with different architectures compared to those among genotypes? Variation in competitive ability among morphotypes could be studied in the context of architectural versus genotypic effects. Competitive ability is linked to morphology, but it is unknown if competitive ability changes as the morphology of a colony is altered. If competitively superior morphs become inferior following a change in morphology, then competitive ability appears more tied to form than genotype. Alternatively, a genotype may be competitively superior regardless of its morphological plasticity.

4.4. Physiological mechanisms underlying morphological plasticity Blackstone demonstrated that ROS-signalling is a mechanism by which environments are sensed through polyp behaviour triggering adaptive morphogenetic plasticity. Redundancy of signalling pathways for plasticity would be advantageous in complex dynamic environments. Where mitochondria perceive anoxia, the presence of oxygen in the cell should prevent signalling of hypoxia-induced compensatory pathways (genes encoding hypoxia-inducing factor 1 [HIF-1] have been identified in Hydra magnipapillata; D. Bridge, personal communication). Under natural conditions redox state and pO2 are correlated (Lesser, 2006). It is unknown whether colonies in nature operate multiple pathways of oxygen-dependent regulation. Hydractiniid hydrozoans, like several Hydractinia species and P. carnea, live on shells occupied by hermit crabs in sandy to muddy marine habitats characterized by variable pO2, redox potential in sediments and temperature (Fenchel and Reidl, 1970). The behaviours of their host hermit crab subject colonies to fluctuations in pO2 ranging from hyperoxia to extreme hypoxia within minutes as the shell inhabited by a colony is buried in reduced sediments (Vasquez,

136

¨bler Dudgeon and Ku

2009). In addition to ROS signalling, it is plausible that colonies operate other signalling pathways to sense environmental pO2 directly. Morphological plasticity of colonies in response to manipulated seawater viscosity suggested shear stress signalling in colony-level morphogenesis. Additional experiments are needed to test that possibility. Previous experiments manipulating the dynamic viscosity of seawater used minimal architectures (single polyp, single stolon). Shear stress could have increased, decreased or remained the same depending on possible changes of transport rates in the gastrovascular system in response to viscosity manipulations. Controlled viscosity manipulations with concurrent measures of transport rates could determine the effect of manipulation on vascular shear stress and consequent morphogenetic effects. Simple observations of correlations between flow rate and the fraction of cells undergoing mitosis (i.e. mitotic index) in unmanipulated colonies suggests a direct link between gastrovascular flow rate and stolon elongation (Fig. 3.11).

4.5. Empirical tests of nutrient-driven polyp behaviour The model presented by Wagner et al. (1998) was based on behaviour of a single polyp fed a defined quantity of food. Manipulating nutrient content could test predictions of this model. Experimental diets can be given to colonies to test specific levels of nutrients that elicit polyp oscillatory behaviour (Dudgeon et al., 2009). If model predictions are consistent with observed polyp behaviours, then scaling up the model to two- and threepolyp cases arranged in various geometries would be possible.

4.6. Colonial hydrozoans as models of vascular development Colonial hydrozoans grow indeterminately, lack a predefined size or shape and continuously develop as they remodel vascular architecture to accommodate changes in state associated with growth, competitive encounters, substrate availability and environmental changes associated with food supply or other factors. Likewise, vascular systems of vertebrates grow indeterminately, lack a predefined size or shape and continuously develop as they remodel vessel architecture to accommodate internal changes in state associated with growth, maturation, reproduction, wound healing and various pathologies, despite the relative constancy of the organism’s external morphology. These parallels make hydrozoans useful models for in vivo study of vascular function. Comparisons between organ systems and colonial organisms, may give insight into common physiological mechanisms to regulate adaptive plasticity of vascular systems. Buss (2001) suggested that genes homologous to vertebrate vascular endothelial growth factors (VEGF), known to act in vascular proliferation, be sought in cnidarians. Since then, two VEGF genes and

The shape of things to come

137

[(Figure_1)TD$IG]

Figure 3.11 Positive covariation between rates of gastrovascular transport and mitosis of stolon cells. (A) Normalized flow rate in two colonies (open or solid circles, respectively) was calculated as the maximum amplitude of lumen oscillations normalized to external stolon diameter, divided by the period (in seconds) of the oscillation cycle. (B) Mitotic index is calculated as the fraction of living cells (indicated by staining with DAPI) that are undergoing mitosis (indicated using the commercially available BrdU/anti-BrdU staining immunoassay). (C) Composite fluorescent image of a stolon showing staining by DAPI (blue; to estimate cell density in a region of stolon) and/or FITC (green; to identify mitotic cells). Image constructed from a Z-series stack of images from bottom to the top of the stolon lumen at 400 magnification. Mitotic rates were estimated in two colonies (distinguished by solid vs. open symbols) along a linear length of stolon proximally near the polyp (circles) and distally near the stolon tip (triangles). Symbols and error bars represent means 95% confidence intervals of n = 4 colonies at each sampling period. (See Plate no.5 in the Color Plate Section.)

138

¨bler Dudgeon and Ku

[(Figure_1)TD$IG]

Figure 3.11

continued

a VEGF receptor have been identified in P. carnea (Seipel et al., 2004; D. Bridge, personal communication). Expression in stolons has not been investigated, but P. carnea VEGF and VEGFR are expressed in the endodermal tissue of tentacles and of the radial canals of the medusa gastrovascular system, consistent with VEGF signalling for morphogenetic processes leading to tube formation (Seipel et al., 2004). Most interestingly, factors known to regulate vertebrate VEGF signalling, redox state, pO2 and vascular shear stress, are the same as those implicated in the adaptive plasticity of hydrozoan colonies living in patchy food, and hypoxic soft sediment, environments (Chen etal., 1999; Osawa etal., 2002; Shay-Salit etal., 2002; Urbich et al. 2002; Holmes and Zachary, 2005; Milkiewicz et al., 2007).

4.7. Conclusions Physiological and anatomical (architectural) mechanisms regulate adaptive plasticity during development of hydrozoan colonies. The potential for rapid, large scale environmental changes call for greater understanding of physiological processes in ecology. Clonal organisms are particularly good models for studies of plasticity in response to environmental change because their development and ecological performance are tightly coupled. As asserted by both Bonner (1965) and Buss and Dick (1992), unravelling the physiological mechanisms that lie on the map between genotype and phenotype, in the ‘middle ground’ of biology, still holds the key to understanding the complexity of phenotypic variation in a changing environment.

ACKNOWLEDGEMENTS We are grateful for discussions with many people over the years, which have contributed to the ideas presented here. We especially thank N. Blackstone,

The shape of things to come

139

D. Bridge, L. Buss, B. Cameron, P. Cartwright, S. Craig, P. Edmunds, R. Grosberg, R. Jackson, J. Kaandorp, M. Lesser, P. Petraitis, R. Vaisnys and A. Wagner. We also acknowledge the contributions made by students in our lab who collected data and contributed ideas to this project, including K. Benes, S. Krueger, C. Marsh, P. Mroz, C. Ryan, C. Slaughter, L. Smith and M. C. Vasquez. Thanks to M. Lesser for the invitation to write this review. This research was supported by grants from the National Institute of Health GMS-MBRS-SCORE programme (NIH-SO6GM48680), National Science Foundation (DEB-1020480, DEB-1059916, OCE-93-15082 [to L. W. Buss]) and the CSUN Sponsored Programs Office. This is contribution number 172 from the CSUN Marine Biology Group.

REFERENCES Anctil, M., Poulain, I., and Pelletier, C. (2005). Nitric oxide modulates peristaltic muscle activity associated with fluid circulation in the sea pansy Renilla koellikeri. J. Exp. Biol. 208, 2005–2017. Beloussov, L. V., Badenko, L. A., Katchurin, A. L., and Kurilo, L. F. (1989). Cytophysiology of growth pulsations in hydroid polyps. J. Exp. Zool. 249, 258–270. Berrill, N. J. (1949). The polymorphic transformations of Obelia. Q. J. Microsc. Sci. 90, 235–254. Blackstone, N. W. (1996). Gastrovascular flow and colony development in two colonial hydroids. Biol. Bull. 190, 56–68. Blackstone, N. W. (1997). A dose-response relationship for experimental heterochrony in a colonial hydroid. Biol. Bull. 193, 47–61. Blackstone, N. W. (1998). Physiological and metabolic aspects of experimental heterochrony in colonial hydroids. J. Evol. Biol. 11, 421–438. Blackstone, N. W. (1999). Redox control in development and evolution: evidence from colonial hydroids. J. Exp. Biol. 202, 3541–3553. Blackstone, N. W. (2001). Redox state, reactive oxygen species, and adaptive growth in colonial hydroids. J. Exp. Biol. 204, 1845–1853. Blackstone, N. W. (2003). Redox signaling in the growth and development of colonial hydroids. J. Exp. Biol. 206, 651–658. Blackstone, N. W. (2009). Mitochondria and redox control of development in cnidarians. Semin. Cell Dev. Biol. 20, 330–336. Blackstone, N., and Bridge, D. (2005). Model Systems for environmental signaling. Integr. Comp. Biol. 45, 605–614. Blackstone, N. W., and Buss, L. W. (1991). Shape variation in hydractiniid hydroids. Biol. Bull. 180, 394–405. Blackstone, N. W., and Buss, L. W. (1992). Treatment with 2,4-dinitrophenol mimics ontogenetic and phylogenetic changes in a hydractiniid hydroid. Proc. Natl. Acad. Sci. U.S.A. 89, 4057–4061. Blackstone, N. W., and Buss, L. W. (1993). Experimental heterochrony in hydractiniid hydroids: why mechanisms matter. J. Evol. Biol. 6, 307–327. Blackstone, N. W., Bivins, M. J., Cherry, K. S., Fletcher, R. E., and Geddes, G. C. (2005). Redox signaling in colonial hydroids: many pathways for peroxide. J. Exp. Biol. 208, 383–390. Blackstone, N. W., and Yund, P. O. (1989). Morphological variation in a colonial marine hydroid: a comparison of size-based and age-based heterochrony. Paleobiology 15, 1–10.

140

¨bler Dudgeon and Ku

Boardman, R. S., Cheetham, A. H., and Oliver Jr., W. A. (1973). Animal Colonies. Dowden, Hutchinson and Ross Publishers, Stroudsburg, Pennsylvania. Bonabeau, E., Theraulaz, G., Deneubourg, J. -L., Aron, S., and Camazine, S. (1997). Selforganization in social insects. Trends Ecol. Evol. 12, 188–193. Bonasio, R., Tu, S., and Reinberg, D. (2010). Molecular signals of epigenetic states. Science 330, 612–616. Bonner, J. T. (1965). Size and Cycle: An Essay on the Structure of Biology. Princeton University Press, Princeton, NJ 219pp. Bradshaw, A. D. (1965). Evolutionary significance of phenotypic plasticity in plants. Adv. Genet. 13, 115–155. Brafield, A. E. (1969). Water movements in the pennatulid coelenterate Pteroidesgriseum. J. Zool. 158, 317–325. Braverman, M. H. (1963). Studies on hydroid differentiation, II. Colony growth and the initiation of sexuality. J. Embryol. Exp. Morphol. 11(1), 239–253. Braverman, M. H., and Schrandt, R. G. (1966). Colony development of a polymorphic hydroid as a problem in pattern formation. Symp. Zool. Soc. Lond. 16, 169–198. Brown, B. E., Sya’rani, L., and LeTissier, M. D. (1985). Skeletal form and growth in Acropora aspera (Dana) from the Pulau Seribu, Indonesia. J. Exp. Mar. Biol. Ecol. 86, 139–150. Bruno, J. F., and Edmunds, P. J. (1997). Clonal variation for phenotypic plasticity in the coral Madracis mirabilis. Ecology 78(7), 2177–2190. Bumann, D., and Buss, L. W. (2008). Nutritional physiology and colony form in Podocoryna carnea (Cnidaria: Hydrozoa). Invertebrate Biology 127, 368–380. Buss, L. W. (1979). Habitat selection, directional growth and spatial refuge: why colonial animals have more hiding places. In ‘‘Biology and Systematics of Colonial Organisms’’ (G. Larwood, and B. R. Rosen, eds.), pp. 459–497. Academic Press, London Systematics Association Special Volume No. 11. Buss, L. W. (1987). The Evolution of Individuality. Princeton University Press, Princeton, NJ 201pp.. Buss, L. W. (2001). Growth by intussusceptions in hydractiniid hydroids. In ‘‘Evolutionary Patterns’’ (J. B. C. Jackson., S. Lidgard, and F. K. McKinney, eds.), pp. 3–26. University of Chicago Press, Chicago, IL. Buss, L. W., and Blackstone, N. W. (1991). An experimental exploration of Waddington’s epigenetic landscape. Phil.Trans. R. Soc. Lond. 332, 49–58. Buss, L. W., and Dick, M. H. (1992). The middle ground of biology. In ‘‘Growing Points in Evolution’’ (P. R. Grant, and H. H. Horn, eds.), pp. 77–97. Princeton University Press, Princeton. Buss, L. W., and Grosberg, R. K. (1990). Morphogenetic basis for phenotypic differences in hydroid competitive behavior. Nature 343, 63–66. Buss, L. W., and Vaisnys, J. R. (1993). Temperature stress induces dynamical chaos in a cnidarian gastrovascular system. Proc. R. Soc. Lond. 252, 39–41. Caswell, H. (2001). Matrix Population Models, 2nd edition. Sinauer Publishers, Sunderland, MA 722pp. Chen, K. -D., Li, Y. -S., Kim, M., Li, S., Luan, S., Chien, S., and Shyy, J. Y. -J. (1999). Mechanotransduction in response to shear stress: roles of receptor tyrosine kinases, integrins and Shc. J. Biol. Chem. 274(25), 18193–18400. Cherry Vogt, KS., Geddes, G. C., Bross, L. S., and Blackstone, N. W. (2008). Physiological characterization of stolon regression in a colonial hydroid. J.Exp.Biol. 211, 731–740. Crowell, S. (1957). Differential responses of growth zones to nutrient level, age, and temperature in the colonial hydroid Campanularia. J. Exp. Zool. 13, 63–90. Crowell, S. (1974). Morphogenetic events associated with stolon elongation in colonial hydroids. Am. Zool. 14, 665–672.

The shape of things to come

141

Davies, P. F., Remuzzi, A., Gordon, E. J., Forbes Dewey Jr., C., and Gimbrone Jr., M. A. (1986). Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro. Proc. Natl. Acad. Sci. U.S.A. 83, 2114–2117. Diggle, P. K. (1995). Architectural effects and the interpretation of patterns of fruit and seed development. Annu. Rev. Ecol. Syst. 26, 531–552. Diggle, P. K. (2003). Architectural effects on floral form and function: a review. In ‘‘Deep Morphology: Toward a Renaissance of Morphology in Plant Systematics’’ (T. Stuessy., E. Horandl, and V. Mayer, eds.), pp. 63–80. Koeltz, Konigstein. Dudgeon, S. R. (2001a). Gastrovascular dynamics of hydractiniid hydrozoans. In ‘‘The Algorithmic Beauty of Corals, Sponges and Seaweeds’’ (J. Kaandorp, and J. E. K€ ubler, eds.), pp. 144–146. Springer-Verlag, Berlin, Germany. Dudgeon, S. R. (2001b). Morphological plasticity in colonial cnidarians. In ‘‘The Algorithmic Beauty of Corals, Sponges and Seaweeds’’ (J. Kaandorp, and J. E. K€ ubler, eds.), pp. 43–48. Springer-Verlag, Berlin, Germany. Dudgeon, S. R. (2001c). Colonial hydrozoans: perturbation experiments of gastrovascular physiology and effects on colony development. In ‘‘The Algorithmic Beauty of Corals, Sponges and Seaweeds’’ (J. Kaandorp, and J. E. K€ ubler, eds.), pp. 155–158. Springer-Verlag, Berlin, Germany. Dudgeon, S. R., Benes, K. M., Krueger, S. A., K€ ubler, J. E., Mroz, P., and Slaughter, C. T. (2009). On the use of experimental diets for physiological studies of hydrozoans. J. Mar. Biol. Assoc. U.K. 89(1), 83–88. Dudgeon, S. R., and Buss, L. W. (1996). Growing with the flow: on the maintenance and malleability of colony form in the hydroid Hydractinia. Am. Nat. 147, 667–691. Dudgeon, S. R., Wagner, A., Vaisnys, J. R., and Buss, L. W. (1999). Dynamics of gastrovascular circulation in the hydrozoan Podocoryne carnea: the one polyp case. Biol. Bull. 196, 1–17. Dustan, P. (1975). Growth and form in the reef-building coral Montastrea annularis. Mar. Biol. 33, 101–107. Falconer, D. S. (1986). Introduction to Quantitative Genetics. Wiley & Sons, New York 438pp. Fenchel, T., and Reidl, R. J. (1970). The sulfide system: a new biotic community underneath the oxidized layer of marine sand bottoms. Mar. Biol. 7, 255–268. Foster, A. B. (1979). Phenotypic plasticity in the reef corals Montastrea annularis (Ellis and Solander) and Siderastrea siderea (Ellis and Solander). J. Exp. Mar. Biol. Ecol. 39, 25–54. Foster, A. B. (1980). Environmental variation in skeletal morphology within the Caribbean reef corals Montastrea annularis and Siderastrea siderea. Bull. Mar. Sci. 30, 678–709. Fulton, C. (1963). Rhythmic movements in Cordylophora. J.Cell.Comp. Physiol. 61, 39–52. Gaten˜o, D., Israel, A., Barki, Y., and Rinkevich, B. (1998). Gastrovascular circulation in an octocoral: evidence of significant transport of coral and symbiont cells. Biol.Bull. 194, 178–186. Gladfelter, E. H. (1983). Circulation of fluids in the gastrovascular system of the reef coral Acropora cervicornis. Biol. Bull. 165, 619–636. Graus, R. R., and McIntyre, I. G. (1982). Variation in growth forms of the reef coral Montastrea annularis (Ellis and Solander): a quantitative evaluation of growth response to light distribution using computer simulation. Smithson. Contrib. Mar. Sci.., 12. Hale, L. J. (1960). Contractility and gastrovascular movements in the hydroid Clytiajohnstonii. Q. J. Microsc. Sci. 101, 339–350. Harper, J. L. (1977). Population Biology of Plants. Academic Press, New York 810pp. Harper, J. L. (1985). Modules, branches and the capture of resources. In ‘‘Population Biology and Evolution of Clonal Organisms’’ (J. B. C. Jackson., L. W. Buss, and R. E. Cook, eds.), pp. 1–33. Yale University Press, New Haven, CT.

142

¨bler Dudgeon and Ku

Harvell, C. D. (1986). The ecology and evolution of inducible defenses in a marine bryozoans: cues, costs, and consequences. Am. Nat. 128, 810–823. Harvell, C. D., and Grosberg, R. K. (1988). The timing of sexual maturity in clonal animals. Ecology 69, 1855–1864. Holmes, D., and Zachary, I. (2005). The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome Biol. 6, 209. Jackson, J. B. C. (1979). Morphological strategies of sessile animals. In ‘‘Biology and Systematics of Colonial Organisms’’ (G. Larwood, and B. R. Rosen, eds.), pp. 499– 555. Academic Press, London Systematics Association Special Volume No. 11. Jackson, J. B. C. (1985). Distribution and ecology of clonal and aclonal benthic invertebrates. In ‘‘Population Biology and Evolution of Clonal Organisms’’ (J. B. C. Jackson., L. W. Buss, and R. E. Cook, eds.), pp. 297–356. Yale University Press, New Haven, CT. Kaandorp, J., and K€ ubler, J. E. (2001). Introduction. In ‘‘The Algorithmic Beauty of Corals, Sponges and Seaweeds’’ (J. Kaandorp, and J. E. K€ ubler, eds.), pp. 1–13. Springer-Verlag, Berlin, Germany. Karlson, A. G., and Marferin, N. N. (1984). Movements of gastrovascular fluid in the colony of the hydroid Dynamena pumila and some other species. J. Gen. Biol. (in Russian) 45, 670–679. Knowlton, N., Weil, E., Weigt, L. A., and Guzman, H. M. (1992). Sibling species in Montastrea annularis, coral bleaching and the coral climate record. Science 255, 330–333. Lange, R. G., and M€ uller, W. A. (1991). SIF, a novel morphogenetic inducer in Hydrozoa. Dev. Biol. 147, 121–132. Lenhoff, H. (1968). Chemical perspectives on the feeding response, digestion, and nutrition of selected coelenterates. In ‘‘Chemical Zoology’’ (B. Scheer, and M. Forkin, eds.), pp. 159–221. Academic Press, New York. Lesser, M. P. (2006). Oxidative stress in marine environments: biochemistry and physiological ecology. Annu. Rev. Physiol. 68, 253–278. Lovett-Doust, L. (1981). Population dynamics and local specialization in a clonal perennial (Ranunculusrepens). I. The dynamics of ramets in contrasting habitats. J. Ecol. 69, 743–755. Lynch, M., and Walsh, B. (1998). Genetics and Analysis of Quantitative Traits. Sinauer Associates, Sunderland, MA 980pp. Mackie, G. O. (1973). Coordinated behavior in hydrozoan colonies. In ‘‘Animal Colonies: Development and Function’’ (R. J. Boardman., A. H. Cheetham, and W. A. Oliver Jr., eds.), pp. 95–106. Dowden, Hutchinson and Ross Publishers, Stroudsburg, Pennsylvania. Mackie, G. O. (1986). From aggregates to integrates: physiological aspects of modularity in colonial animals. Phil.Trans. R. Soc. Lond. B 313, 175–196. Marferin, N. N. (1985). The functioning of the pulsatory-peristaltic type transport system in colonial hydroids. J. Gen. Biol. 46, 153–164. McFadden, C. S., McFarland, M. J., and Buss, L. W. (1984). Biology of hydractiniid hydroids. I. Colony ontogeny in Hydractinia echinata (Flemming). Biol. Bull. 166, 54–67. Meinhardt, H. (1976). Morphogenesis of lines and nets. Di¡erentiation 6, 117–123. Milkiewicz, M., Doyle, J. L., Fudalewski, T., Ispanovic, E., Aghasi, M., and Haas, T. (2007). HIF-1a and HIF-2a play a central role in stretch-induced but not shear stressinduced angiogenesis in rat skeletal muscle. J. Physiol. 583(2), 753–766. M€ uller, W. A., and Plickert, G. (1982). Quantitative analysis of an inhibitory gradient field in the hydrozoan stolon. Wilhelm Roux’s Arch. 191, 56–63. Murdock, G. R. (1978). Digestion, assimilation and transport of food in the gastrovascular cavity of a gorgonian octocoral (Cnidaria: Anthozoa). Bull. Mar. Sci. 28, 354–362.

The shape of things to come

143

Nollert, M. U., Eskin, S. G., and McIntire, L. V. (1990). Shear stress increases inositol triphosphate levels in human endothelial cells. Biochem. Biophys. Res. Commun. 170, 281–287. Osawa, M., Masuda, M., Kusano, K., and Fujiwara, K. (2002). Evidence for a role of PECAM-1 in endothelial cell mechanosignal transduction: is it a mechanoresponsive molecule? J. Cell. Biol. 158, 773–785. Palumbi, S. R. (1984). Tactics of acclimation: morphological changes of sponges in an unpredictable environment. Science 225, 1478–1480. Parker, G. H. (1920). Activities of colonial animals. I. Circulation of water in Renilla. J. Exp. Zool. 31, 342–367. Parrin, A. P., Nethertone, S. E., Bross, L. S., McFadden, C. S., and Blackstone, N. W. (2010). Circulation of fluids in the gastrovascular system of a stoloniferous octocoral. Biol. Bull. 219, 112–121. Pigliucci, M. (1996). How organisms respond to environmental changes: from phenotypes to molecules (and vice versa). Trends Ecol. Evol. 11, 168–173. Plickert, G., Heringer, A., and Hiller, B. (1987). Analysis of spacing in a periodic pattern. Dev. Biol. 120, 399–411. Rees, J. L., Davis, V., and Lenhoff, H. M. (1970). Paths and rates of food distribution in the colonial hydroid Pennaria. Comp. Biochem. Physiol. 34, 309–316. Resnick, N., Yahav, H., Shay-Salit, A., Shushy, M., Schubert, S., Chen, L., Zilberman, M., and Wofovitz, E. (2003). Fluid shear stress and the vascular endothelium: for better and for worse. Prog. Biophys. Mol. Biol. 81, 177–199. Riddinhough, G., and Zahn, L. M. (2010). What is epigenetics? Science 330, 611. Roosen-Runge, E. C. (1967). Gastrovascular system of small hydromedusae: mechanisms of circulation. Science 156, 74–76. Scheiner, S. (1993a). Genetics and evolution of phenotypic plasticity. Annu.Rev.Ecol.Syst. 24, 35–68. Scheiner, S. (1993b). Plasticity as a selectable trait: reply to via. Am. Nat. 142, 371–373. Scheiner, S., and Goodnight, C. J. (1984). The comparison of phenotypic plasticity and genetic variation in populations of the grass Danthoniaspicata. Evolution 38, 845–855. Schierwater, B., Piekos, B., and Buss, L. W. (1992). Hydroid stolonal contractions mediated by contractile vacuoles. J. Exp. Biol. 162, 1–21. Schlichter, D. (1991). A perforated gastrovascular cavity in Leptoserisfragilis: a new strategy to improve heterotrophic nutrition in corals. Naturwissenschaften 78, 467–469. Schlichting, C. D. (1986). The evolution of phenotypic plasticity in plants. Ann.Rev.Ecol. Syst. 17, 667–693. Schlichting, C. D., and Pigliucci, M. (1993). Control of phenotypic plasticity via regulatory genes. Am. Nat. 142, 366–370. Schlosser, G., and Wagner, G. (2004). Introduction: the modularity concept in developmental and evolutionary biology. In ‘‘Modularity in Development and Evolution’’ (G. Schlosser, and G. Wagner, eds.), pp. 1–16. University of Chicago Press, Chicago, IL. Schmitt, J. (1993). Reaction norms of morphological and life-history traits to light availability in Impatiens capensis. Evolution 47, 1654–1668. Sebens, K. P., and Done, T. J. (1992). Water flow, growth form and distribution of scleractinian corals: Davies Reef, (GBR), Australia. Proc.7thInt.CoralReefSymp. 1, 557–568. Seipel, K., Eberhardt, M., M€ uller, P., Pescia, E., Yanze, N., and Schmid, V. (2004). Homologs of vascular endothelial growth factor and receptor, VEGF and VEGFR, in the jellyfish Podocoryne carnea. Dev. Dyn. 231, 303–312. Shay-Salit, A., Shushy, M., Wolfovitz, E., Yahav, H., Breviario, F., Dejana, E., and Resnick, N. (2002). VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 99, 9462–9467. Stearns, S. C. (1989). The evolutionary significance of phenotypic plasticity. Bioscience 39, 436–445.

144

¨bler Dudgeon and Ku

Stearns, S. C. (1992). The Evolution of Life Histories. Oxford University Press, Oxford, UK 249pp. Thompson, J. D. (1991). Phenotypic plasticity as a component of evolutionary change. Trends Ecol. Evol. 6, 246–249. Turing, A. (1952). The chemical basis of morphogenesis. Phil.Trans. R. Soc. Lond. B 237, 37–72. Ulam, S. (1962). On some mathematical problems connected with patterns of growth figures. Proc. Symp. Appl. Math. 14, 215–224. Urbich, C., Stein, M., Reisinger, K., Kaufmann, R., Dimmeler, S., and Gille, J. (2002). Fluid shear stress-induced transcriptional activation of the vascular endothelial growth factor receptor-2 gene requires Sp1-dependent DNA binding. FEBSLett. 535, 87–93. Van Winkle, D. H., and Blackstone, N. W. (1997). Video microscopical measures of gastrovascular flow in colonial hydroids. Invertebr. Biol. 116, 6–16. Vasquez, M. C. (2009). Morphological plasticity in the colonmial hydrozoan Podocoryne carnea. California State University, Northridge, M.Sc. Thesis, 93pp. Via, S. (1993). Adaptive phenotypic plasticity: target or by-product of selection in a variable environment. Am. Nat. 142, 352–365. Via, S., Gomulkiewicz, R., DeJong, G., Scheiner, S. M., Schlichting, C. D., and Van Tienderen, P. H. (1995). Adaptive phenotypic plasticity: consensus and controversy. Trends Ecol. Evol. 10(5), 212–217. Via, S., and Lande, R. (1985). Genotype-environment interaction and the evolution of phenotypic plasticity. Evolution 39(3), 505–522. Vogel, S. (1988). Life’s Devices. Princeton University Press, Princeton, NJ. Vogel, S. (1994). Life in Moving Fluids: The Physical Biology of Flow. Princeton University Press, Princeton, NJ. Waddington, C. H. (1942a). The epigenotype. Endeavour 1, 18–20. Waddington, C. H. (1942b). Canalization of development and the inheritance of acquired characters. Nature 150, 563–565. Wagner, A., Dudgeon, S. R., Vaisnys, J. R., and Buss, L. W. (1998). Non-linear oscillations in polyps of the colonial hydroid Podocoryne carnea. Naturwissenschaften 85, 117–120. Willis, B. L. (1985). Phenotypic plasticity versus phenotypic stability in the reef corals Turbinaria mesenterina and Pavona cactus. Proc.5th Int. Coral Reef Symp. 6, 107–112. Winston, J. E. (2010). Life in the colonies: learning the alien ways of colonial organisms. Integr. Comp. Biol. 50, 919–933. Wyttenbach, C. R. (1968). The dynamics of stolon elongation in the hydroid, Campanularia £exuosa. J. Exp. Zool. 167, 333–352. Wyttenbach, C. R. (1973). The role of hydroplasmic pressure in stolonic growth movements in the hydroid, Bougainvillia. J. Exp. Zool. 186, 79–90. Wyttenbach, C. R. (1974). Cell movements associated with terminal growth in colonial hydroids. Am. Zool. 14, 699–717. Yeh, L. -H., Park, Y. J., Hansalia, R. J., Ahmed, I. S., Deshpande, S. S., GoldschmidtClermont, P. J., Irani, K., and Alevriadou, R. B. (1999). Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS. Am. J. Physiol. 276, C838–C847. Yund, P. O. (1987). Intraspecific competition and the maintenance of morphological variation in the colonial hydroid Hydractinia symbioacadianus. Yale University, New Haven, Connecticut, PhD dissertation. Yund, P. O. (1991). Natural selection on hydroid colony morphology by intraspecific competition. Evolution 45, 1564–1573. Zeeman, E. (1976). Catastrophe theory. Sci. Am. 234, 65–83. Ziegelstein, R. C., Cheng, L., and Capogrossi, M. C. (1992). Flow-dependent cytosolic acidification of vascular endothelial cells. Science 258, 656–659.

C H A P T E R F O U R

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives Miles Lamare*,1, David Burritty and Kathryn Lister*,y

Contents 1. Introduction 1.1 Ultraviolet radiation and the marine environment 1.2 Phylum Echinodermata 1.3 The history of UVR research in echinoderms 2. Responses in echinoderms to UVR exposure 2.1 Avoidance strategies 3. UVR-Induced Damage in Echinoderms 3.1 Cellular 3.2 Organismal damage 4. Future Research 4.1 A greater diversity of UVR research within the echinoderm phyla 4.2 Synergistic effects 4.3 UVR and life histories 5. Conclusions Acknowledgements References

146 146 146 147 148 148 168 168 174 177 177 179 180 180 181 181

Abstract There is general consensus that solar ultraviolet radiation (UVR) negatively impacts many marine species. Echinoderms are ubiquitous within the marine environment, with members of the phyla often long-lived and numerically dominant within the benthic macrofauna, consequently the impact of UVR on the population dynamics of these organisms will influence marine communities and ecosystems. Research to date has shown that

* y

1

Department of Marine Science, University of Otago, Dunedin, New Zealand Department of Botany, University of Otago, Dunedin, New Zealand

Corresponding author.

Advances in Marine Biology, Volume 59 ISSN 0065-2881, DOI 10.1016/B978-0-12-385536-7.00004-2

# 2011 Elsevier LTD. All rights reserved.

145

146

Miles Lamare et al.

exposure of echinoderms to solar UVR can, affect reproduction and development, change behaviour, cause numerous biochemical and physiological changes and potentially cause increased mutation rates, by causing DNA damage. There is also considerable evidence that echinoderms utilise several different mechanisms to protect themselves against excessive UVR and subsequent UVR-induced damage. However, these protective mechanisms may pose conflicting selection pressures on echinoderms, as UVR is an additional stressor in oceans subjected to anthropogenic-induced climate change. This review summarises our knowledge of the effects of UVR on the Echinodermata. We outline the research conducted to date, highlight key studies on UVR that have utilised echinoderms and look to the future of UVR research in a rapidly changing ocean.

1. INTRODUCTION 1.1. Ultraviolet radiation and the marine environment Ultraviolet radiation (UVR) enters the marine environment in the form of UVB (290–315 nm) and UVA (315–400 nm). While UVR is efficiently attenuated through the water column, it can penetrate to depths of greater than 16 m (10% of surface UVB) and 46 m (10% of surface UVA) (reviewed by Tedetti and Sempere, 2006), where it plays a pivotal role in shaping the lives of marine organisms. The biological effects of being exposed to UVR have been extensively studied and comprehensive reviews have outlined the impacts of exposure on marine organisms at the physiological and molecular levels (Dahms and Lee, 2010) and on marine ecosystems (De Mora et al., 2000; H€ader et al., 2007). This review summarises our knowledge of the effects of UVR on the Echinodermata. We outline research to date, highlighting key studies on UVR that have utilised echinoderms and look to the future of UVR research in a rapidly changing ocean.

1.2. Phylum Echinodermata Echinoderms (Phylum Echinodermata) are a diverse, exclusively marine group of invertebrates that consists of over 13,000 extinct species (15 classes) and 7,000 extant described species within five classes (Asteroidea, Echinoidea, Holothuroidea, Ophiuroidea and Crinoidea). Echinoderms are ubiquitous within the marine environment, and members of the phyla can often be long-lived, a numerically dominant component of the benthic macrofauna, and a keystone species within regions they occupy. Consequently, processes that impact on the population dynamics of these organisms can, and do, influence marine communities and ecosystems.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

147

Echinoderms have a complex life cycle, alternating between the long-lived adult stage and the short-lived embryonic and larval stages. Within this general reproductive pattern is a range of life-history strategies, including brooding and vivipary. Echinoderm gametes are readily obtained and have played a pivotal role in enhancing our understanding of reproductive processes such as fertilisation and embryonic development, including a significant contribution to our understanding of the effects of UVR on reproduction.

1.3. The history of UVR research in echinoderms Echinoderms have played a pivotal role in our understanding of the role of UVR on marine organisms. Research concerning the effects of UVR on echinoderms dates back to the early 1900s, with the total number of publications reaching 65 by 2009.The research can be characterised by two main phases: (1) a period in the 1950s and 1960s involving laboratory studies on the role of UVR in developmental biology and (2) during the late 1980s to the present when an appreciation of the ecological importance of UVR and ozone depletion drove an increased number of physiological and field studies (Fig. 4.1). Of the published

[(Figure_1)TD$IG]

Figure 4.1 Meta-analysis ((Web of Science (Thomson Reuters, NY) database search: key words; Echinoderm* OR sea urchin OR starfish OR sea star OR echinoid OR sand dollar OR sea cucumber OR brittlestar OR echinoid OR asteroid OR holothuroid OR ophiuroid OR crinoid OR featherstar AND ultraviolet) of the number of published research papers on UVR in echinoderms. Fig. 4.1 represents the cumulative number of papers from and including the year 1900 through to 2009.

148

Miles Lamare et al.

studies, approximately 80% are on echinoids, with remaining classes the focus of 14 peer-reviewed studies. Furthermore, within this data set, 75% examine UVR responses in the embryological or larval stages, with lesser attention given to the post-settlement life-history stages.

2. RESPONSES IN ECHINODERMS TO UVR EXPOSURE The outcome of exposure to UVR for echinoderms can be in the form of avoidance, damage and repair (summarised in Fig. 4.2). These outcomes may occur at a number of levels, from the molecular, physiological, developmental and behavioural and ultimately through to the population and ecosystem levels. Based on this classification of responses, previous research which examines the effects of UVR in echinoderms is catalogued in Table 4.1, with responses grouped according to echinoderm class. In the following sections, we review the research that has contributed to an understanding of these responses to UVR in the phylum.

2.1. Avoidance strategies 2.1.1. Cellular 2.1.1.1. Mycosporine-like amino acids Sunscreening is an important UVR mitigating strategy, and echinoderms contain a range of molecules capable of absorbing solar radiation and acting as photoprotectants (Table 4.11). Of these the mycosporine-like amino acids (MAAs) are the best studied, and most important group of photoprotective pigments in echinoderms (see reviews by Shick and Dunlap, 2002; Rastogi et al., 2010). Their occurrence has been recorded in all echinoderm classes, and from polar to tropical species (Table 4.2). Within the phyla, eight different MAAs have been reported, with those found in the highest concentrations being mycosporine glycine, Porphyra-334 and shinorine. Concentrations of MAAs vary depending on species and tissue, as well as among developmental stages. For example, in tropical sea cucumbers, MAAs are concentrated in the epidermis and ovaries rather than in the viscera, consistent with the allocation of MAAs to tissues that will experience greater UVR exposure (Shick et al., 1992; Bandaranayake and Rocher, 1999). Similarly, Karentz etal. (1997) and McClintock and Karentz (1997) noted that the greatest MAA concentrations were found in the ovaries of the sea urchin Sterechinus neumayeri, with lower concentrations found in the body wall presumably reflecting the greater exposure of embryos and free-swimming larvae to UVR at the top of the water column compared to the adults found at a greater depth.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

149

[(Figure_2)TD$IG]

Figure 4.2 The major responses to UVR shown by echinoderms. Avoidance and protection (at the behavioural and cellular level) are primary mechanisms of defence and provide organisms with the ultimate safeguard against UVR. When defence is not possible, impairment occurs in the major forms of lipid, protein and DNAdamage.

150

Miles Lamare et al.

Echinoderms obtain MAAs from their diet (Rastogi etal., 2010), and a number of experiments have indicated the importance of diet on the concentration and composition of MAAs within this group. Carroll and Shick (1996), by altering the algal diet of the sea urchin Strongylocentrotus droebachiensis, were able to produce eggs that varied 25-fold in their MAA concentrations and also their composition. Similar findings were shown in later work on S. droebachiensis (Adams and Shick, 1996, 2001) and for Evechinus chloroticus (Lamare et al., 2004). The interconversion of MAAs has been reported in the sea cucumberThelenotaananas (Shick and Dunlap, 2002), and has been proposed to be the result of the action of bacteria within the gut tract hydrolysing shinorine and porphyra-334 to mycosporine glycine. The activity of the bacteria mirrored changes in MAA composition along the gut tract of the animal. MAA concentrations may vary depending on ambient UVR irradiance. Karentz et al. (1997) found significantly higher concentrations of MAAs in the tissues of S. neumayeri individuals collected from intertidal and shallow subtidal depths than in deeper individuals. Lamare etal. (2004) noted that the concentrations of MAAs in the eggs of E. chloroticus varied six-fold along a 40 km fiord gradient, and were correlated with ambient levels of UVB irradiance found along the fiord. These results likely reflect a passive response to changes in MAA concentrations in food sources of the sea urchins and not an active increase in the uptake of MAAs by sea urchins in response to higher irradiances. This conclusion is supported by the observation that, in the laboratory, S. droebachiensis does not accumulate MAAs in response to UVR exposure (Adams et al., 2001). Embryonic and larval stages of echinoderms also contain MAAs (Table 4.2), which are endowed to the eggs maternally (Carroll and Shick, 1996). In the laboratory, MAA concentrations decrease during initial development (i.e. pre-feeding stages) with no evidence that they are supplemented in the larvae by the absorption of MAAs from seawater (Adams and Shick, 1996, 2001). Whether decreases in MAAs in laboratory-reared embryos reflect patterns in situ is unknown, however, it is conceivable that the dietary accumulation of MAAs by feeding larvae occurs in the water column, with their phytoplankton and bacterial food likely to contain MAAs (Sinha et al., 1998).

[()TD$FIG] Figure 4.2 (cont.) Echinoderms are equipped with subsequent repair mechanisms including high-cost strategies such as protein replacement and low-cost strategies such as photoreactivation. When repair is not sufficient, damage can result in impaired embryonic and larval development, slower growth or eventually mortality of individuals. Ultimately, population effects can be seen in reduced recruitment and complex life-history trade-offs leading to largely undocumented impacts on marine communities in which echinoderms play large roles.

Summary table of published studies directly looking at impacts of UVR on echinoderms 1.1 Behavioural responses UVR response

Life-history stage

Collection site/field Location

Source

Echinoidea Strongylocentrotus droebachiensis Paracentrotus lividus

Phototaxis

Adults

Crow Island, Maine, USA (Lab)

(Adams, 2001)

Covering

Adults

Lough Hyne Marine Nature Reserve, County Cork, Ireland (Lab/Field)

Tripneustes ventricosus

Covering

Adults

Strongylocentrotus droebachiensis Lytechinus variegatus

Covering

Adults

Covering

Adults

Asteroidea Pisaster ochraceus

Discovery Bay Marine Laboratory, Jamaica (Lab/Field) Mingan Islands, northern Gulf of St. Lawrence, Canada (Lab) Gulf Specimen Marine Laboratories, Panacea, Florida, USA (Lab)

(Crook et al., 1999; Crook and Barnes, 2001; Verling et al., 2002) Kehas et al. (2005)

Habitat selection

Adults

Ophiuroidea Ophiopholis aculeata

Eastsound, Orcas Island, Washington, USA (Lab/Field)

Burnaford and Vasquez (2008)

Habitat selection

Adults

Mingan Islands, northern Gulf of St. Lawrence, Canada (Lab/Field)

Drolet et al. (2004)

Dumont et al. (2007) Sigg et al. (2007)

151

(continued)

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

Table 4.1

Table 4.1 (continued) 152

1.2 Molecular and cellular responses UVR response

Life-history stage

Collection site/field Location

Source

Strongylocentrotus purpuratus Anthocidaris crassispina

Fertilisation, Egg cleavage Cytokinesis

Eggs

nr (Lab)

Rustad (1959)

Eggs

Ikeda (1965)

Clypeaster japonicus

Cytokinesis

Eggs

Hemicentrotus pulcherrimus Paracentrotus lividus Echinus esculentus Sphaerechinus granularis Hemicentrotus pulcherrimus

Cytokinesis

Eggs

ATPase activity ATPase activity Polyspermy Protein complement, photoreversal Sunscreens (MAAs) Sunscreens (MAAs) Sunscreens (MAAs)

Eggs Eggs Gametes Embryos

Misaki Marine Biological Station, Japan (Lab) Misaki Marine Biological Station, Japan (Lab) Misaki Marine Biological Station, Japan (Lab) Mediterranean (Lab) North Sea (Lab) Roscoff, France (Lab) nr (Lab)

Eggs

Blue Hill Falls, Maine, USA (Lab)

Adams and Schick (1996)

Gonads/Gut/ Test/Spines Gonads/ Digestive tract/Body wall

Blue Hill Falls, Maine, USA (Lab)

Carroll and Shick (1996)

Hero Inlet, Arthur Harbor, Palmer Station, Antarctica (Field)

Karentz et al. (1997)

Ikeda (1965) Petzelt (1974) Petzelt (1974) David et al. (1985) Akimoto and Shiroya (1987a)

Miles Lamare et al.

Strongylocentrotus droebachiensis Strongylocentrotus droebachiensis Sterechinus neumayeri

Ikeda (1965)

1.2 Molecular and cellular responses

Abatus shackletoni

Strongylocentrotus droebachiensis

Collection site/field Location

Source

Sunscreens (MAAs) Sunscreens (MAAs) Sunscreens (MAAs)

Whole animals Whole animals Eggs/ Embryos/ Larvae Eggs/ Embryos/ Larvae Ovaries, Eggs

McMurdo Sound, Antarctica (Field) McMurdo Sound, Antarctica (Field) Pemaquid Point, Maine, USA (Lab)

McClintock and Karentz (1997) McClintock and Karentz (1997) Adams and Schick (2001)

Frazer Point, Maine, USA (Lab)

Adams and Schick (2001)

Crow Island & Pemaquid Point, Maine, USA (Lab)

Adams et al. (2001)

Gonads/ Whole animals Sperm

Doubtful - Thompson Sound complex, Fiordland, New Zealand (Field) Cheung Chau, Hong Kong (Lab)

Lamare et al. (2004)

Sperm

Cheung Chau, Hong Kong (Lab)

Lu and Wu (2005b)

Embryos

North-Western Sicily, Italy (Lab)

Bonaventura et al. (2006)

Coelomocytes

Palermo gulf, Italy (Lab)

Matranga et al. (2006)

Gametes/ Embryos

Bay of Banyuls-sur-Mer, Mediterranean coast, France (Lab)

Nahon et al. (2009)

Echinarachnis parma

Sunscreens (MAAs)

Strongylocentrotus droebachiensis

Sunscreens (MAAs), embryonic development Sunscreens (MAAs)

Evechinus chloroticus Anthocidaris crassispina Anthocidaris crassispina

Paracentrotus lividus Paracentrotus lividus Sphaerechinus granularis

Oxidative stress, fertilisation, sperm damage Mitochondrial function, membrane integrity Heat-shock proteins HsP expression, apoptosis Structural damage, chromatin damage

Lu and Wu (2005a)

(continued)

153

Life-history stage

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

Sterechinus neumayeri

UVR response

154

Table 4.1

(continued) 1.2 Molecular and cellular responses UVR response

Life-history stage

Collection site/field Location

Source

Sphaerechinus granularis Sperm damage

Sperm

Pruski et al. (2009)

Strongylocentrotus droebachiensis Sterechinus neumayeri

Oxidative stress, DNA damage Oxidative stress

Embryos Embryos

Tripneustes gratilla

Oxidative stress

Larvae

Paracentrotus lividus

Stress Proteins

Embryos

Bay of Banyuls-sur-Mer, Mediterranean Coast, France (Lab) Isles of Shoals, Gulf of Maine, USA (Field) Cape Armitage, McMurdo Sound, Antarctica (Field) Aitutaki Lagoon, Cook Islands (Field) Palermo, Sicily, Italy (Lab)

Polyspermy Sunscreens (MAAs) Sunscreens (MAAs) Sunscreens (MAAs) Sunscreens (MAAs)

Gametes Whole animals Whole animals Whole animals Whole animals

Roscoff, France (Lab) McMurdo Sound, Antarctica (Field) McMurdo Sound, Antarctica (Field) McMurdo Sound, Antarctica (Field) McMurdo Sound, Antarctica (Field)

Asteroidea Marthasterias glacialis Diplasterias brucei Odontaster meridonalis

Perknaster fuscus

Lister et al. (2010a) Lister et al. (2010b) Russo et al. (2010) David et al. (1985) McClintock and Karentz (1997) McClintock and Karentz (1997) McClintock and Karentz (1997) McClintock and Karentz (1997)

Miles Lamare et al.

Odontaster validus

Lesser (2010)

1.2 Molecular and cellular responses

Holothuria atra

Stichopus japonicus Crinoidea Promachocrinus kerguelensis

Life-history stage

Collection site/field Location

Sunscreens (MAAs) Sunscreens (MAAs)

Body wall damage

Whole McMurdo Sound, animals Antarctica (Field) Lizard Island group, Epidermal GBR, Australia (Lab) tissue/ Ovaries/ Viscera/Gut Body wall Zhangzidao Island, tissues China (Lab)

Sunscreens (MAAs)

Whole animals

McMurdo Sound, Antarctica (Field)

Source

McClintock and Karentz (1997) Banbaranayake and Rocher (1999) Beiwei et al. (2008)

McClintock and Karentz (1997)

1.3 Development/Growth/Mortality

Echinoidea Strongylocentrotus purpuratus Hemicentrotus pulcherrimus Cypeaster japonicus

Life-history stage

Collection site/field Location

Source

Developmental abnormality

Gametes/ Embryos/ Larvae Embryos

nr (Lab)

Giese et al. (1955)

nr (Lab)

Amemiya et al. (1986)

Eggs

nr (Lab)

Hamaguchi and Hiramoto (1986)

Developmental abnormality during gastrulation Fertilisation, gamete fusion

(continued)

155

UVR response

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

Holothuroidea Cucumaria ferrari

UVR response

(continued)

156

Table 4.1

1.3 Development/Growth/Mortality

Hemicentrotus pulcherrimus Echinometra viridis Lytechinus variegatus Anthocidaris crassispina Strongylocentrotus spp.

UVR response

Life-history stage

Collection site/field Location

Source

Developmental abnormality Mortality Developmental abnormality Sperm motility, fertilisation Carotenoids

Embryos, Larvae Adults Embryos

nr (Lab)

Akimoto et al. (1983)

Lee Stocking Island, Bahamas (Lab) Gulf of Mexico, USA

Reaka-Kudla et al. (1993) Steevens et al. (1999)

Sperm

East Peng Chau, China (Lab)

Au et al. (2002) Lamare and Hoffman (2004) Bonaventura et al. (2005)

Larvae

Vostok Bay, Sea of Japan, Russia

Kipryushina et al. (2010)

Mortality

Adults

Reaka-Kudla et al. (1993)

Ophiocoma echinata

Mortality

Adults

Ophioderma brevispinum

Mortality

Adults

Lee Stocking Island, Bahamas (Lab) Lee Stocking Island, Bahamas (Lab) North Carolina coast, USA (Field)

Paracentrotus lividus Strongylocentrotus intermedius Ophiuroidea Ophionereisreticulata

Developmental abnormality, stress markers Sunscreening, Abnormal development

Reaka-Kudla et al. (1993) Johnsen and Kier (1998)

Miles Lamare et al.

Gonads/Eggs Clallum Bay and the San Juan Islands, Washington State, USA (Field) Embryos North-Western Sicily, Italy (Lab)

Echinoidea Echinarachnius parma Paracentrotus lividus Echinarachnius parma Hemicentrotus pulcherrimus Hemicentrocus pulcherrimus Hemicentrotus pulcherrirnus Hemicentrotus pulcherrimus Hemicentrotus pulcherrimus

UVR response

Life-history stage

Collection site/field Location

Source

Developmental delays, Photoreactivation Photosensitization Photosensitivity, Photoreactivation Chromosome abnormalities, Photoreactivation Developmental abnormalities, Photoreactivation Action spectrum for photoreactivation Thymine dimmer photoreversal, developmental abnormality Developmental abnormality, photoreversal

Gametes/ Embryos Sperm Embryos

nr (Lab)

Cook and Rieck (1962)

nr (Lab) nr (Lab)

Colombo (1967) Cook (1968)

Gametes/ Embryos

nr (Lab)

Ejima and Shiroya (1982a)

Gametes/ Embryos

nr (Lab)

Ejima and Shiroya (1982b)

Eggs

nr (Lab)

Ejima et al. (1984)

Embryos, Larvae

nr (Lab)

Akimoto and Shiroya (1987b)

Embryos

nr (Lab)

Akimoto and Shiroya (1987c)

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

1.4 DNA damage/repair

(continued)

157

(continued)

158

Table 4.1

1.4 DNA damage/repair

Strongylocentrotus droebachiensis Echinarachnius parma Sterechinus neumayeri

Sterechinus neumayeri Evechinus chloroticus Sterechinus neumayeri

Life-history stage

Collection site/field Location

Source

Survivorship, development, CPD accumulation Survivorship, development, CPD accumulation Developmental abnormalities, CPD accumulation

Embryos/Larvae

Isles of Shoals, Gulf of Maine, USA (Lab) Isles of Shoals, Gulf of Maine, USA (Lab) Hero Inlet, Arthur Harbor, Palmer Station, Antarctica (Field) Cape Armitage, McMurdo Sound, Antarctica (Field) Otago Harbour, New Zealand McMurdo Sound, Antarctica (Lab) nr (Lab)

Lesser and Barry (2003)

Developmental abnormalities, CPD accumulation DNA repair rate, photoreactivation DNA repair rate, photoreactivation DNA repair rate, photoreactivation

Embryos/Larvae

Embryos

Embryos

Embryos Embryos Embryos

Lesser and Barry (2003) Karentz et al. (2004)

Lesser et al. (2004) Lamare et al. (2006) Lamare et al. (2006) Lamare et al. (2006)

Miles Lamare et al.

Diadema setosum

UVR response

Table 4.1

(continued) 1.4 DNA damage/repair

Sterechinus neumayeri Evechinus chloroticus Sterechinus neumayeri Tripneustes gratilla Evechinus chloroticus Diadema savignyi Sterechinus neumayeri

Life-history stage

Collection site/field Location

Source

Biological weighting functions, DNA damage Biological weighting functions, DNA damage Biological weighting functions, DNA damage Developmental abnormalities, CPD accumulation Developmental abnormalities, CPD accumulation Developmental abnormalities, CPD accumulation Developmental abnormalities, CPD accumulation DNA repair (Photolyase expression)

Embryos

Isles of Shoals, Gulf of Maine, USA (Lab) McMurdo Sound, Antarctica (Lab)

Lesser et al. (2006)

Embryos

Lesser et al. (2006)

Embryos

Fiordland, New Zealand (Lab)

Lesser et al. (2006)

Embryos

McMurdo Sound, Antarctica (Field)

Lamare et al. (2007)

Embryos

Aitutaki Lagoon, Cook Islands (Field)

Lamare et al. (2007)

Embryos

Otago Harbour, Lamare et al. (2007) New Zealand (Field)

Embryos

Aitutaki Lagoon, Cook Islands (Field)

Lamare et al. (2007)

Gonands/Embryos/ Larvae

McMurdo Sound, Antarctica (Field)

Isley et al. (2009)

159

Information is divided into four major responses: 1.1) behavioural, 1.2) cellular and molecular, 1.3) developmental and 1.4) DNA. Within each response, references are listed chronologically within each class of echinoderm. (nr = location not reported).

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

Strongylocentrotus droebachiensis

UVR response

160

Table 4.2

Published measurement of Mycosporine-like amino acids in echinoderms.

Species

Location

Habitat

MAA type

Tissue(s)

Echinoidea Strongylocentrotus droebachiensis Strongylocentrotus droebachiensis

North-Eastern USA North-Eastern USA

Shallow subtidal Subtidal

Mg, Sh

Eggs

Sterechinus neumayeri

Antarctica

Subtidal

Strongylocentrotus droebachiensis

North-Eastern USA

Shallow subtidal

Strongylocentrotus droebachiensis

North-Eastern USA

Subtidal

Total concentrations

Up to 33.21 nmol mg1 DW Gonads, Gut, Up to Test, Spines 9.56 nmol mg1 DW

Adams and Shick (1996) Carroll and Shick (1996)

<0.08 to 1389 Karentz et al. mg g1 DW (1997) 9 nmol mg1 DW Adams and Shick (2001) 1.75 to 8.86 nmol mg1 DW

Adams et al. (2001) Miles Lamare et al.

Mg, Sh, P-334, M-2-G, Pl, A-330, Pt Body Wall, Mg, Sh, P-334, Pl Ovaries Larvae Sh, P-334, M-2-G, Pl, A-330 Ovaries Sh, P-334, M-2-G, Pl, A-330

Source

Table 4.2 (continued) Location

Habitat

MAA type

Evechinus chloroticus

Southern New Zealand McMurdo Sound, Antarctica North-Eastern USA Southern, New Zealand

Shallow subtidal Subtidal

Sterechinus neumayeri Strongylocentrotus droebachiensis Evechinus chloroticus Holothuroidea Twelve species of tropical Holothuroids

Great Barrier Reef, Australia

Cucumaria ferrari

McMurdo Sound, Antarctica Great Barrier Reef, Australia

Holothuria atra

Total concentrations

Source

Mg, Sh, Gonads/Eggs P-334, Pl Sh, P-334, Embryos Pl

8.54 to 96.65 nmol mg1 DW 6.2 nmol mg1 protein

Lamare et al. (2004) Lesser et al. (2006)

Subtidal

Sh, P-334

Embryos

Subtidal

Mg, Sh

Embryos

650 nmol mg1 protein 155.66 nmol mg1 protein

Lesser et al. (2006) Lesser et al. (2006)

Subtidal

2 m depth

Tissue(s)

Principally in Mg, Sh, the P-334, epidermis M-2-G, Pl, A-330, Pt Ovaries/ Mg, Sh, Brood P-334, Pl, Mg-V Epidermis, Mg, Sh, Viscera, P-334, Gonads Pt, M-2-G, As

21.9 to Shick et al. 2053 nmol mg1 (1992) protein 81–156 (mg g1 DW 0.95 to 4.93 mmol g1 DW

McClintock and Karentz (1997) Banbaranayake and Rocher (1999)

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

Species

(continued)

161

162

Table 4.2 (continued) Species

Location

Habitat

MAA type

Tissue(s)

Total concentrations

Source

Granaster nutrix

Antarctic Peninsula

Intertidal

Whole animal 398 mg g1 DW

Karentz et al. (1991)

Odontaster validus

McMurdo Sound, Antarctica McMurdo Sound, Antarctica

Subtidal

Pl, P-334, Sh, Mg, A-330 Mg, Pl

Ovaries, Body 1-13 mg g1 DW wall

Subtidal

Pl

Body wall

McClintock and Karentz (1997) McClintock and Karentz (1997)

Antarctic Peninsula

Intertidal

Pl

Whole animal 5 mg g1 DW

McMurdo Sound, Antarctica

Subtidal

Mg, Sh, Whole animal 775 mg g1 DW P-334, Pl

Asteroidea

Diplasterias brucei Ophiuroidea Amphiplus a⁄nis Crinoidea

Karentz et al. (1991)

McClintock and Karentz (1997)

Note that the total concentrations are given for a range of units depending on the study. (MAA type abbreviations: Mg = Mycosporine glycine, Sh = Shinorine, P-334 = Porphyra-334, Pl = Palythene, Pt = Palythinol, M-2-G = Mycosprine-2-Glycine, A-330 = Asterina-330, Mg-V = Mycosporine-glycine-valine).

Miles Lamare et al.

Promachocrinus kerguelensis

30 mg g-1 DW

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

163

The key role of MAAs as sunscreens for echinoderms, particularly for their developmental stages, has been demonstrated in a number of elegant experiments undertaken by Adams and Shick (1996, 2001). By dietary manipulation, S. droebachiensis eggs with a range of MAA concentrations were obtained, and exposed in vitro to UVR. The delay in egg cleavage associated with UVR exposure was inversely correlated with MAA concentrations in the eggs, clearly demonstrating the protective role of these sunscreens (Fig. 4.3). The level of photoprotection was estimated by Adams and Shick (1996) based on the size of the eggs and the measured

[(Figure_3)TD$IG]

Figure 4.3 Percentage UVR induced cleavage delay in batches of embryos from S. droebachiensis as a function of MAA concentration and of J (the efficiency for internal self-shading, calculated according to Garcia-Pichel (1994)). Embryos are from adults reared in controlled diets of Laminaria saccharina (~) and Mastocarpus stellatus () or a combination diet of both algae (^). The logarithmic regression of percentage cleavage delay as a function of MAA concentration (y = 27.95 – 13.35 log(x)) and linear regression of percentage cleavage delay as a function of J (y = 30.79 – 26.17x) are both significant (P < 0.05).Figure is reproduced with permission of Dr. Nikki Adams and Professor Malcolm Shick.

164

Miles Lamare et al.

MAA concentration, suggesting that MAAs could account for up to 86% efficiency in UVR screening (Fig. 4.3). Adams and Shick (2001) noted a similar photoprotective role of MAAs in S. droebachiensis embryos, with the proportion of cultured embryos developing normally when exposed to UVR increasing linearly with increasing MAA concentration. 2.1.1.2. Carotenoids Carotenoids have a number of functions in marine organisms, including photoprotection (Rastogi et al., 2010) via antioxidation and as sunscreening agents (Mathew-Roth, 1997). Carotenoids are widespread in echinoderms where their antioxidant role is well established (Matsuno and Tsushima, 2001). However, studies are yet to show that they have a significant UV screening role in sea urchins, but a positive correlation between carotenoid concentration and UVR-induced egg cleavage delay was found to exist in four species of Strongylocentrotus (Lamare and Hoffman, 2004). It was concluded that this was unlikely due to a self-shading effect that they estimated was 1.5 and 4.6% for UVB and UVA, respectively, based on egg carotenoid concentration and size. 2.1.1.3. Melanin A UV screening role of melanin in some echinoderm species is possible, although this is yet to be confirmed. Melanin-like pigments or enzymes (i.e. phenoloxidase) involved in melanin synthesis have been described in echinoderm species across a range of classes, including Diadema antillarum (Jacobson and Millott, 1953; Millott, 1953), Pisaster spp., Stichopus californicus, Thyone briareus (Millott, 1950) and Holothuria spp. (Millott, 1953; Canicatti and Seymour, 1991). Vevers (1963) noted that the integument of echinoderms, including brittlestars, have ‘melanin’, suggesting the pigment is relatively widespread within the phyla. In D. antillarum, melanin occurs in epithelial chromatophores that display light sensitivity (Weber and Dambach, 1974). The process by which these cells respond to light has been described in the Diadematoid, Centrostephanuslongispinus, which also has pigmented, light sensitive chromatophores (Weber and Dambach, 1974). These cells are ovoid when in darkness but extend thin processes on exposure to light, increasing the light absorbing area. Adams (2001) suggested that this process, along with seeking a cryptic habitat during the day, is important for species like D. antillarum to avoid excess solar radiation exposure.

2.1.2. Antioxidants Oxidative stress occurs in echinoderms on exposure to UVR (see Section 3.1.3, Table 4.12). Echinoids contain a range of antioxidant compounds including carotenoids and non-enzymatic antioxidants. Their role in reducing oxidative stress is discussed in Section 3.1.3.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

165

2.1.3. DNA repair mechanisms Repair of UV-damaged DNA can be achieved by nucleotide excision repair (NER) and photoreactivation (PER), and although both occur in echinoderms, only PER has been directly observed within this group (Table 4.14). Photoreactivation, a light-dependent process mediated via photolyase enzymes (EC 4.1.99.3), is the principal mechanism of repairing DNA lesions (CPDs and 6-4 photoproducts). Marshak (1949) was the first to describe photoreactivation in echinoderms, when he noted that the effects of UVR on Arbacia eggs could be reduced by exposure to visible light. Photoreactivation has been observed in the eggs and embryos of number of echinoids (Wells and Giese, 1950; Cook and Rieck, 1962; Cook, 1968), although whether photoreactivation occurs in echinoid sperm is equivocal (i.e. Wells and Giese, 1950; Cook and Rieck, 1962). More recent work showed that photoreactivation prevented the formation of exogastrula or permanent blastulae in Hemicentrotuspulcherrimus embryos UV irradiated at the 8- or 16-cell stage (Akimoto and Shiroya, 1987c). Embryos that were allowed to photoreactivate developed into normal plutei, although the repair was dependent on the time that photoreactivating light was delivered was only successful if the embryos were illuminated up to the onset of the DNA-synthesis phase of the following cell cycle (Akimoto and Shiroya, 1987b,c). Akimoto and Shiroya (1987a) also demonstrated that the quantitative and qualitative changes in the protein complement of the same embryos was mostly reversed by photoreactivation. Lamare et al. (2006) established that PER was the primary means of reversing DNA dimers, and that rates of photoreactivation in embryos and larvae may vary among sea urchin species across latitudes (Lamare etal., 2006), with polar embryos repairing DNA slower than temperature and tropical species. The research showed that temperature affected the rate of repair, both within and between species, which was attributed to the influence of temperature on the photolyase enzyme and on the slower metabolic rates in polar larvae. Based on these findings, Lamare etal. (2007) attributed observed differences in the in situ accumulation of CPDs in embryos in polar, temperate and tropical species to differences in photoreactivation rates. Isely etal. (2009) partially sequenced the photolyase gene from four sea urchins, including S. neumayeri, for which the expression of the photolyase gene was examined during insitu exposures of embryos under a range of illuminations levels (i.e. with depth and sea ice cover). Expression levels were positively correlated with ambient UV dose, implicating that the embryos were responding to CPD accumulation and initiating repair. 2.1.4. Behavioural Echinoderms demonstrate negative phototaxis on exposure to UVR, with the response observed in both larvae and adults (Table 4.11).

166

Miles Lamare et al.

Pennington and Emlet (1986) noted that echinoid larvae (Dendraster excentricus), exposed to UVB in vitro, ceased swimming within minutes and settled out of the water. When UVB was blocked, the same larvae resumed swimming and returned to the water column within 1 h. Similar responses were first observed in larvae of Diademasetosum and Paracentrotus lividus (Fox, 1925). A negative phototaxis response to UVR, as well as covering behaviour (placing objects on dorsal body surfaces) has been documented in adult echinoderms (Fig. 4.4A). Covering behaviour in echinoids has been attributed to a number of stimuli (i.e. predator or desiccation avoidance, water movement) with Sharp and Gray (1962) and Lees and Carter (1972), the first to attribute the covering behaviour to UVR exposure during experimental observations on sea urchins. Adams (2001) found that S. droebachiensis kept in aquaria and exposed to artificial irradiation (containing both PAR and UVR) sought shade and covered themselves significantly more frequently than when irradiated by PAR only. When exposed to ambient irradiation in an outdoor aquarium, a similar covering response was observed depending on time of day, but this was typically less in those individuals exposed to PAR but not UVB. Verling et al. (2002) quantified the effect of UVR on the covering behaviour of P.lividus, and demonstrated that covering rate and duration increased upon exposure to a UVR treatment. The relative importance of UVR in covering

[(Figure_4)TD$IG]

Figure 4.4 Behavioural responses of echinoderms to exposure to UVR. (A) Covering behaviour in the sea urchin S. droebachiensis at a depth of 5–7 m. The animal outlined by the dash line is almost entirely covered by coralline algae encrusted stones, a behaviour induced by exposure to UVB radiation (Dumont et al., 2007). (B) The intertidal seastar, P. orchraceus, seeks shade during low tide. These observations demonstrate the role of solar radiation in determining these small-scale distributions of P. ochraceus. Confirming this, experimental trials showed that the sea stars moved to shade on exposure to solar radiation, although the response was to PAR and not UVR (Burnaford and Vasquez, 2008). Part A is reproduced from Dumont et al. (2007) with permission of Dr. Clement Dumont.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

167

behaviour, compared with other stimuli (predation, wave motion, food, size) in S.droebachiensis was tested in the laboratory (Dumont etal., 2007). While UVR was again found to induce covering, the response was weaker than that caused by other stimuli such as the presence of kelp food and waves. Field observations are also consistent with solar radiation invoking a covering response. Covering has been observed by a number of researchers to be greater during daylight hours (Millott, 1956; Crook etal., 1999), leading to their conclusion that solar radiation may play a role in their behaviour. Kehas et al. (2005) observed covering behaviour in the Caribbean sea urchinTripneustesventricosus, and noted that covering behaviour was greatest at shallower depths. Covering behaviour was reduced when UVR exposure was experimentally decreased by 66%. Interestingly, a small proportion of the population were albinos (4%) and these animal were found to have significantly greater rates of covering compared with pigmented individuals, possibly indicating greater sensitivity to solar radiation. Shade-seeking and covering behaviours also occur in sea stars and brittlestars. Drolet etal. (2004) documented shade-seeking in the brittlestar Ophiopholis aculeata, although they noted that filtering UVR did not alter this behaviour, indicating a general response to solar radiation. Burnaford and Vasquez (2008) observed the intertidal sea star Pisaster ochraceus predominantly (85%) occurred in shaded microhabitats during low tide (Fig. 4.4). Confirming the role of solar radiation in determining these small-scale distributions of P. ochraceus, experimental trials showed the sea stars moved to shade on exposure to solar radiation, although the response was to PAR not UVR. These findings demonstrate the strong behavioural responses that UVR can exert on echinoderms, particularly those found in shallow water and the intertidal zone. Such behaviours should reduce the deleterious effects of UVR, although there may be some indirect costs associated with such strategies. For example, covering and seeking shade may reduce movement and hence the ability to feed (Kehas et al., 2005; Dumont et al., 2007; Burnaford and Vasquez, 2008), may reduce gas exchange and could result in an energetic cost associated with moving and holding objects (Kehas et al., 2005; Sigg et al., 2007). Consequently, reduced movement could decrease the ability of animals to escape predation. Although yet to be quantified, given the key role that echinoderms can play in their environment via movement and feeding, the influence of UVR on behaviour may have wider implications for the community. For example, Burnaford and Vasquez (2008) noted the feeding behaviour, and hence structuring role of the intertidal P. ochraceus may, in part, be controlled by exposure to solar radiation, and at a level greater than has been previously appreciated (Fig. 4.4B).

168

Miles Lamare et al.

3. UVR-INDUCED DAMAGE IN ECHINODERMS 3.1. Cellular 3.1.1. DNA damage Damage to DNA in the form of DNA dimers, strand breaks, linkages, DNA cross-overs and changes in hydration have been observed in echinoderms upon exposure to UV radiation. DNA dimer (CPDs and 6-4 photoproducts) formation is a key response to UVR exposure and has been observed in echinoderms (Table 4.1d). Despite their importance however, DNA dimers have only been quantified in echinoids and only in the embryonic and larval stages. Lesser etal. (2006) explored the effects of UVR (including CPD formation) in the laboratory on the embryos of three echinoid species including two temperate and an Antarctic species. CPD concentrations, expressed as relative absorption units, were quantified in embryos exposed under long-pass filters that provided five UVR treatments within the UVR spectrum. While all three species showed CPD formation in the UVA portion of the spectrum, CPD formation was greatest in the UVB wavelengths and decreased progressively as the shorter wavelengths were blocked. From these data, biological weighting functions (BWFs) were developed from which two important conclusions were drawn: (1) the Antarctic species (S. neumayeri) showed the greatest sensitivity to UVR compared with the temperate species (E.chloroticus and S.droebachiensis) which (2) corresponded with S.neumayeri having the lowest sunscreen compound concentrations. Based on the BWFs and measures of ambient spectral irradiances, the authors concluded that E.chloroticus may receive the greatest biologically effective UVR insitu, although later research by Lamare et al. (2007) showed that CPD accumulation among these species may not reflect these laboratory sensitivities. Lamare et al. (2006) also observed significant accumulation of CPDs in embryos from four species exposed to UVB for 1 h, during in vitro experiments examining DNA photorepair rates. DNA dimer accumulation has also been observed in echinoid larvae exposed to UVR insitu. In experiments aimed at examining the effects of changes in UVB (as a result of ozone depletion) and sea ice coverage on sea urchin embryos, Lesser et al. (2004) measured relative levels of CPD accumulation in larvae of the Antarctic sea urchin, S.neumayeri exposed to ambient UVR under sea ice over two years. CPD accumulation was correlated with the UVB dose received, which was found to be a function of depth, UV-filter treatment and overhead ozone conditions (which varied between the 2 years of the research). Greater rates of embryo abnormality were associated with treatments that resulted in accumulated CPDs. Karentz et al. (2004) also examined the effects of Antarctic ozone

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

169

depletion on the embryos of S. neumayeri, including quantifying the accumulation of CPDs. CPD concentrations were as high as 272 CPDs mb1 DNA after 6 days of exposure. Concentrations of CPDs varied with the UVB dose received, although the relationship between the two was non-linear with a threshold dose of 25 kJ m2. The researchers also identified an ancillary threshold limit of 17 CPDs mb1 DNA, above which normal development of embryos was unlikely. To further understand the relative sensitivity of echinoid embryos to UVR, and to quantify natural levels of CPD accumulation in species from a range of latitudes, Lamare et al. (2007) undertook a series of in situ exposures that included testing the effect of depth and UVR dose on CPD accumulation. Maximum CPD concentrations were greatest in the Antarctic S. neumayeri embryos (30.8 CPDs mb1 DNA) compared with the temperate (E. chloroticus) and tropical (Diadema savignyi) species (16.5 and 3.0 CPDs mb1 DNA, respectively). Within each species, CPD concentration was greater at the shallower depth, and increased with UVB dose (but not UVA dose). In a similar set of experiments, Lesser (2010) explored depth-dependent DNA damage in embryos of the sea urchin, S. droebachiensis in the Gulf of Maine, which included an examination of the relative levels of CPD accumulation and levels of oxidative modification of nucleotide bases (8-hydroxy-20 -deoxyguanosine (8OHdG)). Lesser (2010) showed that DNA damage occurred in embryos exposed to UVR at depths <5 m despite the presence of UVR absorbing sunscreen compounds. By examining different forms of DNA damage, the research highlighted the differential effects of UVB and UVA, which caused direct and indirect damage to DNA, respectively. Importantly, Lesser (2010) noted that DNA damage was mostly the result of exposure to UVA, reflecting its greater irradiances and depth penetration compared to UVB. When biologically weighted for DNA damage (using Lesser et al., 2006), the importance of UVA was even more pronounced. Research by Lesser (2010) highlights the limited range of measurements of DNA damage that have been made in echinoderms. With the exception of 8-OHdG concentrations previously mentioned (Lesser, 2010) and strand excisions in coelomocytes of the sea urchin P. lividus (Schr€ oder et al., 2005), little is known of other indirect effects of UVR on DNA such as cross-linking and singe-strand breaks. In the wider context, measurements of DNA damage have been restricted to echinoids, and with the exception of coelomocytes, within the embryological and larval stages. To fully understand the effects of UVR on DNA damage, a greater suite of measurements may be necessary. The measurements of DNA damage (CPD concentrations) reported in echinoderms are within the range reported for other marine animals exposed to ambient light conditions (i.e. 3–30.8 CPD mb1 DNA,

170

Miles Lamare et al.

Dahms and Lee, 2010). The exception is the high levels (up to 272 CPD mb1 DNA) reported in Antarctic embryos (Karentz et al., 2004; Lamare etal., 2007). Lamare etal. (2007) concluded that the greater accumulation of CPDs in polar embryos may be the result of lower concentrations of sunscreens and slower rates of DNA repair within the species. 3.1.2. Protein and lipid damage Little is known about the impacts of UVR on protein and lipid biochemistry, and metabolism (Table 4.12). Akimoto and Shiroya (1987a) used two-dimensional electrophoresis to determine the patterns of accumulating proteins in normal, UV-irradiated and photoreversed sea urchin (H. pulcherrimus) embryos. In this study many qualitative and quantitative variations in the accumulating proteins were observed. More recently, a proteomic investigation of Strongylocentrotuspurpuratus embryos exposed to UVR has shown differential protein spot migration between UV-protected and UV-treated embryos at 30 and 90 min post-fertilisation (Campanale et al., 2011). The study noted that proteins from multiple pathways were affected by UVR, including cellular stress, protein synthesis, signal transduction and cytoskeletal dynamics. The further application of proteomic, lipidomic and metabolomic profiling techniques could provide, as they have in studies on human cells exposed to UVR (Huang et al., 2005; Reich et al., 2010), useful information on protein and lipid, damage, repair and metabolism in echinoderms exposed to UVR. 3.1.3. Oxidative stress Reactive oxygen species (ROS) are a group of highly reactive oxygen intermediates that include superoxide (O2), hydrogen peroxide (H2O2) and the hydroxyl radical (HO.). ROS are produced by the partial singleelectron reduction of oxygen or by the alteration of oxygen electron spin states by photoactivation resulting in the production of singlet oxygen. In living cells O2 is also produced as a by-product of mitochondrial electron transport, associated with normal respiration (Halliwell and Gutteridge, 2007). Under conditions of normality organisms are exposed to relatively low and steady ROS levels and under these conditions the cellular antioxidant defences, which include non-enzymatic antioxidants such as glutathione and a-tocopherol, and enzymatic antioxidants such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD), are able to regulate ROS levels, minimise any potential oxidative damage, and maintain a balanced and stable cell redox potential. However, exposure to pollutants, rapid changes in environmental condition such as temperature and or oxygen levels, high levels of visible light and exposure to UVR can lead to the over production of ROS and lead to oxidative damage (Smith et al., 2000;

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

171

Burritt and MacKenzie, 2003; Lesser, 2006; Halliwell and Gutteridge, 2007; Burritt, 2008). While most cellular macromolecules are sensitive to oxidation, DNA, proteins and lipids are arguably the most critical cellular macromolecules that can be damaged due to oxidation. Oxidative damage to DNA is often measured as 8-OHdG, to proteins as protein carbonyls and to lipids directly as peroxides or indirectly as malonyldialdehyde (MDA). All the above are considered to be sensitive biomarkers of oxidative stress and have been used to measure the impact of environmental stressors, including UVB, on a wide range on organisms (Lesser, 2006; Halliwell and Gutteridge, 2007). Numerous studies on organisms have demonstrated that exposure to UVR can cause oxidative stress (Lesser, 2006; Halliwell and Gutteridge, 2007). With respect to echinoderms UVR-induced oxidative stress can in theory occur at any stage in the life cycle of the organism, from gametes and larvae through to adults, however due to their relative transparency it is the gametes and larvae that are probably the most sensitive to UVinduced oxidative damage. Lu and Wu (2005a), in a study on the impact of environmentally relevant levels of UVA and UVB on the sperm of the sea urchin Anthocidariscrassispina, showed that UVA and UVB both enhanced the production of ROS and greatly increased LPO. Lu and Wu (2005a) also concluded that the increase in ROS production and oxidative damage due to UVR exposure could account for the decline in sperm motility and fertilisation success that they also observed. They suggested that ROS could directly attack the mitochondrion of the sperm, and could cause significant membrane disruption. They also speculated that elevated UVR levels could pose a significant threat to the ability of natural populations of sea urchins to reproduce successfully in shallow waters. In a field experiment conducted on the embryos of the green sea urchin S. droebachiensis, Lesser (2010) demonstrated the importance of measuring not only direct damage to DNA caused by UVR exposure, in the form of CPDs, but also indirect oxidative damage to DNA, in the form of 8-OHdG. In this study despite embryos containing significant levels of mycosporine-like amino acids that absorb UVR, primarily in the UVA portion of the spectrum, and increased levels of SOD, significant increases in both CPDs and 8-OHdG were observed at depths less than 5 m. Lesser (2010) suggested that screening alone did not provide adequate protection from DNA damage in the field at depths less than 5 m. Perhaps of greater significance was the finding that while exposure to UVA did not result in the formation of CPDs, exposure to UVA greatly increase oxidative DNA damage at depths of 5 m or less. Other studies have also shown UVA can cause oxidative damage to cells (Reinheckel et al., 1999; Hoerter et al., 2008). Two recent studies have shown that oxidative stress caused by UVR occurs irrespective of echinoid species or global location. Lister et al.

172

Miles Lamare et al.

[(Figure_5)TD$IG]

Figure 4.5 Changes in oxidative stress in sea urchin embryos in response to ozone depletion over the Antarctic (from Lister et al., 2010a). (A) Ozone concentrations over the Antarctic during field experiments indicating periods (26 October 2008) of normal ozone concentrations overhead and periods of ozone depletion (3 November 2008) over the experimental site (indicated by a black dot). (B) Experimental racks at 1 and 4 metres depth on which Sterechinus neumayeri embryos were exposed to ambient and filtered (UVB-minus and UVR-minus) light. (C) Oxidative damage (mean (SE) protein carbonyl and lipid hydroperoxide concentrations) in embryos under normal and reduced ozone concentrations. (D) Change in antioxidant enzyme activities (mean (SE) superoxide dismutase and catalase activity) in embryos under normal and reduced ozone concentrations. For all bar graphs, grey bars represent high ozone conditions, and white bars indicate low ozone conditions. Figures reproduced from Lister et al. (2010a). Ozone maps courtesy of NASA (USA). Photograph (B) used with permission of M.F. Barker.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

173

(2010a) conducted an investigation into the influence of increased UVR, caused by the ‘ozone hole’, on the Antarctic sea urchin S.neumayeri over two consecutive 4-day periods in the spring of 2008. They found that the presence of the ozone hole and the corresponding increase in UVB exposure caused an increase in oxidative damage to lipids and proteins, measured as lipid peroxides and protein carbonyls and developmental abnormality in embryos grown in open waters (Fig. 4.5). Their results also showed that while the embryos had a limited capacity to increase the activities of protective antioxidant enzymes, severe oxidative damage occurred following exposure to UVB. More importantly, this study also demonstrated that the effect of the ozone hole was largely mitigated by sea ice coverage, with embryos under the sea ice largely protected from UVinduced oxidative damage. In a second field study Lister et al. (2010b) investigated oxidative damage and antioxidant defences in larvae of the tropical sea urchinTripneustesgratilla, at two depths, in Aitutaki in the Cook Islands. Compared to those shielded from UVB, larvae exposed to UVB held at a depth of 1 m showed increased developmental abnormality and increases in oxidative damage to proteins, despite increased levels of CAT, GPx, GR and SOD. Increased oxidative damage was also observed in larvae held at 4 m, but the amount of damage was much less. However, unlike the Lesser (2010) study, both the above studies provided no conclusive evidence that UVA caused any significant increases in oxidative damage or abnormality. Campanale et al. (2011) provides further support for the importance of antioxidant defences in echinoids exposed to UVR, by showing that proteins involved in redox regulation (e.g. glutathione peroxidase) increased in UV-treated S. purpuratus embryos. 3.1.4. Cell cycle and stress responses genes Lesser and Barry (2003) postulated that cell-cycle arrest at critical DNA check points following UVB-induced DNA damage was a component of the observed developmental delays in S. droebachensis embryos. Their conclusion was based on the observation that survivorship of earlier staged embryos was more sensitive to DNA damage than older embryos, which may reflect lower repair capacities and subsequently greater levels of apoptosis mediated by cell-cycle genes (p53). In support of this, the process of apoptosis in response to UVB exposure has been observed in sea urchins, with coelomocytes from P. lividus (Matranga et al., 2006) displaying apoptotic nuclei after short-term UVB exposure in the laboratory. In the same study, Matranga etal. (2006) showed that levels of the stress protein hsp70 in coelomocytes respond to UVB in a dose-dependent manner. An increase in hsp70 expression on exposure to UVR also occurs in the embryonic stages of echinoids. Bonaventura et al. (2005) noted a dose-dependent increase in the expression of the protein in P. lividus

174

Miles Lamare et al.

blastula, as well as a second stress marker p38 MAPk. Bonaventura et al. (2006) subsequently showed that embryos at the 16-cell stage also increased their levels of hsp70 mRNA and the protein itself in a UVB dose-dependent manner. P.lividus embryos also increase the expression of stress-response proteins from the 14-3-3 protein family after exposure to UVB (Russo etal., 2010). Whole-mount insitu hybridisation of the 14-33 transcripts showed it was locally distributed in the embryonic archenteron and throughout the germ layers. Importantly, these distributions mapped cell-cycle disruption (i.e. failure of the archenteron to elongate). These studies highlight an emerging appreciation of the range of cellular stress responses and regulatory cascades that likely occur in echinoderm tissues and developmental stages on exposure to UVR. These responses are dose-dependent, and their initiation will depend on the ratio of damage to repair within tissues and developmental stages. While studies have quantified levels of damage under ambient conditions (see previous sections), the degree that cell regulatory processes are responsible for the developmental changes seen during in situ experiments, or in the process of mitigating damage, is unknown.

3.2. Organismal damage 3.2.1. Developmental biology The effects of UVR on developmental biology have been examined extensively using echinoderms, and particularly echinoids, as model systems (Table 4.13). Gametes, fertilisation and embryonic and larval development are all sensitive to UVR, with a range of responses observed after exposure to UVB and/or UVA, both in the laboratory and under ambient field conditions. Sperm are the most UVR sensitive gametes in echinoderms with the detrimental effects including impaired swimming (Au etal., 2002; Lu and Wu, 2005a), oxidative stress (Lu and Wu, 2005a), a loss of mitochondrial function and membrane integrity (Lu and Wu, 2005b; Pruski etal., 2009), chromatin and DNA damage (Pruski et al., 2009) and morphological alteration (Pruski et al., 2009). A key outcome of the exposure of sperm to UVR is a reduction in fertilisation rate, which occurs in a dosedependent manner. Lu and Wu (2005b) demonstrated an exponential decrease in fertilisation rates across environmentally relevant doses for UVB (91–98% reductions) and UVA (38–72% reductions). Similarly, Nahon et al. (2009) observed that rates of fertilisation decreased from >90 to 20% when sperm were exposed to a range of environmentally relevant UVR irradiances. The damage seen in sperm reflects a lack of sunscreens, reduced DNA repair and antioxidants, a high surface to volume ratio and high levels of

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

175

polyunsaturated fatty acids (PUFA) in the membranes in these cells (Lu and Wu, 2005a) making them vulnerable to lipid peroxidation (Lu and Wu, 2005a; Pruski et al., 2009). In contrast to sperm, initial fertilisation rates do not decrease when eggs are exposed to UVR prior to insemination (Ikeda, 1965; Nahon et al., 2008, 2009), although the processes during fertilisation can be altered by irradiation. Rustad (1959) noted the lifting of the fertilisation envelope was suppressed on the UV-irradiated side of S.purpuratus eggs, and was inhibited under highest doses, with Whalley etal. (1995) suggesting this represents an interruption exocytosis of the cortical granule. Pronuclear fusion may also be influenced by UVR, with sperm aster and female pronuclei migration responding to regional UV illumination in the fertilised egg (Hamaguchi and Hiramoto, 1986). Cook (1968) noted in earlier work that pronuclear fusion timing was not affected by UVR exposure, although they noted that prophase was delayed. Irradiation can increase rates of polyspermy in sea urchin and sea star eggs (David et al., 1985), which was attributed to the destruction of Na+ channels and a loss of fast-block capacity. Post-fertilisation developmental effects of exposing eggs to UVR are evident and well documented. Outcomes of laboratory irradiances include delays in cleavage rate (Giese, 1939; Cook, 1968; Rustad, 1971; Adams and Shick, 2001; Lamare and Hoffman, 2004; Nahon et al., 2009), reduced survivorship (Lesser and Barry, 2003; Nahon et al., 2009) and increased rates of developmental abnormalities in eggs and larval stages (Akimoto and Shiroya, 1987b,c; Adams and Shick, 2001; Lesser and Barry, 2003; Bonaventura et al., 2006; Lamare et al., 2006; Lesser et al., 2006; Nahon et al., 2009) (Fig. 4.6). Eggs, embryos and larvae undergo a range of detrimental changes on exposure to UVR which may include damage and reduced migration of primary mesenchyme cells (Akimoto and Shiroya, 1987c), impaired skeletal formation (Bonaventura et al., 2005; Nahon et al., 2009) and apoptosis (Lesser et al., 2003). The effects on embryonic development of UV treatment are demonstrable in echinoderms, characterised by reduced development, abnormal cell division, thickening of body walls and cellular packing of the blastocoel (Fig. 4.6). At the molecular level, damage includes DNA lesions (Lesser and Barry, 2003; Lesset et al., 2006; Lamare et al., 2006; Nahon et al., 2008), oxidative stress (Lesser, 2006), reduced expression of a gene (Pl-SM30) coding for skeletal matrix proteins (Bonaventura et al., 2005), expression of heat shock proteins and stress markers (Bonaventura et al., 2005, 2006) and changes in protein profiles (Campanale et al., 2011) including a decrease in actin and tubulin (Akimota and Shiroya, 1987). Many of these processes have been observed in embryos exposed to ambient UVR in field experiments across a range of latitudes. Eggs and

176

Miles Lamare et al.

[(Figure_6)TD$IG]

Figure 4.6 The effects of UVR on the development of P. lividus embryos 24 h after exposure to UVB doses of 200 J m2 (B), 400 J m2 (C), 800 J m2 (D) and no dose (A). Photographs are from Bonaventura et al. (2006), and indicate the typical responses to UVR observed during development, namely reduced size, delayed development, abnormal cell division patterns, thickening of the blastoderm and the accumulation of cells in the blastocoel. Scale bar = 50 mm. Images reproduced with permission of Dr. Rosa Bonaventura.

embryos of the Antarctic sea urchin S. neumayeri show abnormal development (Lesser et al., 2004; Karentz et al., 2004; Lamare et al., 2007), oxidative stress and an increase in antioxidant activities (Lister et al., 2010a), DNA damage (Karentz et al., 2004; Lesser et al., 2004; Lamare et al., 2007) and expression of DNA repair enzymes (Isely et al., 2010). Oxidative stress and DNA damage occur in temperate echinoid embryos and larvae exposed to UVR (Lamare et al., 2007; Lesser, 2010), with similar responses observed in the developmental stages of tropical species exposed in lagoonal environments (Lamare et al., 2007; Lister et al., 2010b).

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

177

These findings point to the important role that UVR may play during reproduction and development of echinoderms. This is despite eggs and embryos being well equipped to mitigate the effects of solar radiation, either via repair and protection mechanisms (DNA repair pathways, heatshock proteins, sunscreens and antioxidants) or possibly by behavioural responses (see previous sections). The often shallow distribution of embryos and larvae in the water column will also contribute to their vulnerability to UVR (Rumrill et al., 1985; Emlet, 1986; Pennington and Emlet, 1986; Pedrotti, 1990; Sewell and Watson, 1993; Miller and Emlet, 1997; Lamare, 1998), with echinoderm larvae probably not able to control their vertical location and therefore more likely to be exposed to damaging levels of UVR in their natural environment. Similarly, reproduction in intertidal and shallow subtidal species will almost inevitably involve exposure of gametes to potentially damaging levels of solar radiation. 3.2.2. Mortality and population level effects Very little is known of the effects of UVR on post-settlement echinoderms in terms of mortality (Table 4.1c). Exposure of the brittlestar Ophiodermabrevispinum to ambient solar radiation in experimental aquaria resulted in almost 100% mortality after 4 days (Johnsen and Kier, 1998). In treatments where UVR was blocked by filters, no mortality occurred. In contrast, Raeka-Kudla et al. (1993) found no effect of ambient UVR on the survival of two brittlestars, Ophionereisreticulata and Ophiocomaechinata, or in the sea urchin Echinometra viridis exposed in experimental aquaria. Species from both studies were tropical, with no information available for how temperate and polar species survive on exposure to UVR.

4. FUTURE RESEARCH In reviewing research on the effects of UVR on echinoderms, a number of patterns in relation to the scope and focus of these investigations have emerged. These patterns are insightful, particularly as a means of providing a guide to future research for echinoderm photobiologists. We give three of the potential directions for this research.

4.1. A greater diversity of UVR research within the echinoderm phyla While a body of literature exists on UVR effects on echinoderms, there are a number of limitations within it. Firstly, of the published studies, approximately 80% are on echinoids, with remaining classes the focus of only 14 peer-reviewed studies. Furthermore, within this literature, 75%

178

Miles Lamare et al.

of studies examined UVR responses in the embryological or larval stages, with little attention given to the post-settlement life-history stages. This bias is for good reason; sea urchins are a widely used model system in developmental research and findings from research on echinoid embryos are generally applicable to a wide range of species, beyond the echinoderm phyla. It does mean, however, that we have a limited understanding of the effects of UVR within the Echinodermata for both non-echinoid species and for their post-settlement stages. An understanding of the effects of UVR on these echinoderms at the population level, both in terms of their dynamics and ecology is also worthy of exploration. This is important as many post-settlement echinoderm populations have a keystone role within their ecosystem. The sea star P.ochraceus is such a species as it can control the vertical distribution of intertidal communities (Paine, 1974), and Burnaford and Vasquez (2008) have shown in the laboratory, that UVR can influence the distribution and movement of this organism. P. ochraceus seeks shelter from solar radiation in the field and, hence by influencing the seastar, UVR contributes to key prey/predator interactions in this habitat. Similar interactions in echinoids and sea cucumbers (important grazers and bioturbators) probably occur (i.e. Dumont et al., 2007), along with a suite of other ecological processes associated with movement for which UVR may influence yet we know little of. Similarly, there has been little linkage or application of developmental studies to post-settlement processes. While many laboratory and field studies have shown that UVR is detrimental to development, the implications for recruitment and the population dynamics of echinoderm species is yet to be quantified. A good example is the Antarctic, where research has focused on the effects of ozone depletion and increased UVR on embryos and larvae of S.neumayeri. While this work has shown that the ozone hole results in DNA damage, oxidative stress, mortality and developmental delays, there is, as yet, no evidence to suggest whether there have been changes at the population level during 40 years of ozone depletion. Researchers are aware of these issues; Lesser et al. (2004) discussed the potential for UVR-induced recruitment failure of S.neumayeri in McMurdo Sound over this period and suggested monitoring of populations over the long term would shed light on this interesting question. A second limitation is in the range of responses examined in echinoderms. As model organisms, echinoderms can readily be used to quantify the effects of UVR across a suite of molecular and cellular processes. For example, research by Lesser (2010) highlights the limited range of measurements of DNA damage that have been made in echinoderms and notes that damage can mostly be the result of exposure to UVA and 8-OHdG formation. Similarly, repair of DNA can be via photoreactivation, DNA glycosylases and NER, the latter two of which have not been

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

179

examined in echinoderms. The same limitations could be noted for a range of possible molecular and cellular responses to UVR such as cell signalling, stress responses and apoptosis. Insight into these processes could be forthcoming with the application of modern molecular techniques such as proteomics (Campanale et al., 2011), genomics and DNA microarrays (i.e. Hofmann and Gaines, 2008) now possible with the DNA sequencing of species within the Echinodermata (i.e. S. purpuratus).

4.2. Synergistic effects Research on the effects of UVR on echinoderms has generally been conducted in isolation and does not consider the potential impacts of other environment parameters. In reality, UVR acts in concert with a range of abiotic and biotic factors that could be independent of, or interactive with UVR, either in an additive, synergistic or antagonistic manner. The cumulative impacts on marine systems from human activities have shown these interactions occur at approximately equal proportions when two or more stressors are examined (Crain et al., 2008). Understanding such interactions is especially relevant when considering future climate change scenarios (Todgham and Hofmann, 2009), where increases in UVR may be accompanied by ocean acidification, rising sea temperatures, reduced sea ice cover, altered salinities and changes in productivity (reviewed by Hoegh-Gulberg and Bruno, 2010). H€ader et al. (2007) provided a perspective on the effects of UV radiation on aquatic systems and interactions with climate change, noting that at the organism level UVR often acts as an additional stressor while at the ecosystem level the effect of UVR and climate change processes can be more pronounced, particularly on community and trophic structure. Indeed, research on the interactive effect of UVR and ocean acidification is underway on other marine groups (Gao et al., 2009; Gao and Zheng, 2010), and past studies have shown that exposure to UVR exacerbates the detrimental effects of reduced pH (and vice versa) on marine algae. Echinoderms may provide excellent model systems to further examine these processes in marine metazoans. Equally important is the interaction of UVR with pollutants. Steevens et al. (1999) examined the combined effects of UVB and polycyclic aromatic hydrocarbons (PCBs) on Lytechinus variegatus embryos, noting an additive toxicological effect. In contrast, the amount of DNA strand breakage in sea urchin coelomocytes following UVR exposure was, unexpectedly, reduced when combined with toxic concentrations of cadmium (Schr€ oder et al., 2005). The interactive effect of UVR and pollutants may be intrinsic (i.e. cadmium-induced inhibition of the DNA excision repair enzyme, endonucleases in the Schr€ oder et al., 2005 study) or extrinsic (i.e. changes in the pollutant chemistry and its

180

Miles Lamare et al.

toxicity by UVR-induced photochemical processes). The toxicological effects of a range of pollutants such as metals, hydrocarbons and PCBs have been studied extensively in echinoderm gametes and embryos (Pagano et al., 1997; Coteur et al., 2004; Manzo et al., 2010). Such approaches, combined with UVR as a factor, provide an opportunity to expand our understanding of the interaction of these pollutants under real-world conditions were UVR could play an important synergistic role.

4.3. UVR and life histories UVR has had a major influence on the evolution of life, including its role in promoting oxygen toxicity (i.e. oxidative stress) in metazoans (Lesser, 2006). In recent years ecological research has focused on the relevance of antioxidants and oxidative stress in the evolution of life-history strategies and physiological trade-offs in animals. Managing oxidative stress is likely to be a major determinant of life histories, as virtually all metabolic activities generate ROS, the levels of which also increase in organisms exposed to abiotic and/or biotic stressors (Monaghan et al., 2009). Important considerations are that the levels of oxidative stress incurred by organisms are not constant but vary with developmental stage, environmental conditions and levels of activity (Monaghan et al., 2009). Echinoderms have been used extensively to theorise on life-history strategies (Hart, 2002) and could provide ideal model systems to address questions relating to the role of UVR and oxidative stress in shaping developmental patterns.

5. CONCLUSIONS The influence of UVR on echinoderms has been researched for more than 100 years and has increased our understanding of the role of UVR in the marine environment. While focused mainly on echinoids, and particularly their developmental stages, this research has added to our knowledge in a number of areas:

The effect of UVR on developmental biology, and specifically on gametes, embryos and larval stages. The physiological responses and mitigating strategies occurring in animals exposed to UVR, particularly the role of sunscreens, DNA damage and repair and oxidative damage. The types and level of UVR damage occurring in situ, including an understanding of the effects of ozone depletion on marine ecosystems in polar regions.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

181

Looking forward, future research on echinoderms has the potential to make a significant contribution to photobiology at a number of levels:

Echinoderms, particularly echinoids, as model systems for developmental biology will continue to add to our understanding of the effects of UVR on development, particularly with the application of new molecular techniques. In a changing marine environment, the role of UVR and its synergistic relationship with other climate change agents, will need to be addressed. With a good general knowledge of the effects of UVR on echinoderms coupled with a broad knowledge of developmental and physiological processes in the phyla, echinoderms could be at the forefront of this research. Fundamental and theoretical biological questions relating to photobiology (such as the role of UVR shaping life histories) would be ideally asked in the Echinodermata.

ACKNOWLEDGEMENTS We acknowledge the help of Sara Cumming and Matthew Baird when preparing this review. Dr. Nikki Adams and Professor Malcolm Shick generously provided their data for Fig. 4.3. Photographs were also provided by Associate Professor Mike Barker and Dr. Clement Dumont. The University of Otago provided financial support to aid in the production of the review.

REFERENCES Adams, N. L. (2001). UV radiation evokes negative phototaxis and covering behavior in the sea urchin Strongylocentrotus droebachiensis. Mar. Ecol. Prog. Ser. 213, 87–95. Adams, N. L., and Shick, J. M. (1996). Mycosporine-like amino acids provide protection against ultraviolet radiation in eggs of the green sea urchin Strongylocentrotus droebachiensis. Photochem. Photobiol. 64, 149–158. Adams, N. L., and Shick, J. M. (2001). Mycosporine-like amino acids prevent UVBinduced abnormalities during early development of the green sea urchin Strongylocentrotus droebachiensis. Mar. Biol. 138, 267–280. Adams, N. L., Shick, J. M., and Dunlap, W. C. (2001). Selective accumulation of mycosporine-like amino acids in ovaries of the green sea urchin Strongylocentrotus droebachiensis is not affected by ultraviolet radiation. Mar. Biol. 138, 281–294. Akimoto, Y., Shiroya, T., and Egami, N. (1983). Abnormal morphogenesis of sea urchin embryo induced by UV partial irradiation given at cleavage stage. J. Radiat. Res. 24, 197–202. Akimoto, Y., and Shiroya, T. (1987a). Changes in the protein complement following UV irradiation and their photoreversal in sea urchin (Hemicentrotuspulcherrimus) embryos. Photochem. Photobiol. 45, 809–814. Akimoto, Y., and Shiroya, T. (1987b). Photoreversibility of UV-induced thymine dimers and abnormal morphogenesis in sea-urchin embryos. Photochem. Photobiol. 45, 403–406.

182

Miles Lamare et al.

Akimoto, Y., and Shiroya, T. (1987c). Photoreversal of abnormal morphogenesis in seaurchin embryos caused by UV-irradiation. Photochem. Photobiol. 45, 407–412. Amemiya, S., Yonemura, S., Kinoshita., and Shiroya, T. (1986). Biphasic stage sensitivity to UV suppression of gastrulation in sea urchin embryos. Cell Di¡erentiation 18, 45–49. Au, D. W. T., Chiang, M. W. L., Tang, J. Y. M., Yuen, B. B. H., Wang, Y. L., and Wu, R. S. S. (2002). Impairment of sea urchin sperm quality by UV-B radiation: predicting fertilization success from sperm motility. Mar. Pollut. Bull. 44, 583–589. Bandaranayake, W. M., and Rocher, A. D. (1999). Role of secondary metabolites and pigments in the epidermal tissues, ripe ovaries, viscera, gut contents and diet of the sea cucumber Holothuria atra. Mar. Biol. 133, 163–169. Beiwei, Z. H. U., Zheng, J., Zhang, Z., Xiuping, D., Lulu, Z., and Mikiro, T. (2008). Autophagy plays a potential role in the process of sea cucumber body wall ‘‘melting’’ induced by UV irradiation. Wuhan UniversityJ. Natural Sciences 13, 232–238. Bonaventura, R., Poma, V., Costa, C., and Matranga, V. (2005). UVB radiation prevents skeleton growth and stimulates the expression of stress markers in sea urchin embryos. Biochem. Biophys. Res. Commun. 328, 150–157. Bonaventura, R., Poma, V., Russo, R., Zito, F., and Matranga, V. (2006). Effects of UV-B radiation on development and hsp70 expression in sea urchin cleavage embryos. Mar. Biol. 149, 79–86. Burnaford, J. L., and Vasquez, M. (2008). Solar radiation plays a role in habitat selection by the sea star Pisaster ochraceus. Mar. Ecol. Prog. Ser. 368, 177–187. Burritt, D. J. (2008). The polycyclic aromatic hydrocarbon phenanthrene causes oxidative stress and alters polyamine metabolism in the aquatic liverwort Riccia£uitans L. Plant Cell Environ. 31, 1416–1431. Burritt, D. J., and MacKenzie, S. (2003). Antioxidant metabolism during the acclimation of Begonia xerythrophylla to high-light. Annals of Botany 91, 783–794. Campanale, J. P., Tomanek, L., and Adams, N. L. (2011). Exposure to ultraviolet radiation causes proteomic changes in embryos of the purple sea urchin, Strongylocentrotus purpuratus. J. Exp. Mar. Biol. Ecol. 397, 106–120. Canicatti, C., and Seymour, J. (1991). Evidence for phenoloxidase activity in Holothuria tubulosa (Echinodermata) brown bodies and cells. Parasitol. Res. 77, 50–53. Carroll, A. K., and Shick, J. M. (1996). Dietary accumulation of UV-absorbing mycosporine-like amino acids (MAAs) by the green sea urchin (Strongylocentrotus droebachiensis). Mar. Biol. 124, 561–569. Colombo, G. (1967). Photosensitization of sea-urchin sperm to long-wave ultraviolet light by psoralen. Experimental Cell Research 48, 167–169. Cook, J. S. (1968). On the role of DNA in the ultraviolet-sensitivity of cell division in sand dollar zygotes. Exp. Cell Res. 50, 627–638. Cook, J. S., and Rieck, A. F. (1962). Studies on photoreactivation in gametes and zygotes of the sand dollar, Echinarachnius parma. J. Cellular and Comparative Physiology 59, 77–84. Coteur, G., Corriere, N., and Dubois, Ph. (2004). Environmental factors influencing the immune responses of the common European starfish (Asterias rubens). Fish Shell¢sh Immunol. 16, 51–63. Crain, C. M., Kroeker, K., and Halpern, B. S. (2008). Interactive and cumulative effects of multiple human stressors in marine systems. Ecol. Lett. 11, 1304–1315. Crook, A. C., and Barnes, D. K. A. (2001). Seasonal variation in the covering behaviour of the echinoid Paracentrotuslividus (Lamarck). Marine Ecology 22, 231–239. Crook, A. C., Verling, E., and Barnes, D. K. A. (1999). Comparative study of the covering reaction of the purple sea urchin, Paracentrotus lividus, under laboratory and field conditions. J. Mar. Biol. Assoc. U.K. 79, 1117–1121.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

183

Dahms, H. U., and Lee, J. -S. (2010). UV radiation in marine ectotherms: molecular effects and responses. Aquat.Toxicol. 97, 3–14. David, C., Moreau, M., Vilain, J. P., and Guerrier, P. (1985). U.V. irradiation inhibits the electrical block to polyspermy in echinoderms. Dev. Growth Di¡er. 27, 333–348. De Mora, S., Demers, S., and Vernet, M. (2000). The Effects of UV Radiation in the Marine Environment. Cambridge University Press, Cambridge, UK. Drolet, D., Himmelman, J. H., and Rochette, R. (2004). Effect of light and substratum complexity on microhabitat selection and activity of the ophiuroid Ophiopholis aculeata. J. Exp. Mar. Biol. Ecol. 313, 139–154. Dumont, C. P., Drolet, D., Desch^enes, I., and Himmelman, J. H. (2007). Multiple factors explain the covering behaviour in the green sea urchin, Strongylocentrotus droebachiensis. Anim. Behav. 73, 979–986. Ejima, Y., and Shiroya, T. (1982a). Photoreactivation associated with morphological abnormality in sea urchin embryos induced by ultraviolet-irradiated sperm. Photochem. Photobiol. 35, 175–180. Ejima, Y., and Shiroya, T. (1982b). Photoreactivation associated with chromosomal abnormality in sea urchin eggs fertilised with ultraviolet-irradiated sperm. Photochem. Photobiol. 36, 37–41. Emlet, R. B. (1986). Larval production, dispersal, and growth in a fjord: a case-study on larvae of the sand dollar Dendraster excenticus. Mar. Ecol. Prog. Ser. 31, 245–254. Fox, H. M. (1925). The effect of light on the vertical movement of aquatic organisms. Proc. Cambridge Philos. Soc. Biol. Sci. 1, 219–224. Gao, K., Ruan, Z., Villafan˜e, V. E., Gattuso, J. -P., and Helbling, E. W. (2009). Ocean acidification exacerbates the effect of UV radiation on the calcifying phytoplankter Emiliania huxleyi. Limnol. Oceanogr. 54, 1855–1862. Gao, K., and Zheng, Y. (2010). Combined effects of ocean acidification and solar UV radiation on photosynthesis, growth, pigmentation and calcification of the coralline alga Corallina sessilis (Rhodophyta). Global Change Biol. 16, 2388–2398. Garcia-Pichel, F. (1994). A model for internal self-shading in planktonic organisms and its implications for the usefulness of ultraviolet sunscreens. Limnol. Oceanogr. 39, 1704–1717. Giese, A. C. (1939). The effects of ultraviolet radiation of various wavelengths upon cleavage of sea urchin eggs. Biol. Bull. 75, 238–247. Giese, A. C., Inverson, R. M., Sanders, R. T., and Bors, J. P. (1955). Peroxide and ultraviolet radiation effects. Experimental Cell Research 8, 369–386. H€ader, D. -P., Kumar, H. D., Smith, R. C., and Worrest, R. C. (2007). Effects of solar UV radiation on aquatic ecosystems and interactions with climate change. Photochem. Photobiol. Sci. 6, 267–285. Halliwell, B., and Gutteridge, J. M. C. (2007). Free Radicals in Biology and Medicine, Edition 4. Oxford University Press, Oxford. Hamaguchi, M. S., and Hiramoto, Y. (1986). Analysis of the role of astral rays in pronuclear migration in sand dollar eggs by the colcemid-UV method. Dev. Growth Di¡er. 28, 143–156. Hart, M. W. (2002). Life history evolution and comparative developmental biology of echinoderms. Evol. Dev. 4, 62–71. Hoegh-Gulberg, O., and Bruno, J. F. (2010). The impact of climate change on the world’s marine ecosystems. Science 328, 1523–1528. Hoerter, J. D., Ward, C. S., Bale, K. D., Gizachew, A. N., Graham, R., Reynolds, J., Ward, M. E., Choi, C., Kagabo, J. L., Sauer, M., Kuipers, T., Hotchkiss, T., Banner, N., Chellson, R. A., Ohaeri, T., Gant, L., and Vanderhill, L. (2008). Effect of UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts. Int. J. Biol. Sci. 4, 63–70.

184

Miles Lamare et al.

Hofmann, G. E., and Gaines, S. D. (2008). New tools to meet new challenges: emerging technologies for managing marine ecosystems for resilience. Bioscience 58, 43–52. Huang, C. M., Xu, H., Wang, C. C., and Elmets, C. A. (2005). Proteomic characterization of skin and epidermis in response to environmental agents. Expert Review of Proteomics 2, 809–820. Ikeda, M. (1965). Behaviour of sulfhydryl groups of sea urchin eggs under the blockage of cell division by UV and heat shock. Exp. Cell Res. 40, 282–291. Isely, N., Lamare, M., Marshall, C., and Barker, M. (2009). Expression of the DNA repair enzyme, photolyase, in developmental tissues and larvae, and in response to ambient UV-R in the Antarctic sea urchin Sterechinus neumayeri. Photochem. Photobiol. 85, 1168–1176. Isely, N., Lamare, M., Marshall, C., and Barker, M. (2010). Expression of the DNA repair enzyme, photolyase, in developmental tissue and larvae, and in response to ambient UV-R in the Antarctic sea urchin Sterechinus neumayeri. Photochem. Photobiol. 85, 1168–1176. Jacobson, F. W., and Millott, N. (1953). Phenolases and melanogenesis in the coelomic fluid of the echinoid Diadema antillarum Phillippi. Proc. R. Soc. Lond. Biol. Sci. 141, 231–247. Johnsen, S., and Kier, W. M. (1998). Damage due to solar ultraviolet radiation in the brittlestar Ophioderma brevispinum (Echinodermata: Ophiuroidea). J. Mar. Biol. Assoc. U.K. 78, 681–684. Karentz, D., Bosch, I., and Mitchell, D. M. (2004). Limited effects of Antarctic ozone depletion on sea urchin development. Mar. Biol. 145, 277–292. Karentz, D., Dunlap, W. C., and Bosch, I. (1997). Temporal and spatial occurrence of UVabsorbing mycosporine-like amino acids in tissues of the Antarctic sea urchin Sterechinus neumayeri during springtime ozone-depletion. Mar. Biol. 129, 343–353. Karentz, D., McEuen, F. S., Land, M. C., and Dunlap, W. C. (1991). Survey of mycosporine-like amino acid compounds in Antarctic marine organisms: potential protection from ultraviolet exposure. Marine Biology 108, 157–166. Kehas, A. J., Theoharides, K. A., and Gilbert, J. J. (2005). Effect of sunlight intensity and albinism on the covering response of the Caribbean sea urchinTripneustes ventricosus. Mar. Biol. 146, 1111–1117. Kipryushina, Y. O., Lukyanov, P. A., and Odintsova, N. A. (2010). Effect of mytilan on the UV-radiation resistance of marine invertebrate and human lymphocytes. Russian J. Marine Biology 36, 305–310. Lamare, M. D. (1998). Origin and transport of larvae of the sea urchin Evechinuschloroticus (Echinodermata: Echinoidea) in a New Zealand fiord. Mar. Ecol. Prog. Ser. 174, 107–121. Lamare, M. D., and Hoffman, J. (2004). Natural variation of carotenoids in the eggs and gonads of the echinoid genus, Strongylocentrotus: implications for their role in ultraviolet radiation photoprotection. J. Exp. Mar. Biol. Ecol. 312, 215–233. Lamare, M. D., Lesser, M. P., Barker, M. F., Barry, T. M., and Schimanski, K. B. (2004). Variation in sunscreen compounds (mycosporine-like amino acids) for marine species along a gradient of ultraviolet radiation transmission within Doubtful Sound, New Zealand. N. Z. J. Mar. Freshwater Res. 38, 775–793. Lamare, M. D., Barker, M. F., Lesser, M. P., and Marshall, C. (2006). DNA photorepair in echinoid embryos: effects of temperature on repair rate in Antarctic and nonAntarctic species. J. Exp. Biol. 209, 5017–5028. Lamare, M. D., Barker, M. F., and Lesser, M. P. (2007). In situ rates of DNA damage and abnormal development in Antarctic and non-Antarctic sea urchin embryos. Aquat. Biol. 1, 21–32. Lees, D. C., and Carter, G. A. (1972). Covering response to surge, sunlight, and ultraviolet-light in Lytechinus anamesus (Echinoidea). Ecology 53, 1127–1133.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

185

Lesser, M. P. (2006). Oxidative stress in marine environments: biochemistry and physiological ecology. Ann. Rev. Physiol. 68, 253–278. Lesser, M. P. (2010). Depth-dependent effects of ultraviolet radiation on survivorship, oxidative stress and DNA damage in sea urchin (Strongylocentrotus droebachiensis) embryos from the Gulf of Maine. Photochem. Photobiol. 86, 382–388. Lesser, M. P., and Barry, T. M. (2003). Survivorship, development, and DNA damage in echinoderm embryos and larvae exposed to ultraviolet radiation (290–400 nm). J. Exp. Mar. Biol. Ecol. 292, 75–91. Lesser, M. P., Barry, T. M., Lamare, M. D., and Barker, M. F. (2006). Biological weighting functions for DNA damage in sea urchin embryos exposed to ultraviolet radiation. J. Exp. Mar. Biol. Ecol. 328, 10–21. Lesser, M. P., Kruse, V. A., and Barry, T. M. (2003). Exposure to ultraviolet radiation causes apoptosis in developing sea urchin embryos. J. Exp. Biol. 206, 4097–4103. Lesser, M. P., Lamare, M. D., and Barker, M. F. (2004). Transmission of ultraviolet radiation through the Antarctic annual sea ice and its biological effects on sea urchin embryos. Limnol. Oceanogr. 49, 1957–1963. Lister, K. N., Lamare, M. D., and Burritt, D. J. (2010a). Sea ice protects the embryos of the Antarctic sea urchin Sterechinus neumayeri from oxidative damage due to naturally enhanced levels of UV-B radiation. J. Exp. Biol. 213, 1967–1975. Lister, K. N., Lamare, M. D., and Burritt, D. J. (2010b). Oxidative damage in response to natural levels of UV-B radiation in larvae of the tropical sea urchinTripneustes gratilla. Photochem. Photobiol. 86, 1091–1098. Lu, X., and Wu, R. (2005a). UV induces reactive oxygen species, damages sperm, and impairs fertilisation in the sea urchin Anthocidaris crassispina. Mar. Biol. 148, 51–57. Lu, X. Y., and Wu, R. S. S. (2005b). Ultraviolet damages sperm mitochondrial function and membrane integrity in the sea urchin Anthocidaris crassispina. Ecotoxicol. Environ. Saf. 61, 53–59. Manzo, S., Buono, S., and Cremisini, C. (2010). Cadmium, lead and their mixtures with copper: Paracentrotuslividus embryotoxicity assessment, prediction, and offspring quality evaluation. Ecotoxicology 19, 1209–1223. Marshak, A. (1949). Recovery from ultraviolet-induced delay in cleavage of Arbacia eggs by irradiation with visible light. Biol. Bull. 97, 315. Mathew-Roth, M. M. (1997). Carotenoids and photoprotection. Photochem. Photobiol. 65, 148–151. Matranga, V., Pinsino, A., Celi, M., Di Bella, G., and Natoli, A. (2006). Impacts of UV-B radiation on short-term cultures of sea urchin coelomocytes. Mar. Biol. 149, 25–34. Matsuno, T., and Tsushima, M. (2001). Carotenoids in sea urchins. In ‘‘Edible Sea Urchins: Biology and Ecology’’ (J. M. Lawrence ed.), Elsevier Science, Amsterdam, The Netherlands. McClintock, J. B., and Karentz, D. (1997). Mycosporine-like amino acids in 38 species of subtidal marine organisms from McMurdo Sound, Antarctica. Antarctic Sci. 9, 392–398. Miller, B. A., and Emlet, R. B. (1997). Influence of nearshore hydrodynamics on larval abundance and settlement of sea urchins Strongylocentrotus franciscanus and S.purpuratus in the Oregon upwelling zone. Mar. Ecol. Prog. Ser. 148, 83–94. Millott, N. (1950). Integumentary pigmentation and the coelomic fluid of Thyonebriareus (Lesueur). Biol. Bull. 99, 343–344. Millott, N. (1953). Observations on the skin pigment and amoebocytes, and the occurrence of phenolases in the coelomic fluid of Holothuria forskali Delle Chiaje. J. Mar. Biol. Assoc. U.K. 31, 529–539. Millott, N. (1956). The covering reaction of sea urchins. 1. A preliminary account of covering in the tropical echinoid Lytechinus variegatus (Lamarck) and its relation to light. J. Exp. Biol. 33, 508–523.

186

Miles Lamare et al.

Monaghan, P., Metcalfe, N. B., and Torres, R. (2009). Oxidative stress as a mediator of life history trade-offs: mechanisms, measurements and interpretation. Ecol. Lett. 12, 75–92. Nahon, S., Charles, F., and Pruski, A. M. (2008). Improved comet assay for the assessment of UV genotoxicity in Mediterranean sea urchin eggs. Environ. Mol. Mutagen. 49, 351–359. Nahon, S., Castro Porras, V. A., Pruski, A. M., and Charles, F. (2009). Sensitivity to UV radiation in early life stages of the Mediterranean sea urchin Sphaerechinus granularis (Lamarck). Sci.Total Environ. 407, 1892–1900. Pagano, G., Bonassi, S., De Biase, A. D., Degan, P., Deeva, I. B., Doronin, Y. K., Iaccarino, M., Oral, R., Warnau, M., and Korkina, L. G. (1997). L-Methionine induces stagedependent changes of differentiation and oxidative activity in sea urchin embryogenesis. Pharmacol.Toxicol. 81, 134–143. Paine, R. T. (1974). Intertidal community structure: experimental studies on the relationship between a dominant competitor and its principal predator. Oecologia 15, 93–120. Pedrotti, M. L. (1990). Etude des processus biologiques et facteurs physiques responsables de la dispersion et du recrutement des larves meroplanctoniques. Modele: les larves d’Echinodermes. These de doctorat de l’Universite Pierre et Marie Curie en Sciences de la Vie, Paris. Pennington, J. T., and Emlet, R. B. (1986). Ontogenetic and diel vertical migration of a planktonic echinoid larva, Dendrasterexcentricus (Eschscholtz): occurrence, causes, and probable consequences. J. Exp. Mar. Biol. Ecol. 104, 69–95. Petzelt, C. (1974). UV-irradiation of sea urchin eggs changes the course of the Ca2+-activated ATPase activity. Experimental Cell Research 86, 404–408. Pruski, A. M., Nahon, S., Escande, M. -L., and Charles, F. (2009). Ultraviolet radiation induces structural and chromatin damage in Mediterranean sea-urchin spermatozoa. Mutat. Res. Genet.Toxicol. Environ. Mutagen. 673, 67–73. Raeka-Kudla, M.L., O’Connell, D.S., Regan, J.D., Wicklund, R.I. (1993). Effects of temperature and UV-B on different components of coral reef communities from the Bahamas. In R.N. Ginsburg, Ed. Proceedings, Colloquium on Global Aspects of Coral Reefs: Health, Hazards, and History, Miami, FL, USA, June 10–11, 1993, pp. 126–131. Rastogi, R. P., Richa., Singh, S. P., Hader, D. P., and Sinha, R. P. (2010). Mycosporinelike amino acids profile and their activity under PAR and UVR in a hot-spring cyanobacterium Scytonemasp HKAR-3. Aust. J. Bot. 58, 286–293. Reich, A., Schwudke, D., Meurer, M., Lehmann, B., and Shevchenko, A. (2010). Lipidome of narrow-band ultraviolet B irradiated keratinocytes shows apoptotic hallmarks. Experimental Dermatology 19, E103–E110. Reinheckel, T., Bohne, M., Halangk, W., Augustin, W., and Gollnick, H. (1999). Evaluation of UVA-mediated oxidative damage to proteins and lipids in extracorporeal photoimmunotherapy. Photochem. Photobiol. 69, 566–570. Rumrill, S. S., Pennington, J. T., and Chia, F. S. (1985). Differential susceptibility of marine invertebrate larvae: laboratory predation of sand dollar, Dendraster excentricus (Eschscholtz), embryos and larvae by zoeae of the Red Crab, Cancerproductus Randall. J. Exp. Mar. Biol. Ecol. 90, 193–208. Russo, R., Zito, F., Costa, C., Bonaventura, R., and Matranga, V. (2010). Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B. Cell Stress Chaperones 15, 1–9. Rustad, R. C. (1959). Consequences of unilateral ultraviolet radiation of sea urchin eggs. Biol. Bull. 116, 294–303.

Ultraviolet Radiation and Echinoderms: Past, Present and Future Perspectives

187

Rustad, R. C. (1971). Radiation responses during the mitotic cycle of the sea urchin egg. In ‘‘Developmental Aspects of the Cell Cycle’’ (I. Cameron., G. Padilla, and A. Zimmerman, eds.), pp. 127–159. Academic Press, New York. Schr€ oder, H. C., Di Bella, G., Janipour, N., M€ uller, W. E. G., Bonaventura, R., Russo, R., and Matranga, V. (2005). DNA damage and developmental defects after exposure to UVand heavy metals in sea urchin cells and embryos compared to other invertebrates. In ‘‘Echinodermata’’ (W. E. G. M€ uller., P. Jeanteur., Y. Kuchino., A. MacieiraCoelho, and R. E. Rhoads, eds.), pp. 111–137. Springer, Berlin, Heidelberg. Sewell, M. A., and Watson, J. C. (1993). A source for asteroid larvae–recruitment of Pisaster ochraceus, Pycnopodia helianthoides and Dermasterias imbricata in Nootka Sound, British-Columbia. Mar. Biol. 117, 387–398. Sharp, D. T., and Gray, I. E. (1962). Studies on factors affecting local-distribution of two sea-urchins, Arabacia punctulata and Lytechinus variegatus. Ecology 43, 309–313. Shick, J. M., and Dunlap, W. C. (2002). Mycosporine-like amino acids and related gadusols: biosynthesis, accumulation, and UV-protective functions in aquatic organisms. Annu. Rev. Physiol. 64, 223–262. Shick, J. M., Dunlap, W. C., Chalker, B. E., Banaszak, A. T., and Rosenzweig, T. K. (1992). Survey of ultraviolet radiation-absorbing mycosporine-like amino-acids in organs of coral-reef holothuroids. Mar. Ecol. Prog. Ser. 90, 139–148. Sigg, J. E., Lloyd-Knight, K. M., and Boal, J. G. (2007). UV radiation influences covering behaviour in the urchin Lytechinus variegatus. J. Mar. Biol. Assoc. U.K. 87, 1257–1261. Sinha, R. P., Klisch, M., Groniger, A., and Hader, D. P. (1998). Ultraviolet-absorbing/ screening substances in cyanobacteria, phytoplankton and macroalgae. J. Photochem. Photobiol. B Biol. 47, 83–94. Smith, J. L., Burritt, D. J., and Bannister, P. (2000). Shoot dry weight, chlorophyll and UVB-absorbing compounds as indicators of plants’ sensitivity to UV-B radiation. Annals of Botany 86, 1057–1063. Steevens, J. A., Slattery, M., Schlenk, D., Aryl, A., and Benson, W. H. (1999). Effects of ultraviolet-B light and polyaromatic hydrocarbon exposure on sea urchin development and bacterial bioluminescence. Mar. Environ. Res. 48, 439–457. Tedetti, M., and Sempere, R. (2006). Penetration of ultraviolet radiation in the marine environment. A review. Photochem. Photobiol. 82, 389–397. Todgham, A. E., and Hofmann, G. E. (2009). Transcriptomic response of sea urchin larvae Strongylocentrotus purpuratus to CO2-driven seawater acidification. J. Exp. Biol. 212, 2579–2594. Verling, E., Crook, A. C., and Barnes, D. K. A. (2002). Covering behaviour in Paracentrotuslividus: is light important? Mar. Biol. 140, 391–396. Vevers, G. (1963). Pigmentation of the echinoderms. Proc.16thInt.Cong.Zool. 3, 120–129. Weber, W., and Dambach, M. (1974). Light-sensitivity of isolated pigment cells of the sea urchin Centrostephanus longispinus. CellTissue Res. 148, 437–440. Wells, P. H., and Giese, A. C. (1950). Photoreactivation of ultraviolet light injury in gametes of the sea urchin Strongylocentrotus purpuratus. Biol. Bull. 99, 163–172. Whalley, T., Terasaki, M., Cho, M. S., and Vogel, S. S. (1995). Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs. J. Cell Biol. 131, 1183–1192.

[(Plate_1)TD$FIG]Plate 1

Phylogenetic tree of the p53 superfamily. Sequences corresponding to the conserved DNA binding domain were used to generate an Unrooted Phylogenetic Tree using the Splits Tree 4 program (Huson and Bryant, 2006). Abbreviations used are Ag, Anopheles gambiae; Ap, Acyrthosiphon pisum; Bf, Branchiostoma floridae (Amphioxus); Cbr, Caenorhabditis brenneri; Cbri, Caenorhabditis briggsae; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Co, Capsaspora owczarzaki; Cp, Capitella sp.; Cq, Culex quinquefasciatus; Cre, Caenorahbditis remanei; Cs, Ciona savignyi; Dp, Daphnia pulex; Dm, Drosophila melanogaster; Dr, Danio rerio (Zebra fish); Hr, Helobdella robusta; Hs, Homo sapiens; Lf, L. forbesi; Lg, Lottia gigantea; Ma, M. arenaria; Mb, Monosiga brevicollis; Me, M. edulis; Nav, Nasonia vitripenes; Nv, N. vectensis; Ph, Pediculus humanus; Pp, Pristionchus pacificus (Elephant shark); Leuconraja erinacea (Little skate); Sp, Strongylocentrotus purpuratus; Ta, Trichoplax sp.; Tc, Tribolium castaneum; Tp, Trichonella sp.; TET, tetramerization domain; SAM, sterile alpha motif; TS, tetramerization domain and SAM domains. Reprinted from Rutkowski et al. (2010) with permission from CSH Press.

[(Plate_1)TD$FIG]

Plate 2 (a) Genomic structure of p53/63/73 in M. arenaria. Boxes represent exons and V indicates intron regions (not drawn to scale). The 50 -UTR is grey, the core sequence for both clam p53 and p63/73 proteins is green, the p53 extension is blue and the p63/73 extension is red. The yellow arrow denotes the location of a predicted DN isoform; (b) amino acid sequences for clam p53 and p63/73 with intron/exon boundaries marked (!, clam p53; , human p53). Functional domains are indicated by horizontal lines above the sequence. Conserved amino acids implicated in Mdm2 binding are underlined. Sequence highlighted in yellow denotes the predicted DN isoform. Genomic DNA was purified from flash-frozen gill tissue of M. arenaria as previously described (Barker et al., 1997). DNA template was prepared using the Universal Genome Walker Kit according to the manufacturer’s instructions (Clontech). Clam sequence-specific PCR primers were designed based on the clam p53 and p63/73 coding sequences (GenBank Accession numbers AF253323 and AF253324, respectively) and used in conjunction with adapter primers (Clontech). Amplified products were cloned into the pCRII vector (Invitrogen) and sequenced in both directions at the University of Maine DNA Sequencing Facility. Contiguous sequences were assembled using Sequencher version 4.1.2 software (Gene Codes Corp., Ann Arbor, MI) and the open reading frames identified; sequence for genomic DNA was deposited under GenBank Accession number FJ041332 and (c) sequence for M. edulis DNp63/73 (DQ865151.1) was aligned to FJ041332 using Spidey (http:// www.ncbi.nlm.nih.gov/spidey/) to elucidate a sequence and intron/exon boundaries for a predicted DN isoform in M. arenaria, and was found to be 87% identical to M. edulis at the nucleic acid level and 100% at the protein level and located in intron 3. ~

[(Plate_1)TD$FIG]

Plate 3 Structural analysis of M. arenaria p53 shows conservation of a putative Mdm2 binding site. Representation of a portion of the human Mdm2 protein (ribbon structure) bound to the minimal binding site of the human p53 protein (stick structure). Modified from Kussie et al. (1996) using RasMol software. Four residues critical for hydrophobic binding interactions (F19, L22, W23 and L26, shown in green) are conserved between human p53 and clam p53. Two potentially important changes are the substitutions in M. arenaria p53 of glutamic acid and tyrosine, for S20 (red) and D21 (blue) in human p53, respectively (Holbrook et al., 2009).

]GIF$DT)1e_talP([

Plate 4 Clam haemocyte cancer: (a) interference image of freshly collected cancerous clam haemocytes; notice dumb-bell shaped haemocyte involved in cytokinesis near the centre; (b) Cytospin of CCH stained with oil red O; notice mitotic division figure on the left of the image; (c) Quantum-dot immunocytochemical localization in cytoplasmically sequestered p53 in an individual CCH; (d) quantum-dot immunocytochemical localization in cytoplasmic mortalin in the same CCH; (e) dual quantum-dot immunocytochemical localization of cytoplasmically sequestered p53 and mortalin in the same CCH—yellow indicates colocalization of the two proteins (scale bar in (a) and (b) = 20 mm and in (c–e) = 10 mm) and (f) co-immunoprecipitation of clam p53 and mortalin in normal (NCH) and CCH. Std, protein standard; C1 and C2, first and second elute from CCH lysates; N1 and N2, first and second elude from NCH lysates; NA, first elude from gel loaded with NCH and CCH lysate mix (negative control); Q, first elude from quenched gel loaded with NCH and CCH lysate mix (negative control); Ccyt, cytoplasmic protein extract from CCH and Cnuc, nuclear protein extract from CCH.

[(Plate_1)TD$FIG]

Plate 5 Positive covariation between rates of gastrovascular transport and mitosis of stolon cells. (A) Normalized flow rate in two colonies (open or solid circles, respectively) was calculated as the maximum amplitude of lumen oscillations normalized to external stolon diameter, divided by the period (in seconds) of the oscillation cycle. (B) Mitotic index is calculated as the fraction of living cells (indicated by staining with DAPI) that are undergoing mitosis (indicated using the commercially available BrdU/anti-BrdU staining immunoassay). (C) Composite fluorescent image of a stolon showing staining by DAPI (blue; to estimate cell density in a region of stolon) and/or FITC (green; to identify mitotic cells). Image constructed from a Z-series stack of images from bottom to the top of the stolon lumen at 400 magnification. Mitotic rates were estimated in two colonies (distinguished by solid vs. open symbols) along a linear length of stolon proximally near the polyp (circles) and distally near the stolon tip (triangles). Symbols and error bars represent means 95% confidence intervals of n = 4 colonies at each sampling period.

TAXONOMIC INDEX

A Abatus shackletoni, 153 Acyrthosiphon pisum, 4 Amphiplus a⁄nis, 162 Anopheles gambiae, 4 Anthocidaris crassispina, 152–153, 156, 171 Arabidopsis, 89 Ascophyllum nodosum, 62, 66, 75 B Branchiostoma £oridae, 4, 11, 13 C Caenorahbditisremanei, 4 Caenorhabditis, 3 Caenorhabditis brenneri, 4 Caenorhabditis briggsae, 4 Caenorhabditis elegans, 4 Campanularia £exuosa, 123 Capitella spp., 4, 11, 13 Capsaspora owczarzaki, 4 Centrostephanuslongispinus, 164 Cerastoderma edule, 24 Ciona intestinalis, 4, 11, 13 Ciona savignyi, 4 Clypeaster japonicus, 152 Crassostrea gigas, 9, 13 Crassostrea virginica, 23 Cucumaria ferrari, 155, 161 Culexquinquefasciatus, 4 Cypeaster japonicus, 155 D Daniorerio, 4 Daphnia pulex, 4, 11, 13 Diadema antillarum, 164 Diadema savignyi, 159, 169 Diadema setosum, 158, 166 Diplasterias brucei, 154, 162 Drosophila, 3 Drosophila melanogaster, 3, 4, 11, 13

E Echinarachnis parma, 153 Echinarachnius parma, 157–158 Echinometra viridis, 156, 177 Echinus esculentus, 152 Eirene viridula, 121 Evechinus chloroticus, 150, 153, 158–159, 161, 168–169 F Fucus ceranoides, 58, 63 Fucusdistichus, 45–46, 48, 51, 57–60, 62, 65, 67, 75, 77, 83 Fucus evanescens, 47, 53, 84 Fucus evanescens (=F. distichus), 62, 77 Fucus gardneri, 67 Fucusradicans, 58, 61, 68, 84–85 Fucus serratus, 45, 48–50, 52–53, 55, 57–58, 62–63, 65–66, 68, 75, 77, 79–80, 82–85 Fucus spiralis, 47, 57–58, 62, 66, 68, 71 Fucus spp., 39, 41–42, 59, 61, 64, 76, 88 Fucus vesiculosus, 45–55, 57–59, 61–71, 75, 77–81, 83–88 Fucus virusoides, 46 G Gammaruslocusta, 80 Gracilaria, 63 Gracilaria vermiculophylla, 63 Granaster nutrix, 162 H Haliotistuberculata, 26 Helobdella robusta, 4 Hemicentrotus pulcherrimus, 152, 155–157, 165, 170 Holothuria atra, 155, 161 Holothuria spp., 164 Homosapiens, 4, 11 Hydra magnipapillata, 135

189

190

Taxonomic Index

Hydractinia echinata, 131 Hydractinia polyclina, 113 Hydractinia spp., 113, 120, 133 Hydractinia symbiolongicarpus, 110, 113, 119–120, 122, 124 I Idotea baltica, 63, 78–79 L Laminaria saccharina, 163 Leuconraja erinacea, 4 Loligo forbesi, 4 Loligoforbesi, 9 Lottia gigantea, 4, 13 Lytechinus variegatus, 151, 156, 179 M Macoma baltica, 22 Marthasterias glacialis, 154 Mastocarpus stellatus, 163 Meloidogyne arenaria, 9–12, 14, 17–23 Mercenaria spp., 19 Monosiga brevicollis, 4, 11, 13 Mya arenaria, 4–5, 8, 19 Mytilus edulis, 4–5, 9, 18, 22 Mytilus galloprovincialis, 26 Mytilus spp., 9, 26 Mytilus spp., 19, 21 Mytilus trossulus, 2, 9, 11, 18 N Nasonia vitripenes, 4 Nematostella vectensis, 3–4, 13 O Odontaster meridonalis, 154 Odontaster validus, 154, 162 Ophiocoma echinata, 156, 177 Ophioderma brevispinum, 156, 177 Ophionereisreticulata, 156, 177

Ophiopholis aculeata, 151, 167 P Paracentrotus lividus, 151–154, 156–157, 166, 173–174 Pediculus humanus, 4 Pelvetia canaliculata, 62 Perknaster fuscus, 154 Pisaster ochraceus, 151, 166–167, 178 Pisaster spp., 164 Podocorynecarnea, 113, 118, 120–121, 123–124, 126–127, 135, 138 Pristionchus paci¢cus, 4 Promachocrinus kerguelensis, 155, 162 S Sphaerechinus granularis, 152–154 Spisula solidissima, 9 Sterechinus neumayeri, 148, 150, 152–154, 158– 161, 165, 168–169, 172–173, 176, 178 Stichopus californicus, 164 Stichopus japonicus, 155 Strongylocentrotus droebachiensis, 150–154, 158– 161, 163–164, 166–169, 171, 173 Strongylocentrotus intermedius, 156 Strongylocentrotuspurpuratus, 4, 11, 13, 152, 155, 170, 173, 175, 179 Strongylocentrotus spp., 156 T Thelenota ananas, 150 Thyone briareus, 164 Tribolium castaneum, 4 Trichonella spp., 4 Trichoplaxadhaerens, 10, 13 Trichoplax spp., 4 Tripneustes gratilla, 154, 159, 173 Tripneustes ventricosus, 151, 167 U Uniopictorum, 22

SUBJECT INDEX

A Abiotic stress, in Fucus, 64–71 biotic modulation of, 81 detection of adaptation to direct, 84–86 indirect, 83–84 meta-analysis of, 71 protection against, 76–77 simultaneous, 69–71 single stresses, 64–69 desiccation, 67 irradiance, 64–66 osmotic stress, 68 pollution, 67–68 temperature stress, 66–67 water motion, 68–69 Aclonal organisms vs. colonial organisms., 109 gene expression in response to environment, 133 Adaptation to stress, in Fucus, 83–86 direct detection, 84–86 indirect detection, 83–84 Antioxidants, 164 Architectural vs. genotypic effects, phenotypic plasticity, 135 B Biotic interactions, in Fucus emersion effects on, 79 eutrophication effects on, 80–81 impact on abiotic stress, 81 irradiance effects on, 77–78 salinity effects on, 79 temperature effects on, 78–79 water motion effects on, 79–80 Biotic stress, in Fucus, 71–76 detection of adaptation to direct, 84–86 indirect, 83–84 meta-analysis, 75–76 protection against, 76–77

simultaneous, 75 single stresses, 71–75 competition, 73–74 epibionts, 74 herbivory, 74–75 parasitism, 75 C Carotenoids, 164 Cell cycle and stress responses genes, UVRinduced damage to, 173–174 Chemical defence, in Fucus, 76–77 Clonal organisms development of, 109–110 gene expression in response to environment, 133 as model systems for studying development, 111 morphological plasticity, 110 morphological variation in, 115 Cnidarian colonies canalization of phenotype, 133 genotypic classes in, phenotypic outcomes for, 131–133 Cnidarians gastrovascular dynamics, 116 gastrovascular system, 115–119 genetic programmes of, 131 genotypic classes in, 131–132 Colonial organisms. vs. aclonal (or unitary) organisms, 109 morphological patterning of, 108–109 Colony development as dynamical system, 131–133 vascular architecture role in, 127, 129 Competition, in Fucus, 73–74 Congeneric introduction, in Fucus, 63 Conspecific introduction, in Fucus, 63 D Defence mechanisms, in Fucus, 76–77 Desiccation stress, in Fucus, 67

191

192

Subject Index

Developmental biology and UVR-induced damage, 177 changes in eggs, embryos and larvae, 175–176 fertilisation rates, 175 sperm damage, 174 Developmental plasticity of colony morphology, 123 DNA damage, 168–170 DNA repair mechanisms, 165 E Echinoderms avoidance strategies for UVR exposure to antioxidants, 164 carotenoids, 164 DNA repair mechanisms, 165 melanin, 164 mycosporine-like amino acids, 148, 150, 161–164 negative phototaxis response, 165–167 behavioural responses to exposure to UVR, 165–168 characterization of, 146–147 history of UVR research in, 147–148 response to UVR exposure, 149, 151–162 UVR-induced damage in cell cycle and stress responses genes, 173–174 DNA damage, 168–170 effect on developmental biology, 174–177 future research in, 177–180 mortality and population level effects, 177 oxidative stress, 170–173 protein and lipid damage, 170 Emersion effects, on biotic interactions, 79 Environmental signalling of morphological pattern, 132, 133 Epibionts, in Fucus, 74 Epigenetics, 129 Epitheliomuscular cells (EMCs), 121–122 Eutrophication effects, on biotic interactions, 80–81 F Fucus spp., stress ecology of, 37–91 background and monodisciplinary research on, 41–42 chemical defence in, 76–77 climate change effects, 89–90 dispersal in, 61 diversification, 57–58 rapid ecotypic, 58

genetic levels of stress response, 81–86 detection of adaptation, 83–86 sensitivity vs. genetic diversity, 82 genomic introduction and invasion, 62–63 global distribution of, 57–58 latitudinal and zonational shifts of, 61–62 life history and demography, 58–61 meta-analysis, 42–57 calculation of effect sizes, 43–57 data acquisition, 43 data search for, 42–43 effects of stresses based on, 72 stress groupings used in, 43, 56 stress interactions study used for, 44–55 modulation of stresses, 77–81 emersion effects on biotic interactions, 79 eutrophication effects on biotic interactions, 80–81 irradiance effects on biotic interactions, 77–78 salinity effects on biotic interactions, 79 temperature effects on biotic interactions, 78–79 water motion effects on biotic interactions, 79–80 mortality in, 59–61 photoinhibition in, 65 protection against stresses, 76–77 reproduction in, 58–59 stressful environment effects, 64–81 abiotic stress, 64–71 biotic stress, 71–76 survivorship curve of, 60 zonation pattern of, 40 G Gastrovascular flow and polyp behaviour changes in polyp volume, 117 environmental conditions, 119 following ingestion and digestion, 117, 119 oscillatory behaviour, 116–117 Gastrovascular system hydrozoan colony integration by, 115–119 physiology and colony form changes in colony form, 120–121 environmental signalling, 119–120 experimental replicates, 123 hydromechanical forces, 122–123 ROS signal, 121–122 vascular architecture, 120 and polyp behaviour. See Gastrovascular flow and polyp behaviour

193

Subject Index

Gastrovascular transport, 116 and mitosis of stolon cells, covariation between, 137–138 morphogenetic events mechanisms, 121 in P. carnea, 118, 121 quantitative models for, 119 Genetic effects and their descriptions, abbreviations for, 112 Genetic population structure, in Fucus, 83 Genomic introduction, in Fucus, 62–63 Gracilaria vermiculophylla, 63 Grazing pressure, Fucus and, 74–75 H Herbivory, in Fucus, 74–75 Hybridization, in Fucus, 58 Hydractinia echinata colony development as dynamical system, 131 Hydractinia spp., vascular architecture of runner colonies of, 120 Hydractinia symbiolongicarpus gastrovascular transport among stolons, 122 morphological plasticity variation in, 124, 126 reticulate vasculature, 120 Hydractiniid hydrozoans ecological traits of interest and colony morphology, correlation between, 115 gastrovascular transport, 120 Hydra magnipapillata, 135 Hydrozoan colonies heritability of, 134 morphological plasticity, 111, 124, 135–136 morphological variants, 111 nutritional status and colony morphology, link between, 122 perturbations to redox state of, 121 principal elements of, 110 ROS signalling in morphogenetic events, 121 polyp behaviour, 122 Hydrozoan colony form and gastrovascular flow rate, co-variation between, 120–121 genetic and environmental components of, 112, 113, 115 genetic basis of, 111 clonal repeatability in Hydractinia spp., 113–115 integration by gastrovascular system, 115–119 morphological variation of, 111, 115 polyps and stolons, 111

Hydrozoans as models of vascular development, 136–138 I Idotea baltica, 63 Introgression, in Fucus, 58 Invasion, in Fucus, 62–63 Invasive species, in Fucus, 62–63 Irradiance effects, on biotic interactions, 77–78 Irradiance stress, in Fucus, 64–66 M Melanin, 164 Meta-analysis database search, 147 Meta-analysis of stress in Fucus, 42–57 of abiotic stresses, 71 calculation of effect sizes, 43–57 data acquisition, 43 data search for, 42–43 stress groupings used in, 43, 56 stress interactions study used for, 44–55 MMAs. See Mycosporine-like amino acids Morphological plasticity architectural vs. genotypic effects, 135 heritability of, 134–135 physiological mechanisms underlying, 135–136 range of, 124 runner morphology in food replete environment, 126–129 in polyp/stolon investment, 126 vascular architecture and constraints on, 124–131 Mortality and population level effects, UVR-induced damage to, 177 Multicellular clonal organisms, 108 Mycosporine-like amino acids concentrations, 148, 150 occurrence in echinoderm classes, 148 sources of, 150 as sunscreens for echinoderms, 163–164 N Nutrient-driven polyp behaviour, empirical tests of, 136 Nutritional status and colony morphology, link between, 122 O Ophiopholis aculeata, covering behaviours of, 167

194

Subject Index

Osmotic stress, in Fucus, 68 Oxidative stress ROS levels, 170–171 in sea urchin embryos, 171–173 P Paracentrotuslividus embryos, UVR effect on development of, 176 Parasitism, in Fucus, 75 Pelvetia canaliculata, 62 Photoinhibition, in Fucus, 65 Phototaxis response, negative, 165–167 Pisaster ochraceus, shade-seeking and covering behaviours of, 167 Podocoryne carnea experimental replicates of, 123 gastrovascular flow rate in, 120, 121 mechanism signalling morphogenetic events in, 121 runner morphology plasticity assessment, 126 single polyp/single stolon systems of, 124 Pollution stress, in Fucus, 67–68 Polyp behaviour characterization of, 116 and gastrovascular flow changes in polyp volume, 117 environmental conditions, 119 following ingestion and digestion, 117, 119 oscillatory behaviour, 116–117 pumping, 119 Polyp formation and stolon branching, hydromechanical forces role in, 123 Protein and lipid damage, 170 R Reactive oxygen species (ROS) signalling associated with polyp behaviour, 122 signalling morphogenetic events, 121 S Salinity effects, on biotic interactions, 79 Salinity stress, in Fucus, 68 Species introduction, in Fucus, 62–63 Stolon diameter, 120 Stolon regression, 121 Stress defined, 39–41 extrinsic components of, 40

intrinsic components of, 40–41 Strongylocentrotus droebachiensis embryos covering behaviour in, 166–167 UVR-induced cleavage delay in batches of, 163 Synergistic stress, effect on Fucus, 71 T Temperature effects, on biotic interactions, 78–79 Temperature stress, in Fucus, 66–67 metabolic rates and, 66 U Ultraviolet radiation (UVR) and marine environment, 146 research in echinoderms, history of, 147–148 Unitary organisms. See Aclonal organisms UVR-induced damage in echinoderms cell cycle and stress responses genes, 173–174 cleavage delay in batches of S. droebachiensis embryos, 163 DNA damage, 168–170 effect on developmental biology, 177 changes in eggs, embryos and larvae, 175–176 fertilisation rates, 175 sperm damage, 174 future research in at population level, 177–178 post-settlement processes, 178 synergistic effects, 179–180 UVR and life histories, 180 mortality and population level effects, 177 oxidative stress ROS levels, 170–171 in sea urchin embryos, 171–173 Protein and lipid damage, 170 V Vascular architecture definition, 127, 129 importance in colony development, 127, 129 for ontogenetic stage, 133 structural state of, 129, 131 W Water motion effects, on biotic interactions, 79–80 Water motion, in Fucus, 68–69