Bulgarian Antarctic Research: Life Science (Life Sciences)

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BULGARIAN ANTARCTIC RESEARCH ‹ VOLUME 5

LIFE SCIENCES VOLUME

5

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BULGARIAN ANTARCTIC RESEARCH Life Sciences Vol. 5

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Bulgarian Antarctic Institute

Bulgarian Academy of Sciences

BULGARIAN ANTARCTIC RESEARCH Life Sciences Volume 5

Edited by

Acad. V. Golemansky & Dr. N. Chipev

Sofia - Moscow 2006

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Bulgarian Academy of Sciences

© PENSOFT Publishers ISBN-10: 954-642-263-0 (Vol. 5) ISBN-13: 978-954-642-263-7 (Vol. 5) First published 2005 All rights reserved

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form by any means, electronic, mechanical, photo copying, recording or otherwise, without the prior written permission of the copyright owner.

Pensoft Publishers, Geo Milev str., No. 13a, 1111 Sofia, Bulgaria E-mail: [email protected], www.pensoft.net

Printed in Bulgaria, April 2006

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Contents POLYMORPHISM OF THE POPULATION IN GENTOO PENGUINS (PYGOSCELIS PAPUA AVES: SPHENISCIFORMES) FROM LIVINGSTON ISLAND, SOUTH SHETLANDS. P. Zehtindjev, R. Metcheva, A. Savov, Y. Yankov, A. Ludwig, D. Lieckfeldt ............................ 1

PARAMETERS OF CHROMOSOMAL INSTABILITY OF PYGOSCELIS PAPUA K. Afanasieva, S. Rushkovsky, V. Bezrukov ................................................................................. 9

CORRELATION BETWEEN CHROMOSOMAL INSTABILITY AND SOME MORPHOPHYSIOLOGYCAL CHARACTERISTICS OF GENTOO PENGUINS’ NESTLINGS S. Rushkovsky, K. Afanasieva, V. Bezrukov ............................................................................... 15

RAPD ANALYSIS OF GENTOO PENGUINS’ POPULATIONS G. D. Telegeev, A. S. Dranitsina, M. V. Dybkov, A. S. Savov, B.Vachev, S. S. Maliuta, V. F. Bezrukov, O. Georgiev ................................................................................................................... 23

STUDYING TELOMERES LENGTH OF GENTOO PENGUINS ON THE ANTARCTIC PENINSULA G. D. Telegeev, A. S. Dranitsina, M. V. Dybkov, E. E. Melnikova-Khablo, A. S. Savov, B.Vachev, S. S. Maliuta, V. F. Bezrukov, O. Georgiev .................................................................................. 31

BILL COLLORATION OF GENTOO (PYGOSCELIS PAPUA ELLSWORTHII) PART I: A MARKER FOR GENTOO POPULATION STUDIES R. Metcheva, P. Zehtindjiev, V. Bezrukov, A. Savov, Y. Yankov .............................................. 37

BILL COLLORATION OF GENTOO. PART II:A REVIEW OF THE POSSIBLE CAUSES K. Dimitrov, M. Beltcheva, R. Metcheva, V. Bezrukov ............................................................. 45

STUDY OF YEAST ISOLATED FROM PENGUIN PLUMAGE FROM WESTERN ANTARCTICA K. Pavlova, R. Metcheva, I. Savova, V. Bezrukov, Y.Yankov, L.Woodworth ........................ 51

A STUDY OF ANTARCTIC YEASTS FOR PROTEASE PRODUCTION K.Pavlova, A.Koleva, I.Savova, L.Koleva, I.Pishtiyski, R.Metcheva ..................................... 63

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CONTENTS

HEAVY METAL CONTENT IN GENTOO PENGUIN FEATHERS AT PETERMANN ISLAND AND LIVINGSTON ISLAND A. V. Andreev, A. A. Andreev, M. O. Kursa, R. P. Metcheva, G. P. Milinevsky, S. E. Shnyukov, V. R. Morozenko ............................................................................................................................ 75

MONITORING OF ELEMENTAL CONTENTS IN PENGUIN FEATHER USING EDXRFA E. Nikolova, S. Aleksandrova ...................................................................................................... 83

A MULTICRITERIA APPROACH TO THE ESTIMATION OF THE SURVIVAL OF PENGUINS B. Vachev, R. Metcheva ................................................................................................................... 93

CHROMOSOMAL INSTABILITY OF WINTERERS BEFORE AND AFTER ANTARCTIC EXPEDITIONS K. Afanasieva, S. Rushkovsky, V. Bezrukov ............................................................................. 101

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Introduction The Republic of Bulgaria acceded to the Antarctic Treaty in 1978. In 1992, the first biological project started, dealing with the biodiversity and ecology of living organisms on Livingston Island (South Shetland Islands). The National Fund of the Ministry of Education and Science has supported these Bulgarian activities so far and we are grateful to their understanding and funding. Later the Bulgarian biological and ecological investigations were enhanced and developed, and some of them became funded by the European Community as well. A good example is the international INTAS-funded Research Project Comparative Population and Monitoring Research on Gentoo Penguins (Pygoscelis papua) in Antarctica (2001 –2005). Scientists from Bulgaria, Ukraine and Switzerland take part in this project. Within the framework of the project, the Second International INTAS Workshop was organized in Sofia between 08 July and 11 July, 2005. (The first one took place in Kiev in June 2001). This Workshop was convened to give new impulses to the development of the study of one the most interesting and poorly documented Antarctic animals – the Gentoo penguin (Pygoscelis papua). The present Volume 5 of Bulgarian Antarctic Research contains the results reported at the workshop sessions. We hope that these results constitute new contributions to the study of the live history and the ecology of Antarctica and they will be of interest to all Antarctic researchers. All the participants and the editors take the opportunity to thank INTAS, the National Fund for Scientific Investigations, the Bulgarian Antarctic Institute and the Institute of Zoology at the Bulgarian Academy of Sciences, for their attendance, support and for their encouragement before and during the Workshop. The editors and the authors express their gratitude to Pensoft Publishing House for the excellent quality of the book. Sofia, November, 2005

The Editors

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© PENSOFT Publishers Bulgarian Antarctic Research ... POLYMORPHISM OF THE POPULATION IN GENTOO PENGUINS 1 Sofia – Moscow Life Sciences, vol. 5: 1-8, 2006

Polymorphism of the population in Gentoo penguins (Pygoscelis papua Aves: Sphenisciformes) from Livingston Island, South Shetlands. PAVEL ZEHTINDJEV1, ROUMIANA METCHEVA1, ALEXEY SAVOV2, YORDAN YANKOV3, ARNE LUDWIG4, DIETMAR LIECKFELDT4 1 2

Institute of Zoology, Bulgarian Academy of Sciences, 1000 Sofia, Bulgaria

Laboratory of Molecular Pathology, the Medical University Sofia, 2 Zdrave Street, 1431, Sofia, Bulgaria 3

The Bulgarian Antarctic Institute, 15 1 Tzar Osvoboditel Boul., 1504 Sofia, Bulgaria 4

Institute for ZOO and Wildlife Research, Berlin, Germany

ABSTRACT The levels of genotypic variability in the mitochondrial DNA (D-loop) of Gentoo penguins within and between colonies were studied at Livingston Island (the South Shetland Islands) 62°38’29’’ S and 60°24’53’’ W. Approximately 1000 ml of blood was drawn from the cubital vein using a syringe and placed into K3 EDTA. All the penguins examined were marked using glass-encapsulated TROVAN identification electronic transponders. DNA was extracted using a standard phenol-chloroform method. Results of 30 samples from Livingstone Island have been compared with data from Nelson Island and Falklands. Low polymorphism of the mitochondrial genome has been found in both colonies. Regular gene flow and the low number of breeding birds support the temporal status of the local colony. The high exchange rate resulting in a regular gene flow allow us to consider that there exists a large population in the studied territory that includes these two colonies and probably the colonies on the whole territory of South Shetland Islands. KEY WORDS Gentoo penguins, DNA sequence, D-loop, Livingston Island

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INTRODUCTION This study was stimulated by the ever-increasing interest to penguins, which are an important part of the Antarctic ecosystem. At present their biology is one of the hottest fields in Antarctic investigations of the international community. Genetic differentiation, speciation and the understanding of the biodiversity creation process in Gentoo penguins (Pygoscelis papua), which is one of the four penguin species breeding in Antarctica were investigated. Despite the rich literature on penguins, data on many aspects of their biology and ecology remain insufficiently studied. Gentoo (Pygoscelis papua) is one of the most poorly documented species. Gentoo penguins breed on subantarctic islands and on the Antarctic Peninsula. There are two subspecies of Gentoo: P. papua papua (J.R. Forster, 1781) and P. papua ellsworthi (Murphy, 1947). The first subspecies inhabit the sub-Antarctic area south up to 60° of South Latitude. The second subspecies lives in the Antarctic region – South Sandwich Island and the Antarctic Peninsula (Martinez I., Ed: del Hoyo et al. 1992). The scattered populations show considerable variation on weight and measurements (Stonehouse, 1970). Nowadays Gentoo attract the close attention of Antarctic scientists. Gentoo is one of the species-indicator for the evaluation of the impact of global environmental changes in the Antarctic ecosystem (Croxall & Williams 1991, Le Maho et al. 1993), the estimation of the tourist impact on this ecosystem (Coblley & Shears, 1999, Erwin, 1989), etc. Very important aspects of their study are the structure of local populations and the relations among them. The most common and generally used parameters for describing the population structure are age and sex distributions, the gene pool, and the level of variation. The study of population gene pools is possible with different kinds of genetic markers. Each kind of marker has its own advantages and disadvantages. For the study of the biology of wild-living vertebrates, several genetic techniques are widely used (mtDNA, microsatellites, RAPD, AFLP). Most of them are able to find genetic variability, and in some cases to obtain markers of populations (Wenink & Baker 1996, Wennerberg 2001). Genetic methods are more precise if they allow to identify particular populations. For the determination of the most probable origin of the population found on migration, the so-called assignment tests can be used (Waser & Strobeck 1998). Mitochondrial DNA has a higher mutation rate than other parts of the genome and is strongly influenced by the genetic drift (Quinn & Wilson 1993, Avise 1994, Ingman et al. 2000). This predetermines it as a useful tool for following recent divergent events on the intra-specific level (Baker & Marshall 1997). Genetic markers allowed a deep insight into the gene flow in a population and the reconstruction of the relationship between parental and offspring generations.

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The present study is part of an international project (INTAS grant Ref. # 2001 0517) which is a complex investigation of Gentoo penguins on its traditional territories in Antarctica. The key scientific questions in the biology of Gentoo penguins were addressed through an interdisciplinary approach by the development and refinement of hypotheses, field sampling and data collection, application of molecular methods, data analysis and hypothesis testing. The only genetic study on Gentoo available, from two colonies at Crozet and Kerguelen islands, revealed unexpected differentiation, pointing to the isolation between the colonies (Viot, 1987). Thus, the key questions addressed were the analysis of genetic variability and the genetic structure of the populations, the level of genetic diversity, adaptation to the Antarctic environment, and the changes of the genetic structure of the population under an ecological impact. The main objectives of this study were to evaluate the level of genotypic variability of Gentoo penguins within and between colonies by DNA markers. We predicted to find a high exchange rate of the genes between the colonies studied in the large territory. This prediction is based on the data from individual marking and the long term monitoring of the colony at Livingstone Island suggesting low philopatry and high dispersion rate of the juveniles (own unpublished data). We predicted that the small colonies like the studied one at Livingstone Island are temporary and the future development of such colonies will highly depend on the breeding success of the bigger colonies in the region. MATERIAL AND METHODS Blood samples were collected from adult, non-moulting Gentoo penguins (Pygoscelis papua) inhabiting Livingston Island (South Shetland Islands) 62º38´29´´ S and 60º24´53´´ W during three Antarctic summer seasons over the period December – February (2001 – 2003). Blood samples collected from 40 birds are used in the present study. Approximately 1000 ml of blood was drawn from the cubital vein using a syringe and placed into K3 EDTA CCAMLR (2003). All the penguins examined were individually marked using glass-encapsulated TROVAN identification electronic transponders. Subcutaneously implanted (Clarke and Kerry 1998) transponders have demonstrated to be reliable means of identifying individual penguins. Transponders were injected in the back of the neck or between the shoulder blades. DNA was extracted using a standard phenol-chloroform method, as described by Sambrook et al. (1989). DNA samples were re-suspended in 10mM Tris, 1 mM EDTA pH 8.0 and kept at minus 20° C.

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RESULTS AND DISCUSSION The phylogenetic relationships of the Livingstone Island Gentoo colony presented here is likely to be robust because the study incorporated data from a small number of individuals identifying a close out-group consisting genes from all available sequences in the GenBank (Figure 1).

Fig. 1. A phylogenetic tree using the DNA sequence data of the D-loop of 40 Pygoscelis papua from Livingstone Island and 28 specimens from the GenBank. Abriviations: P, PENG – penguins from Livingstone Island; Nelson – birds from Nelson Island; Falkland – birds from the Falkland Islands; Ppp – Pygoscelis papua papua; Ppe - Pygoscelis papua ellsworthi

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First, the Livingstone colony is currently paraphyletic because of the presence of other genotypes within the clade of the colony studied. We described genotypes close to those from Nelson Island (Figure 2) and Falkland Islands colonies, already published in GenBank. Second, several life histories and morphological traits are informative for comparisons among colonies. A low level of polymorphism has already been demonstrated in bill, flipper and body weight within the colonies of Gentoo (Metcheva et al. 2005). Thus, life-history traits and morphological characters in the colony studied must evolve rapidly, and similar traits can be convergent. Third, the major clade coincides with the group of Livingstone Island colony, smallest within the breeding groups sampled in this study. These results underscore the pressing need to sample and include a far greater proportion of the genotypes of this species to uncover the more complete evolutionary history in Gentoo in the large scale of sub-Antarctica. However, to explain the present results and to check our prediction we analyzed the gene flow in the colony at Livingstone Island. The nearest big Gentoo colony is one located at Nelson at a distance of 70 kilometers (Figure 2). The number of breeding Gentoo in this colony is relatively high. The number of Gentoo at Nelson Island remains constantly around 3500 pairs (Silva, 1998). The genotypes of this big

Fig. 2. Location of the Nelson Island population

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colony are of the same proportion as genotypes from the Falkland Islands located more than 1000 km northward. Probably the exchange of genes with these two big colonies takes place in different periods of the annual cycle. It can happen during the wintering period when the most southern colonies move to the north and winter probably together, or it can be a result of dispersion. There is a high number of nonbreeding sub-adult birds appearing at Livingstone Island every year (not published own data) and consisting probably of birds from different colonies. This model is supported by the mitochondrial DNA data in our study (Figure 3). It supposes a onedirection gene drift, from a pool of a bigger colony to the smaller, temporary and peripheral colonies like the one studied at Livingstone Island. Alternatively, there exists an option of a sample effect in the data analyzed. The small sample size and particularly the low number of samples presenting genotypes of big colonies from Nelson and Falkland Islands, where thousands of Gentoo breed annually, can skip the specific genotype of a small group of Livingstone’s birds. As a result we can simply not have a chance to describe this small proportion of genotypes in a sample of published in GenBank sequences, but this possibility cannot be excluded either. However, the high exchange rates resulting in a regular gene flow allow us to consider the existence of one large population in the territory studied, including these two colonies and probably the whole territory of the South Shetland Islands.

Fig 3. Scheme of the gene flow based on the philogenetic tree of the DNA sequence data (Dloop). Black circles: Nelson Island hyplotypes; grey circles – Falkland Island hyplotypes; Transparent circles: Livingston Island hyplotypes

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ACKNOWLEDGEMENT This work was funded through grant INTAS Res. Project, Ref. # 2001 - 0517 REFERENCES AVISE J. C. 1994: Molecular markers, natural history and evolution. Chapman & Hall, London. BAKER A. J. & MARSHALL H. D. 1997: Mitochondrial control region sequences as tools for understanding evolution. In: Mindell D. P. (ed.): Avian Molecular Evolution and Systematics. Academic Press, London: 51–82. CCAMLR. 2003. Standard methods for monitoring studies. Convention for the conservation of Antarctic marine living resources, Hobart, Tasmania, Australia CLARKE J., K. KERRY. 1998. Implanted transponders in penguins: Implantation, reliability, and long-term effects. J.Field Ornithology, 69 (2): 149 – 159. COBLEY N. D., J. R. SHEARS. 1999. Breeding performance of Gentoo penguins (Pygoscelis papua) at a colony exposed to high levels of human disturbance. Polar biology, 21: 335 – 360. CROXALL J.P., T.D. WILLIAMS 1991. The Gentoo penguin as a candidate species for the CCAMLR ecosystem monitoring program. INGMAN M., KAESSMANN H., PÄÄBO S. & GYLLENSTEN U. 2000: Mitochondrial genome variation and the origin of modern humans. Nature 408: 708–713. METCHEVA, R., P.ZEHTINDJIEV, A. SAVOV, Y.YANKOV, I.SIRENKO, I. KOZERETSKA, S.PAVLOVICH, V.BEZRUKOV. 2005. Sex determination and sex-related morphological and esterase variation in Gentoo penguins from Livingston and Petermann Islands. Proceed.II Int. INTAS Workshop, Sofia. QUINN T. W. & WILSON A. C. 1993: Sequence evolution in and around the mitochondrial control region in birds. J. Mol. Evol. 37: 296–310. VIOT, C.R. 1987. Differenciation et isolement entre populations chez le Manchot royal (Aptenodytes patagonicus) at le Manchot papou (Pygoscelis papua) des iles Crozet et Karguelen. L’Oiseau et R.F.O., v. 57, (3), 251 – 259. WASER P. M. & STROBECK C. 1998: Genetic signatures of interpopulation dispersal. Trends Ecol. Evol. 13: 43–44. WENINK P. W. & BAKER A. J. 1996: Mitochondrial DNA lineages in composite flocks of migratory and wintering Dunlins (Calidris alpina). Auk 113: 744–756. WENNERBERG L. 2001: Breeding origin and migration pattern of Dunlin (Calidris alpina) revealed by mitochondrial DNA analysis. Mol. Ecol. 10: 1111–1120. CHRISTOPHER-ROBIN VIOT 1987 Identification of the sex of a wide range of Carinatae birds by PCR using primer set selected from chicken EE0.6 and its related sequences. The journal of Heredity. 92 (4).

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ERWIN, R.M. 1989. Responses to human intrusion by birds nesting in colonies: experimental results and management guidelines. Colonial Waterbirds 12: 104 – 108. LE MAHO J. P.GENDER, E. CHALLET, C.A. BOST, J. GILLES, C.VERDON, C. PLUMERE, C. ROBIN, Y. HANDRICH.1993. Undisturbed breeding penguins as indicators of changes in marine resources. Marine Ecology Progress Ser. 95:1-6. MARTINEZ I., 1992. Handbook of the birds of the world1del Ed: Hoyo, J., Elliott, A. R., SARGATAL, J., Lynx Editions., ICBP, Barcelona, vol. SAMBROOK J., E. F. FRITSCH, T. MANIATIS. 1989. Molecular cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. STONEHOUSE B. 1970. Geographic variation in Gentoo penguins. Ibis 112: 52 – 57. SILVA, M.P., FAVERO, M., CASAUX, R. & BARONI, A. 1998. The status of breeding birds at Harmony Point, Nelson Island, Antarctica in summer 1995/96. Marine Ornithology 26: 75–78.

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© PENSOFT Publishers Bulgarian PAPUA Antarctic Research9 PARAMETERS OF CHROMOSOMAL INSTABILITY OF PYGOSCELIS Sofia – Moscow Life Sciences, vol. 5: 9-13, 2006

Parameters of Chromosomal Instability of Pygoscelis Papua KATERYNA AFANASIEVA, STANISLAV RUSHKOVSKY, VLADIMIR BEZRUKOV Taras Shevchenko National University of Kyiv 64 Volodymyrska Street, Kyiv, 01033, Ukraine ([email protected])

ABSTRACT The analysis of parameters of chromosomal instability of Pygoscelis papua was carried out. The rates of micronuclei (MN) and other nuclear anomalies (NA) in mature peripheral blood erythrocytes were selected as main end-points of chromosomal instability. The most frequently registered NA were classified as ”budding” nucleus (BN), ”tailed” nucleus (TN), two-lobe nucleus (TLN) and nucleus with cavity (NC). Micronuclei are widely accepted parameters of chromosomal instability, NA we propose as additional end-points to characterize chromosomal instability. The possible mechanisms of formations and applications of studied parameters of Pygoscelis papua are discussed. KEY WORDS Pygoscelis papua, chromosomal instability, micronuclei, nuclear anomalies

INTRODUCTION The main purpose of genetic monitoring is the estimation of the influence of environmental conditions. Such investigations are necessary for the native Antarctic species because of the global climate changes. Pygoscelis papua is one of the most conservative species that has lived in stationary environmental conditions for millions of years. Thus Gentoo penguins may be a marker species for studying the influence of the Antarctic environmental alteration on the genome. The in vivo micronucleus (MN) test in peripheral blood erythrocytes is a universally recognized method for birds’ genetic monitoring (ZUNIGA, 1996). This assay detects the effect of mutagenic agents on chromosomes by the identification of acentric fragments and legging chromosomes. The MN test is a simple and precise method for detecting induced and spontaneous chromosomal instability.

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It is known that the karyotype of birds’ is extremely stable in different conditions. The frequency of micronuclei (MNi) is very low for most bird species (ZUNIGAGONZALES, 2001). Consequently, it is necessary to detect some additional parameters of chromosomal instability and to estimate their initial level. This article deals with the detection and classification of parameters of chromosomal instability, useful for the genetic monitoring of Gentoo penguins. MATERIALS AND METHODS. We obtained 70 peripheral blood samples from adult Gentoo penguins and 112 peripheral blood samples from nestlings. The blood samples of birds were collected at Piterman island during the austral summer of 2002. Blood was obtained by incision of the pounce of the leg’s 4-th rudimentary finger. Smears were made on precleaned microscope slides, fixed in 96% ethanol for 30 min and stained with a combination of Leishman and Giemsa stains. The frequencies of MNi and nuclear anomalies (NA) were manually. scored in mature erythrocytes through an immersion objective lens. Studied parameters were counted per 10.000 mature erythrocytes for each individual. RESULTS AND DISCUSSION. The average rates of MNi were 0.03±0.01 ‰ for the adult penguins and 0.11±0.01 ‰ for the nestlings (fig. 1). Micronuclei originate from chromosome fragments or whole chromosomes that lag behind at anaphase during mitotic division (TOLBERT, 1992). MNi reflect action in both clastogenic and aneugenic environmental factors. On the other hand, MN rate may depend on genotype (DNA repair and chromosomal segregation deficiency), immune status, sex and age of individual (ZUNIGAGONZALES, 2001). Our data show higher MNi level for nestlings (p<0.05). This result may be explained by two reasons: either adult penguins are more adapted and resistant to environmental factors or nestlings with an extremely high MNi rate do not survive after the first winter.

Figure 1. Erythrocyte with MN A – foto, B - scheme

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Figure 2. Erythrocyte with “budding” nucleus. A – foto, B - scheme

Our data showed that micronuclei in erythrocytes of Gentoo appear quite rarely. Therefore, we also used other nuclear anomalies as additional parameters of chromosomal instability. We detected and classified more frequently registered NA into a “budding” nucleus (BN), “tailed” nucleus (TN), two-lobe nucleus (TLN) and nucleus with a cavity (NC). A “budding” nucleus is a nucleus with two parts, one of which no more than 1/ 3 of the main nucleus. The width of a nucleus in the constriction area equals approximately 1/3 of the diameter of the main nucleus (fig. 2). The most probable reason for the BN appearance is gene amplification (FENECH, 2002). Amplified DNA localizes selectively to specific sites at the periphery of the nucleus and is eliminated via nuclear budding. Since different chemicals may induce DNA amplification, BNi reflects their mutagenic impact on the organism. The average levels of BNi were 0.05±0.01 ‰ for adult penguins and 0.03±0.01 ‰ for nestlings. The “tailed” nucleus has a sharply narrowed and extended end (fig. 3). TNi is the result of nucleoplasmic bridges breakage during cytokinesis. The nucleoplasmic bridges form by dicentric chromosomes that miss-segregated to opposite cell poles (FENECH, 2002). The nucleoplasmic bridges and dicentric chromosomes are markers of ionizing radiation effects (FEDORTSEVA, 1998). The average levels of TNi were 0.06±0.01 ‰ for adult penguins and 0.04±0.01 ‰ for nestlings. Two-lobe nucleus is a nucleus with a constriction dividing it into two approximately equal parts (fig. 4). The most probable mechanism of TLN formation is

Figure 3. Erythrocyte with “tailed” nucleus. A – foto, B - scheme

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Figure 4. Erythrocyte with two-lobe nucleus. A – photo, B - scheme

Figure 5. Erythrocyte with a nucleus with a cavity. A – photo, B - scheme

incomplete amitotic division of the nucleus. Amitosis is not the typical way for normal erythrocytes’ nuclear division. Factors induced TLN formations are unknown in contrast to the anomalies described above. The average levels of TLNi were 0.09±0.02 ‰ for adult penguins and 0.02±0.004 ‰ for nestlings. A nucleus with a cavity is a nucleus with a clear deep cavity, approximately up to 1/3 of the diameter of the main nucleus with non-adjoined edges (fig. 5). Mechanisms and factors induced NC formation are unknown. The average levels of NC were 0.12±0.01 ‰ for adult penguins and 0.03±0.01 ‰ for nestlings. The pooled data of the levels of described parameters are shown in the table. The rates of all NA were higher for adult Gentoo penguins. The differences between NA levels for adult penguins and nestlings were statistically significant only for TLNi and NC. Additional investigations of the parameters described should be conducted for different birds species in order to explain this result.

Table 1. The pooled data of the levels of described parameters of chromosomal instability of Gentoo penguins. MN, ‰ Adult penguins Nestlings

BN, ‰

0.03±0.01 0.05±0.01 0.11±0.01 0.03±0.01

TN, ‰

TLN, ‰

NC, ‰

0.06±0.01 0.04±0.01

0.09±0.02 0.02±0.004

0.12±0.01 0.03±0.01

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CONCLUSIONS. We estimated the initial levels of micronuclei and detected nuclear anomalies. We propose that our data and the parameters of chromosomal instability described be used in the genetic monitoring of Gentoo penguin populations. REFERENCES FENECH M., CROTT J. W. 2002. Micronuclei, nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes – evidence for breakage – fusion – bridge cycles in the cytokinesis – block micronucleus assay. – Mutation Research, 504:131 - 136. TOLBERT P., SHY C., ALLEN J. 1992. Micronuclei and other nuclear anomalies in buccal smears: methods development. – Mutation Research, 271:69 - 77. ZUNIGA G., TORRES-BUGARIN O., RAMIREZ-MUNOS M.P., et al. 1996. Spontaneous micronuclei in peripheral blood erythrocytes from 35 mammalian species. – Mutation Research, 369:123 – 127. ZUNIGA-GONZALES G., TORRES-BUGARIN O., ZAMORA-PEREZ A. et al. 2001. Differences in the number of micronucleated erythrocytes among young and adult animals including humans spontaneous micronuclei in 43 species. – Mutation Research, 494:161 - 167. FEDORTSEVA. F., KRAVTSOV V. JU., STARKOVA E. V. et al. 1998. “Hvostatye” jadra kak vozmozhnji ekspress-indokator nestabil’nosti genoma pri obluchenii. – Epidemiologicheskie aspekty Chernobilskoj katastrofy: Sb. tez. 33.

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©C PENSOFT Publishers Bulgarian Antarctic Research ORRELATION BETWEEN CHROMOSOMAL INSTABILITY AND SOME MORPHOPHYSIOLOGYCAL ... 15 Sofia – Moscow Life Sciences, vol. 5: 15-21, 2006

Correlation Between Chromosomal Instability and Some Morphophysiologycal Characteristics of Gentoo Penguins’ Nestlings STANISLAV RUSHKOVSKY, KATERYNA AFANASIEVA, VLADIMIR BEZRUKOV Department of General and Molecular Genetics, Kyiv Taras Shevchenko National University. 64 Volodymyrska Street, Kyiv, 01033, Ukraine ([email protected])

ABSTRACT The correlation between cytogenetic parameters of genome instability and some morphophysiologycal characteristics of Gentoo nestlings from Peterman Island was studied. Blood samples were collected and measurements were made during the austral summer of the years 2002-2003. The sum of the various anomalies of erythrocyte nuclei was chosen as a parameter of genome instability. The nestling’s body mass and heterophil-lymphocyte ratio (an indicator of stress for the birds) were selected as morphophysiologycal characteristics. The average rate of the nuclei anomalies for the group of birds studied was 0.22±0.02 ‰. The average nestlings’ body mass was 2.55±0.07 kg, the average heterophil-lymphocyte ratio was 1,35±0,08. The correlation analysis revealed existence of significant linkages between the morphophysiologycal and the cytogenetic parameters studied. The coefficients of correlation between body mass and the rate of nuclei anomalies and the heterophil-lymphocyte ratio were negative. The correlation between the heterophil-lymphocyte ratio and the rate of the nuclei anomalies was positive. All of these coefficients of correlation were statistically significant (ð<0,05).

INTRODUCTION Pygoscelis papua is a species well adapted to Antarctic conditions. However, Gentoo penguins have a high death-rate, especially during their first year of life. The rough calculation displays approximately 90% of offspring to be lost. Only 10% of nestlings survive and contribute to the general reproductive pool. Such high nestlings’ mortality may change the genetic structure of the population during one generation.

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It is necessary to know some parameters, which will allow us to discriminate between more and less successful nestlings. The major indicators of individuals’ “well-being” are body mass, proportions, immune status, stress level and the level of genome instability. These indexes characterize the relative fitness of individuals and of the whole population. It is obvious that individuals with low levels of stress and genome instability and good immunity will be the first to survive. Here we describe the correlations between the parameters of individuals’ “wellbeing”: end-points of genome instability, body mass and stress of Gentoo nestlings. MATERIALS AND METHODS. We obtained 112 samples of peripheral blood from nestlings of Gentoo penguins. The blood samples of birds were collected at Peterman Island during the austral summer 2002. Blood was obtained by skin incision of the web of the 4-th rudimentary finger. Smears were made on pre-cleaned microscope slides, fixed in 96% ethanol for 30 min and stained with a combination of Leishman and Giemsa stains. The frequencies of micronuclei (MN) and nuclear anomalies (NA) were selected as parameters of chromosomal instability. We scored more frequently registered NA – ”budding” nucleus (BN), ”tailed” nucleus (TN), two-lobe nucleus (TLN) and nucleus with a cavity (NC) (AFANASIEVA et al., 2006 this issue). Studied parameters were counted per 10000 mature erythrocytes for each individual. Differential white blood cell counting was performed by using the standard method (MCDONALD, 2005) The nestling’s body mass and the heterophil-lymphocyte ratio (an indicator of stress for birds (GROSS, 1983) were selected as morphophysiologycal characteristics. RESULTS AND DISCUSSION. Chromosomal instability of Gentoo nestlings. The levels of all selected parameters of chromosomal instability were rather low (Figure 1). The most frequently occurring end-point was MN. The lowest rate was observed for TLN. As an example, the MN is the rarest end-point occurrence for adult Gentoo penguins (AFANASIEVA et al., 2005, this issue). It suggests that the low rate of MN may be a major parameter of chromosomal instability that determinates the nestlings’ “well-being”. All of these parameters reflect the influence of genome damaging factors and occur with low frequencies. Thus we decided to make use of the frequency of the sum of MN and NA (SMN) in our subsequent investigations. The level of SMN varied from 0 ‰ to 1 ‰ with a mean of 0.22±0.02 ‰.

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Figure 1. The average values of the selected parameters of Gentoo nestlings’ genome instability

White blood cells count of Gentoo nestlings. The level of genome instability depends on the individual’s genotype, the influence of environmental factors, the immune status and the general health (ILYINSKIKH, 1986; RACINE, 1992). The white blood cell (WBC) count is a good, sensitive indicator of the bird’s general health and immune status. A change in the hemogram can be noted when no other abnormalities are detected (FUDGE, 2005). We can suppose that nestlings with a normal hemogram are more successful. The normal WBC count differs among bird species. Thus it was necessary to estimate the normal WBC count and the WBC ratio for Gentoo nestlings. The data for the white blood cells count per 10000 erythrocytes of Gentoo nestlings are shown in the table. The white blood cells ratio of Gentoo nestlings is summarized in Figure 2. In general, this WBC count is within the normal limits defined by veterinary standards for domestic birds (PHILLIPS, 2005). Table 1. White blood cells count per 10000 erythrocytes of Gentoo nestlings

WBC Heterophils Esinophils Basophils Lymphocytes Monocytes Total

Amount per 10000 erythrocytes min - max mean 3 – 294 1 – 36 0–7 7 – 131 0–6 43 – 380

71± ±3.62 8.25± ±0.68 0.68± ±0.11 58.78± ±2.18 1.66± ±0.15 140.05± ±5.16

Variance ( σ 2) 1471.55 52.37 1.36 532.81 2.64 2987.10

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The high variability of the white blood cells count per 10000 erythrocytes indicates the possible presence of sick individuals in the group. Nestlings with a number of leukocytes more or less than X ± σ ( X is a mean value, σ is the standard deviation) were presumably ill. We suppose the normal leukocyte values for nestlings are 85 – 194 per 10000 erythrocytes. The nestlings with leukocyte number less then 85 per 10000 erythrocyte have leukopenia. The most common causes of leukopenia include acute viral infections and bone marrow failure (FUDGE, 2005; MCDONALD, 2005). We found 15 nestlings with leukopenia among the 112 inspected. The nestlings with a leukocyte number less than 194 per 10000 erythrocytes have leukocytosis. Leucocytosis occurs primarily due to the increasing number of circulating heterophils (heterophilia).

Figure 2. The white blood cells ratio of Gentoo nestlings. H – heterophils, E – eosinophils, B – basophils, L – lymphocytes, M – monocytes.

Heterophilia is generally caused by bacterial and fungal infection, trauma, toxicities, and cancers such as leukemia or stress (FUDGE, 2005; MCDONALD, 2005). We found 14 nestlings with leukocytosis. Correlation between chromosomal instability and the morphophysiologycal characteristics of Gentoo penguins’ nestlings. As mentioned above, the level of genome instability depends on the individual’s genotype, the influence of environmental factors and the morphophysiologycal parameters. We estimated the correlation between the level of SMN and some morphophysiologycal characteristics. The heterophil-lymphocyte (H/L) ratio and body mass were selected as the main morphophysiologycal indexes. The H/L ratio is an indicator of stress for the birds and their immune status (GROSS, 1983) . The body mass of the nestlings varied from 0.75 kg to 5.3 kg (mean 2.55±0.07 kg). The H/L ratio fluctuated from 0.08 to 6.71 (a mean of 1,35±0,08).

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We found a significant negative correlation between the body mass and the level of SMN (Pearson’s coefficient of correlation r = -0.22, p<0.05) (Figure 3). A significant negative correlation between the body mass and the H/L ratio was observed too (r = -0.29, p<0.05) (Figure 4) and a significant positive correlation between the H/L ratio and the SMN level (Figure 5) (r = 0.23, p<0.05).

Figure 3. The correlation of the nestlings’ body mass with the SMN.

The results of the analysis suggest the existence of a certain relationship between cytogenetic manifestations of genome instability and selected morphophysiologycal parameters. But we cannot determine a causal relationship. It is known that a high

Figure 4. The correlation of the nestlings’ body mass with the heterophil-lymphocyte ratio.

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Figure 5. The correlation of the stress parameter with the level of genome instability of Gentoo nestlings

level of stress may cause a decrease in body mass. On the other hand, an individual with a low body mass may have a high level of susceptibility to stress. The same is true of the relationship between chromosomal instability and the H/L ratio. High stress level and pour immune status lead to an increase of genome instability health (ILYINSKIKH, 1986). However, a genetically specified high rate of genome instability may cause immune and nervous system failure, which were observed for chromosomal breakage syndromes of human and laboratory animals (HOWELL, 1991). CONCLUSIONS. The level of SMN varied from 0 ‰ to 1 ‰ with a mean of 0.22±0.02 ‰. The white blood cells ratio of Gentoo nestlings corresponds to veterinary norms for domestic birds. Significant negative correlations between the body mass and the level of SMN, and the body mass and the H/L ratio were observed. A significant positive correlation between the H/L ratio and the level of SMN were observed too. REFERENCES AFANASIEVA K., RUSHKOVSKY S., BEZRUKOV V. 2006. Parameters of chromosomal instability of Pygoscelis papua. - Bulgarian Antarctic Research, vol. 5:9 - 13 GROSS, W.B. SIEGEL H.S. . 1983. Evaluation of the heterophil/lyphocyte ratio as a measure of stress in chickens. - Avian Diseases, 27:972 - 979. FUDGE A.M. 2005. Avian blood panel interpretation. - http://www.californiaavianlab.com/ bldpanedff.pdf ILYINSKIKH N N., ILYMSKIKH I. N., BOCHAROV E.F. 1986. Cytogenetic homeostasis and immunity. – Novosibirsk, Nauka: 256 pp. (in Ruusian, summary in English)

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MCDONALD S. 2005. Complete Blood Count. - http://www.parrottalk.com/cbc.html PHILLIPS K M. 2005. Psittacine Blood Collection and Hematology: Basics for the Veterinary Practitioner. - http://www.vet.uga.edu/ivcvm/1999/Phillips/ phillips.htm RACINE R. R., MATTER B. E. 1992. The micronucleus test as an indicator of mutagenic exposure. – Mutation Research, 271:23-34 HOWELL R.T., TAYLOR A.M.R. 1991. Chromosome instability syndromes. - In “Human Cytogenetics. Volume II. Malignancy and acquired abnormalities. A practical approach.” Ed. by D.E. Rooney and B.H. Czepulkowski. - Oxford, New York, Tokyo: Irl press: 209-234.

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© PENSOFT Publishers Bulgarian Antarctic Research RAPD ANALYSIS OF GENTOO PENGUINS’ POPULATIONS 23 Sofia – Moscow Life Sciences, vol. 5: 23-29, 2006

RAPD analysis of Gentoo penguins’ populations G. D. TELEGEEV1, A. S. DRANITSINA 1, 4, M. V. DYBKOV 1, A. S. SAVOV 2, B.VACHEV 3, S. S. MALIUTA 1, V. F. BEZRUKOV 4 , O. GEORGIEV5 1

Institute of Molecular Biology and Genetics of NAS of Ukraine, Kyiv 2 3

Institute for Nuclear Research and Nuclear Energy, Sofia, Bulgaria 4

5

The Medical University Sofia, Sofia, Bulgaria

Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

Institut für Molekularbiologie, Univerity Zurich-Irchel, Switzerland

ABSTRACT The possibility for the use of the molecular-genetics RAPD-PCR method for studying penguins’ populations was estimated. Based on the similarity indexes (S), intragroup similarity indexes (Sw) were calculated between specimens in every group and a high level of relationship between penguins of two Antarctic Islands was detected (Fst = 7,89%). The most informative primer (OPM-02) was chosen for interspecific comparison of the genetic diversity of penguins. The characteristics obtained may be used for the description of the genomic profiles of penguins. KEY WORDS Penguins, markers, PCR, RAPD, primer, polimorphysm.

INTRODUCTION The Antarctic environment is characterized by extreme conditions, widely changing. Indeed, understanding the molecular, physiological and behavioral mechanisms by which Antarctic organisms adapt to them would provide information concerning the evolutional and biological basis of the development of the animal world and the survival of animals in extreme polar conditions. Penguins are an important part of the Antarctic ecosystem and the study of their biology is an important contemporary issue of international Antarctic research communities. There are two species of penguins that live in Antarctica (on the continent). Ukrainian Antarctic station “Academic Vernandsky” is located on the Antarctic Peninsula. Five species

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of penguins live there, and three of them nest (Adelie, Gentoo and Chinstrap) (Cabot J. et al. 1992; Williams 1995). The research of Gentoo penguins (Pygoscelis papua) is carried out with the support of the international INTAS project and the Ukrainian Antarctic Centre. The aim of the present work was to evaluate the use of the RAPD analysis for penguins’ population research. MATERIAL AND METHODS 2.1. SOURCE OF GENOMIC DNA Blood samples of penguins were collected at Piterman and Livingston Islands by the Ukrainian team during the 8 Antarctic expeditions in the summers of 2002-2004. The blood samples with heparin were kept at -20°C. Genomic DNA was isolated by the modified salt extraction method (Maliuta et al. 1996). Also 20 probes were extracted by the standard phenol-chlorophorm method. We did not find any differences in DNA analysis obtained by both methods. Therefore, for routine extraction we used the salt extraction method as an inexpensive and simple one. The concentration of DNA probes was determined using a spectrophotometer based on absorbance at 260 and 280 nm, respectively, and was defined more accurately by electrophoresis in 0,8% agarose gel. 2.2. AMPLIFICATION CONDITIONS A RAPD-PCR analysis was carried out as the modified method of Operon. A series of random primers 10 bp each has 60-70% GC contents (OPA, OPM, OPP (Operon Technologies, Alameda, CA, USA)) (Mishta et al. 2002) and an original primer (11 mer) were used for the RAPD analysis (Mishta et al. 2002). A reaction mixture was prepared in a volume of 25 ml containing 100 ng of genomic DNA, PCR buffer (67 mM Tric-HCl, pH 8,8; 16 mM (NH4)2SO4; 0,1% Tween-20; up to 0,1 M b-mercaptoethanol; up to 5 mM MgCl2; 30 pmol primer; 200 mM each of dNTP and 1 U of Taq DNA polymerase (Fermentas). Amplification was performed in a AMPLY 4 Biokom programmed for 4 cycles of 1 min 20 sec at 94°C, 1 min at 40°C, 2 min at 72°C; then for 36 cycles of 1 min at 94 °C, 1 min at 36°C, 2 min at 72°C with the last cycle of 5 min at 72°C, using the fastest available transitions between each temperature. 2.3. AGAROSE GEL ELECTROPHORESIS AND VISUALIZATION OF BAND PATTERNS Amplified products were analyzed by electrophoresis in 2-2,5% agarose gel and detected by staining with ethidium bromide (Maniatis et al. 1982). Plasmid pUC 19 digested by restriction endonuclease MvaI and plasmid pUC 19 digested by MspI

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were used as a molecular size standard. A negative control without any DNA template was included in each amplification batch. 2.4. STATISTICAL ANALYSIS OF AMPLIFIED PRODUCTS RAPD profiles were analyzed by RFLP Scan 3.12. (Scanalytics). Each band was considered as a RAPD marker. All the amplifications were repeated two times for analysis and only reproducible bands were considered. The amplification products of 150-1500 bp were scored across all samples as a binary matrix 1 when present or 0 when absent. Accordingly to obtained results the following indexes were estimated for every group of Gentoo penguins: 1.)

Mean number of amplified fragments (N)

N=

1 n ∑ xi , xi – overall number of fragments in individual i, n – overall number n i =1

of individuals in a group; 2.) Percentage of polymorphic loci (P)

1 n ∑ pi ⋅ 100 n i =1 , where N – mean number of fragments in a group, n – overall P= N number of individuals in a group, pi - overall polymorphic loci number in individual i of the group. 3.) Similarity indexes (index Chekanowski) (S) S=2Fab (Fa + Fb), where Fab – number of the same fragments of specimens a and b; Fa and Fb – overall fragments number of individuals a and b. 4.) Based on the values S intragroup similarity indexes APS (Sw) and intergroup indexes (Sb) (Maliuta et al. 1996; Morozova et al. 2002) were calculated between individuals of Gentoo penguins in every group;

Sw = ∑ S / n , n – number of specimens in a group; Sb=

∑ Sw / n , n – number of groups;

5.) Coefficient of populations’ subdivision: Fst =

(1 − Sb) (2 − Sb − Sw)

Values are expressed as means ± SD. Statistical significance was accepted at values of P < 0,05.

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RESULTS AND DISCUSSION To study the molecular-genetic structure of Gentoo penguins’ populations 3 primers (OPA–10, OPP–12, OPM–02) were chosen, which allowed revealing the most polimorphic and reproducible profiles and detecting from 3 to 13 RAPD-markers (300– 1100 bp at the average). DNA polymorphism was observed not only in the presence or absence of RAPDpatterns fragments with certain size but also in intensity changes of amplification fragments of the same size in different individuals. But this parameter concerning low reproducibility level wasn’t taken into account. A DNA amplification with OPM–02 primer revealed 13 polymorphic RAPD-patterns (polymorphism Ð=23%), and another 10 amplification products present in all individuals of a given group, that is they were monomorphic. The same result was achieved with primers OPA-10 and OPP12. Thus 11 marker bands were revealed using primer OPA-10: 4 of them were polymorphic (Ð=36,3%). The use of OPP-12 primer allowed to detect 9 RAPDbands and 3 of them were polymorphic (P=33,3%). To investigate further the molecular-genetic structure in Gentoo penguin’s populations, a RAPD-PCR was carried out with 2 groups of birds: ¹ 1 - 20 specimens from Livingston Island and ¹ 2 - 18 penguins from Petermann Island. Similarly to previous RAPD-analyses of Gentoo penguins’ DNA, which suggested an approximately small variability, the DNA of related species was investigated: Adelie penguins (3 individuals) and Chinstrep penguins (4 individuals).

Picture 1.

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Picture 1 represents one of the genomic profiles, which was obtained by means of a RAPD-analysis using a OPM-02 primer. 3 of 9 bands of Chinstrep penguins were polymorphic, 4-5 of 13 bands of Gentoo penguins’ No 1, and 3 of 13 for group No 2 of Gentoo penguins. An electrophoregramm of RAPD-PCR: DNA samples of different penguin species (primer OPM – 02). 1, 10 - molecular weight marker - ðUÑ19 plasmid, digested by MvaI restriction endonuclease and ðUÑ19 plasmid digested by MspI restriction endonuclease; 2 - positive control (890 bp PCR fragment); 3 - DNA of Adelie penguin; 4,6 - DNA of Chinstrep penguins; 5 - negative control of PCR; 7 - 9 - DNA of Gentoo penguins (group ¹ 2); 11 - 14 - DNA of Gentoo penguins (group ¹ 1).

Table 1. Comparative analysis of the intra-specific variability using RAPD markers (ÎÐÌ – 02 primer) Mean value Sw and Sb (±SD)

95% Confidence interval Sw

Percentage of polymorphic loci (±SD)

20

Mean number of amplified fragment per individual (± SD) 11,33 ± 0,685

0,886 ± 0,041

(0,682-1,144)

28,4 ± 0,585

18

12,25 ± 0,622

0,918 ± 0,046

(0,708-1,129)

24,50 ± 0,521

38

11,79 ± 0,453

0,906 ± 0,042

(0,755-1,165)

25,45 ± 0,348

Nuber of individuals

Sample №

Pygoscelis papua (Gentoo) 1. Sample №1 (Livingston Island) 2. Sample №2 (Petermann Island) 3. №1+№2

The mean number of amplified fragment per individual (N), the percentage of polymorphic loci (P) and similarity indexes (Sw) didn't differ significantly in groups of Gentoo penguins (primer ÎÐÌ-02) (Table 1). The variability range was 11,33 – 12,25 (N), 28,4 – 24,5 (P) and 0,886 – 0,918 (Sw). Thus we suggest a relatively low polymorphism level between individuals in each group and among groups Fst =

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7,89% (Kulikova et al. 2002, Morozova et al. 2002). A relatively lower Sw value and a higher Ð value in Group 1 was observed, that may be due to the higher nesting area of the particular group.

ОР А-10 ОР М -02 ОР А-10 ОР М -02 ОРА-10 ОР М -02 P ri m e rs

Table 2. Comparison of the inter-specific similarity

Adelie

Chinstrap

Gentoo, sample №1 (Petermann Island)

0,323 ± 0,081

0,344 ± 0,047

Gentoo, sample №1 (Petermann Island)

0,482 ± 0,073

0,493 ± 0,069

Gentoo, sample №2 (Livingston Island)

0,354 ± 0,038

0,373 ± 0,013

Gentoo, sample №2 (Livingston Island)

0,508 ± 0,067

0,507 ± 0,054

Adelie

_

0,429 ± 0,07

Chinstrap

0,509± ± 0,158

_

Species, S

w

(± ± SD)

The Sw comparison of ÎÐÀ-10 and ÎÐÌ-02 primers at the inter-specific level has shown a higher variability rate of the ÎÐÌ-02 primer (table 2). The variability range of the ÎÐÌ-02 primer amounted to 0,323 - 0,429, and was higher than that of ÎÐÀ-10 (0,482 - 0,509) (Morozova et al. 2002; Semyenova et al. 2002). CONCLUSIONS Thus the self-descriptiveness of the RAPD markers for the molecular-genetic analysis of penguins’ populations was determined. Accordingly to the obtained results there were no significant differences between two groups of Gentoo penguins

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at all indices. Variability rate with Sw≅0,9, are typical mostly for intra-population variability Fst = 7,89%. Therefore, high rate of relationship between specimens of two Antarctic islands could be suggested. Besides, primer ÎÐÌ-02 in comparison to primer ÎÐÀ-10 revealed a higher rate of polymorphism upon further analysis of individuals in three species (0,2<Sw<0,6), this rate are mostly typical of the inter-specific variability rate. ACKNOWLEDGMENTS This research was partly supported by grant of INTAS 2001-0517. REFERENCES CABOT J., CARBONERAS C., A. E., et al. (1992). Handbook of the birds of the world. Barcelona, Spain: Lynx Editions. 696 p. DE WOLF H., BLUST R.,BACKELJAU T. (2004) The use of RAPD in ecotoxicology. - Mutat Res, 566(3): 249-262. KULIKOVA I.V., CHELOMANA G.N.,GURAVLEV U.N. (2002) RAPD – PCR analysis of genetic variability in Phasanus colchicus. - Genetic (Moscow), 38(6): 836 – 841. (In Russian, Abstract in English) MALIUTA S.S., DYBKOV M.V.,TELEGEEV G.D. (1996) Study of DNA polymorphism in Ukrainian human population detected by M13 phage as a probe. - Cytology and genetic (Kiev), 33(1): 31-35. (In Russian, Abstract in English) MISHTA M., DUBEY N.,TOTEY S.M. (2002) Phylogenetic relationships and genetic polymorphisms in wild Indian mice. - Biomolecular Engineering, 18: 281 – 288. MOROZOVA E.V., RYSKOV A.P.,SEMYENOVA S.K. (2002) RAPD Variation in two trematode species (Fasciola hepatica and Dicrocoelium dendriticum) from a single cattle populations. - Genetic, 38(8): 1155 – 1162. (In Russian, Abstract in English) SEMYENOVA S.K., VASILYEV V.A., MOROZOVA E.V., et al. (2002) Fingerprinting and genetic diversity of American bison (Bison bison), Aurochs (Bison bonasus), and Gray Ukrainian cattle (Bos Taurus). - Genetic, 36(11): 1535–1545. (In Russian, Abstract in English) WILLIAMS T.D. (1995). The Penguins. Oxford University Press, Oxford. 295 p.

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© PENSOFT BulgarianPENINSULA Antarctic Research TELOMERES LENGTH OF GENTOO PENGUINS ON THE ANTARCTIC STUDYINGPublishers 31 Sofia – Moscow Life Sciences, vol. 5: 31-35, 2006

Studying Telomeres Length of Gentoo Penguins on the Antarctic Peninsula G. D. TELEGEEV 1, A. S. DRANITSINA 1, 4, M. V. DYBKOV 1, E. E. MELNIKOVA-KHABLO 1, A. S. SAVOV 2, B.VACHEV 3, S. S. MALIUTA 1, V. F. BEZRUKOV 4, O. GEORGIEV5 1

Institute of Molecular Biology and Genetics of NAS of Ukraine, Kyiv 2 3

4 5

The Medical University, Sofia, Bulgaria

Institute for Nuclear Research and Nuclear Energy, Sofia, Bulgaria Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

Institut für Molekularbiologie, Univerity Zurich-Irchel, Switzerland

ABSTRACT In this report we present the first results of telomere length measuring in knownaged family individuals of Penguins Gentoo (Pygoscelis papua) to determine whether telomeres from the nucleated blood cells (mainly erythrocytes) of these birds shorten during aging and how the rate of shortening varies with maximum lifespan. We determined the telomere restriction fragment length in erithrocyte DNA. A special telomeric probe was constructed by ligating telomeric repeats with the following cloning in pUC19. It was labeled by P32dCTP and used for hybridization. The obtained results are discussed in connection with the data on other species. KEY WORDS Penguins, Gentoo, telomeres, pUC19.

INTRODUCTION Ukrainian Antarctic station “Academic Vernandsky” is located on the Antarctic Peninsula [1,2]. Five species of penguins live there and three of them nest (Adelie, Gentoo and Chinstrap). Gentoo penguins are a very important part of the Antarctic ecosystem. They are different from other taxonomic groups and provide possible surviving and adaptation in Antarctica. The research of Gentoo penguins (Pygoscelis papua) is carried out with the support of the international INTAS project and the Ukrainian Antarctic

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Centre. Telomeres are short tandem repeated sequences of DNA found at the ends of eukaryotic chromosomes [3]. The repeats consist of a short G-rich sequence; in vertebrates, the telomeric repeat (TTAGGG)n is conserved. The function of the repeat is in stabilizing chromosomal end integrity [4]. Telomeric repeats are lost during each cell cycle because DNA polymerase is unable to replicate the 3’ end completely [5]. In vivo studies of somatic tissue of mammals and birds have shown a correlation between telomere length and organismal age within species, and correlations between telomere shortening rate and lifespan among species. Maximum telomere length and the telomere length rate of change differ among species, as does maximum lifespan among species. Haussmann and colleagues found [6, 7] that telomere length at a given life stage did not correlate with lifespan but telomere length rate of change correlated with lifespan in birds and mammals. Telomere length and telomere length rate of change vary among individuals of the same species and among tissues from an individual. Inter-species, interindividual and intertissue differences are caused mainly by factors such as different rates of cell replication, levels of telomerase activity and levels of oxidative stress [8]. Thus telomere length could be used to provide the much needed information on age, ageing and survival in natural populations where such studies are lacking. The aim of the present work was to evaluate the possibility of telomere length measuring by means of a special telomeric probe labeled by P32dCTP in knownaged family individuals for Gentoo penguins’ population research. MATERIAL AND METHODS 2.1. SOURCE OF GENOMIC DNA Blood samples of penguins were collected at Pitermann Island by the Ukrainian team during the 8 Antarctic expeditions in the summers of 2002-2004. The blood samples with heparin were kept at -20°C. Genomic DNA was isolated by the modified salt extraction method [9]. Concentration of DNA probes was determined using a spectrophotometer based on absorbance at 260 and 280 nm, respectively, and was defined more accurately by electrophoresis in 0,8% agarose gel. 2.2. LABELING OF THE TELOMERE PROBE For obtaining probes with (TTAGGG)n repeat the corresponding oligonucleotide (TTAGGG)3 and complementary them oligonucleotide (TAACCC)3 were synthesized. The primers were prepared for anneling and then ligated into a pUC19 plasmid vector. The cloned fragment (450 bp.) was labeled with P32dCTP using forward and reverse standard primers in a polymerase chain reaction.

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2.3. AGAROSE GEL ELECTROPHORESIS AND VISUALIZATION OF PATTERNS After obtaining by standard salt-extraction alcohol-precipitation, DNA probes were digested by Hinf I for 16 h, separated on agarose gel and blotted on nylon. Approximately 3 and 10 µg of digested DNA were analyzed by electrophoresis in 2-2,5% agarose gel and detected by staining with ethidium bromide and transferred to nylon filters [10]. For hybridization we used PCR product labeled with P32dCTP (see above). Detection was performed by X-ray film and exposition was carried out during 5-7 days. RESULTS AND DISCUSSION There are a number of techniques available for measuring telomere length. The telomere (terminal) restriction fragment (TRF) analysis and related methods such as the telomere amount and length assay (TALA) are relatively easy methods and are probably the most widely used in telomere research. In these methods, average lengths of TRFs (created by particular restriction enzymes and hybridized with a radioactive oligonucleotide) are measured [11].

Picture 1. Southern blotting of telomere restriction fragments of blood from Gentoo penguins. 1 – Plasmid marker with telomere repeats; 2, 4 – Samples with concentration of DNA 10 µg 3, 5 – Samples with concentration of DNA 3 µg

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To study telomere length in Gentoo penguin’s populations, a special telomeric probe was constructed by ligating telomeric repeats with the following cloning in pUC19. The probe was labeled by P32dCTP and used for hybridization. The average observed telomere length of Gentoo penguins was approximately 8000 bp in our research (Picture 1). Our preliminary results can be used only with data obtained on penguins Adelie (Haussmann et al. 2003; Nakagawa et al. 2004) where the maximum observed telomere length was 9500 bp. Thus we intend to use our data for further research on Gentoo penguins populations with regard to penguins’ age and distribution. CONCLUSIONS Thus, the self-descriptiveness of determination of telomere restriction fragment length in Gentoo penguins erithrocyte DNA by a special telomeric probe constructed by means of ligating telomeric repeats with the following cloning in pUC19 was approved. The results obtained are discussed in connection with the data on other species. ACKNOWLEDGMENTS This research was partly supported by grant of INTAS 2001-0517. REFERENCES AVIV A. 2002. Telomeres, sex, reactive oxygen species, and human cardiovascular aging. - Journal of Molecular Medicine, 80: 689–695. BLACKBURN E.H. 1991. Structure and function of telomeres. - Nature, 350: 569–573. CABOT J., CARBONERAS C., A. E., et al. 1992. Handbook of the birds of the world. Barcelona, Spain: Lynx Editions. 696 p. HAUSSMANN M.F., WINKLER D.W., O’REILLY K.M., et al. 2003. Telomeres shorten more slowly in long-lived birds and mammals than in short-lived ones. - Proc Biol Sci, 270(1522): 1387-1392. MALIUTA S.S., DYBKOV M.V.,TELEGEEV G.D. 1996. Study of DNA polymorphism in Ukrainian human population detected by M13 phage as a probe. - Cytology and genetic (Kiev), 33(1): 31-35. (In Russian, Abstract in English) MANIATIS T., FRITSCH E.,SAMBROOK J. 1982. Molecular cloning. Cold Spring Harbour Labaratory. 480 p. MEYNE J., RATLIFF R.L.,MOYZIS R.K. 1989. Conservation of the human telomere sequence (TTAGGG)n among vertebrates. - Proceedings of the National Academy of Sciences USA, 86: 7049-7053.

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NAKAGAWA S., GEMMELL N.J.,BURKE T. 2004. Measuring vertebrate telomeres: applications and limitations. - Mol Ecol, 13(9): 2523-2533. VLECK C.M., HAUSSMANN M.F.,VLECK D. 2003. The natural history of telomeres: tools for aging animals and exploring the aging process. - Experimental Gerontology, 38: 791– 795. WATSON J. 1972. Origin of concatameric T4 DNA. - Nature, 239: 197–201. WILLIAMS T.D. 1995. The Penguins. Oxford University Press, Oxford. 295 p.

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© PENSOFT B Publishers Bulgarian ILL COLLORATION OF GENTOO (PYGOSCELIS PAPUA ELLSWORTHII )...Antarctic Research 37 Sofia – Moscow Life Sciences, vol. 5: 37-43, 2006

BILL COLLORATION OF GENTOO (Pygoscelis papua ellsworthii) PART I: A MARKER FOR GENTOO POPULATION STUDIES R. METCHEVA1, P. ZEHTINDJIEV1, V. BEZRUKOV2, A. SAVOV3, Y. YANKOV4 1 2 3

Institute of Zoology, Bulgarian Academy of Sciences, 1000 Sofia, Bulgaria

Taras Shevchenko National University of Kyiv, 64 Volodymyrska Street, Kyiv, 01033, Ukraine.

Laboratory of Molecular Pathology, the Medical University, Sofia, 2 Zdrave Street, 1431, Sofia, Bulgaria 4

The Bulgarian Antarctic Institute, 151 Tzar Osvoboditel Boul., 1504 Sofia, Bulgaria.

ABSTRACT During the morphological investigations in two populations inhabiting Livingston Island (62° 38’S) (South Shetlands) Wiencke ( 64o 52’ S) and Petermann Island (65° 10’ S) a new feature in bill colloration of penguins was discovered. A clear yellow-orange spot in the base of the upper part of the bill, not described in the literature before, appears in a significant part of the individuals. The yellow spot of the bill is a characteristic feature of female and male individuals. The results show clearly that the colloration is not sexdependent. KEY WORDS Pygoscelis papua, sex determination, bill colloration

INTRODUCTION Penguins are a very special group of seabirds. All the species are really quite similar, both in structure and colloration. Gentoo belong to pygoscelids, which are the most common representatives of the avian fauna of the sea-zones of Antarctica. From a taxonomic point of view, two subspecies belong to Gentoo (Pygoscelis papua): Pygoscelis papua papua (J.R. Forster, 1781), found in the sub-Antarctic to 60 ° S, and Pygoscelis papua ellsworthi (Murphy, 1947), (South Sandwich Island and the Antarctic

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Figure 1. Gentoo without a colloration on the upper part of the bill

Figure 2. Gentoo with a clear yellow-orange spot on the upper part of the bill

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Peninsula). Race ellsworthi was separated on smaller size and proportion of bill (del Hoyo et al. 1992). The present study deals with the territory inhabit by Gentoo supposed to belongs to P .p. ellsworthi. During the morphological investigations among populations inhabiting the South Shetland Islands and Petermann and Wiencke Islands, a new feature in the bill colloration of penguins was observed (Figure 1 and Figure 2). A clear yellow-orange spot in the base of the upper part of the bill, not described in the literature before, appears in a significant part of the individuals. The distribution of the yellow spot in the two remote populations and its sex dependence is the aim of this study. MATERIALS AND METHODS Populations studied The present study concerns the territory inhabited by P. p. ellsworthi. Field measurements and blood sampling for sex identification were carried out on Livingston Island, Wiencke and Petermann Islands. Handling and marking of birds The birds were captured using a hand net, inspected and marked. On Livingston Island the studied rookery was a small one (84 nests) and all the penguins examined here were marked with glass-encapsulated TROVAN identification electronic transponders. Subcutaneous implanted transponders have demonstrated to be reliable means of identifying individual penguins. Transponders were injected at the back of the neck or between the shoulder blades. These locations were chosen because the skin of the birds there is the loosest. The TROVAN system enables automatic detection of birds for a long time; also for birds’ identification the TROVAN reader should be sufficiently close to the bird (30-50 cm). On Peterman and Wiencke Islands, having large colonies (more than 2000 nests), we used temporary marks – white dots and strips on the bills and black or blue numbers on the feet. For white painting the correction pen was used and for black (blue) – a laboratory marker with permanent waterproof ink. These marks remained distinguishable for three-four days. Blood sampling, DNA extraction and sex identification Blood samples for sex determination were collected in two different ways according to the CCAMLR EMP Standard methods recommendations. Approximately 1 ml of blood was drawn from the cubital vein with a syringe and placed into

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tubes with K3EDTA or into heparinzed tubes, plasma was separated by centrifugation, erythrocytes were suspended in equal volume of TE buffer (TRIS, EDTA), frozen and stored (under -20 °C) until usage. DNA was extracted using a standard phenol-chloroform method as described by Sambrook et al. (1989). DNA samples were resuspended in 10 mM TRIS, 1 mM EDTA pH 8.0 and kept at either 4 or -20 C. Sex determination was performed by PCR, as described by Itoh et al. (2001). The PCR products in volume of 20 µl were separated on 2% agarose gel and visualized by ethidium bromide staining. RESULTS AND DISCUSSION The yellow spot and its variation During the inspections of Gentoo on Petermann, Wiencke and Livingston Islands we found birds with a clear spot on the upper side of the bill. The spot was variable in size and color: its size ranged from very small (1 – 2 mm) to quite large (20 – 25 mm, spreading over almost all the upper surface of the bill). The color of the spot varied from white (yellowish) to red with yellow, orange and pink intermediate forms. No other visible peculiarities of the body or the feather colloration of yellowspotted Gentoo were revealed. The reason for the appearance of the spot is the main question to be answered if one attempts to use this trait as a marker for population studies. The possible reasons may be age or physiological peculiarities. On Petermann and Wiencke Islands, the yellow spot was observed in adults and in the chicks inspected, so it is not related to the age of the birds. It was also observed in birds independently of whether they Table 1. Distribution of the yellow spot in populations

Population

Yellow spot

Total

Frequency

Present

Absent

Petermann

61

139

200

0,305

Wiencke

58

56

114

0,509

Livingston

31

126

157

0,197

All

150

321

471

0,318

Significance of differences Comparing p* populations Petermann – 3,4×10-4 Wiencke Wiencke – 1,1×10-7 Livingston Petermann – 1,4×10-2 Livingston

*Probability of chance that the difference is a random deviation calculated with the exact Fisher test. In the table, a two-tailed probability is given.

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were molting or not. The other reason for the appearance of the spot could be sexual differences among individuals. The testing of this possibility could be performed after the identification of the sex of Gentoo. SEX IDENTIFICATION AND DISTRIBUTION OF THE TRAIT BETWEEN MALES AND FEMALES Gentoo are monomorphic, that is why sex identification was performed using DNA markers from blood samples. A convenient and simple method for sex determination is the DNA analysis of specific loci on W and Z chromosomes by multiplex PCR reaction. In homogametes males, the reaction of amplification displayed a product with a size of 252 bp. The heterogametes females displayed one additional band with a size of 190 bp. The DNA was extracted from the blood samples of birds from Livingston and Petermann Islands. The method applied (PCR-testing) allowed us to identify the sex of birds more accurately than the discrimination on the bill dimensions. After electrophoresis, the sex of individual birds could be easily identified (Figure 3). The analysis revealed that in the 89 samples from Livingston, 37 birds were females and 52 males. In the sample from Petermann Island we found 49 females and 99 males. Regarding this circumstance, we analyzed the possible dependence of the yellow spot on the sex of Gentoo only for the Livingston population, where all collected blood samples were tested.

Figure 3. Sex identification in Gentoo penguins Track 1 - 100 bp ladder, Tracks 2,4,5,9,10,11,12,14,15,16,18 – males Tracks 3, 6, 7, 13, 17, 19, 20 - females

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The distribution of the yellow spot among males and females does not differ significantly. This means that the yellow spot trait may not be related to the sex of Gentoo. The analysis revealed that on Livingston Island 37 of the birds investigated were female and 52 male. On Petermann Island 49 penguins were identified as female and 99 as male. Altogether, 86 of the individuals are female and the rest 151 are male. The distribution of the yellow spot divided by sex in the two investigated Gentoo populations from Livingston and Petermann Islands is shown in Table 2 and Table 3. Table 2. Frequency by sex of the character in adult Gentoo, Livingston Island

sex Female Male Total

black 75,68% 73,08% 74,16%

yellow spot 24,32% 26,92% 25,84%

Table 3. Frequency by sex of the character in adult Gentoo, Petermann Island

Sex Female Male Total

black 85,71% 72,73% 77,36%

yellow spot 14,29% 27,27% 22,64%

The results show that the female and male individuals of the two remote penguin populations have a very similar frequency of appearance of this specific bill coloration. CONCLUSIONS The presence of the newly described colloration of the bill as a yellow spot is recorded in a substantial part in adult Gentoo from the two populations studied. The yellow spot of the bill is a characteristic feature of female and male individuals alike. The results show clearly that the coloration is not sex-dependent. ACKNOWLEDGEMENT This work was funded through grant INTAS Res. Project, Ref. # 2001 - 0517 REFERENCES CLARKE J., K. KERRY 1998. Implanted transponders in penguins: Implantation, reliability, and long-term effects. J.Field Ornthology, 69 (2): 149 – 159.

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DEL HOYO, J., ELLIOTT, A. R., J. SARGATAL 1992. Handbook of the birds of the world. Lynx Edicions, . ICBP, Barcelona, vol.1. ITOH Y., M. SUZUKI, A.OGAWA, I.MUNECHIKA, K.MURATA, S.MIZUNO 2001. Identification of the sex of a wide range of Carinatae birds by PCR using primer set selected from chicken EE0.6 and its related sequences. The journal of Heredity. 92 (4): 315-321. SAMBROOK J., E. F. FRITSCH, T. MANIATIS 1989. Molecular cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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© PENSOFT Bulgarian Antarctic Research COLLORATION OF GENTOO. PART II: A REVIEW OF THE POSSIBLE CAUSES BILLPublishers 45 Sofia – Moscow Life Sciences, vol. 5: 45-49, 2006

Bill Colloration of Gentoo. Part II:A Review of the Possible Causes K. DIMITROV1, M. BELTCHEVA1, R. METCHEVA1, V. BEZRUKOV2 1 Institute of Zoology, Bulgarian Academy of Sciences, 1000 Sofia, Bulgaria 2 Taras Shevchenko Kiev University

ABSTRACT A new morphologic feature of Gentoo has been described – variation of the bill colloration. This is an evidence for the greater morphologic variability of P. p. ellsworthi among Pygoscelidae penguins. Such a variation provides an opportunity to investigate a morphologic feature using a multidisciplinary approach. We offer a theoretical background for the presence of this unique characteristic of Gentoo and propose a set of realistic approaches to investigate it. The first question to be answered is if the variation is directly heritable. Using marking of parents and chicks with identification transponders and applying multilocus DNA-fingerprinting, followed by a parental analysis, it is possible to give the answer. Such data give the opportunity to assess the possibility that the feature is determined by a genetic predisposition too. The next hypothesis is that the variation of the colloration is environmentaly iduced. It could be dietdetermined, and monitoring of the carotenoid pigments in blood plasma (lutein, zeaxanthin, anhydrolutein, and â-cryptoxanthin) using HPLC should be used. A next possibillity is that endoparasitism effects the expression of carotenoid - and melanin-based coloration. Using a variety of approaches, it is possible to determine to what degree bill colloration is either genetically or environmentally induced in a relatively monomorphic bird genus. KEY WORDS Gentoo, bill colloration, carotenoids, endoparasitism, melanin, UV-radiation

INTRODUCTION The reason for the appearance of the yellow spot on the Genoo’s bill is the main question to be answered if one attempts to use this trait as a marker for population

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studies. In part I (Metcheva et al.,2006, this volume) it was clarified that at Petermann and at Wiencke Islands the yellow spot is observed in adults and in chicks, thus it is not age-related. In addition, it was detected in birds independently of the molting status. The results show clearly that the coloration is not sex-dependent. The two general questions for its appearance are why does a bill colloration variation exist in Gentoo and does such a variation have a genetic basis or it is environmental induced? The possible reasons for the appearance of the yellow spot might be formulated by the answers of the following questions: is the feature heritable or the predisposition for its presence is heritable; is it disease-dependent; is it carotenoid - or/and melanindependent; is it UV-radiation-dependent.

DISCUSSION Is the color variation of Gentoo’s bill heritable? Marking penguins with identification transponders (Metcheva et al., 2005) provides a base to test the heritable character of the variation in mating pairs and their chicks. Long lasting marking and quick identification are some of the most important advantages of this approach. It is possible to use DNA fingerprinting and parentage analysis to establish not only the heritable character of the yellow spot, but to prove the absence of sex dependence of the appearance of the yellow spot. This approach reveals possible extra-pair fertilizations but requires intensive laboratory work and is relatively expensive. There are already advances in the individual profiling of Gentoo by RAPD (Telegeev et al., 2006, this volume) and parentage analysis is expected to be realistic in the near future. If one has proven that the variation of beak colloration in Gentoo has a genetic basis, than it is worth while to have a deep insight in it. There are more than 100 target genes potentially involved in the bird melanin based colloration and its variation. Remarkably, however, recent studies have shown that a single amino acid difference in the first candidate gene examined (Mundy et al., 2004), melanocortin- 1 receptor (MC1R), correlates perfectly with color variation. The repeated implication of this same gene suggests that there may be a more limited number of genetic mechanisms to produce dark color in natural bird populations than is suggested by genetic studies of mammals. The circulating melanocyte-stimulating hormone (MSH) binds to MC1R, turning on the cell’s melanin-making machinery in the membrane of the melanocytes, which are the site of melanin synthesis in birds and investigating the sequence variation of MC1R gene in Gentoo looks perspective. As long as Pygoscelis genome is far from being sequenced and functionally analyzed, we propose to concentrate further investigations on a biochemical level at this point (see bellow).

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The role of carotenoids. Within the various classes of natural pigments, carotenoids are the most widespread and structurally diverse pigmenting agents. They are responsible, in combination with proteins, for many of the brilliant yellow to red colors in crustaceans, fish and birds. The general distribution and metabolic pathways of carotenoids have been investigated previously in detail (Goodwin 1984, Davis 1985, Matsuno and Hirao, 1989). Gentoo penguin’s diet from about 50 to 80% in different seasons consists in crustaceans – mainly krill (Euphasia superba) - a basic source of carotenoids (Berrow, at all., 1999). Most crustaceans contain a mixture of carotenoids in the carapace as well as the blood, eyes, midgut gland, and ovary. Vertebrates are unable to produce carotenoids de novo, only plants and protists synthesizing them, so birds must ingest carotenoids from their diet. In the natural aquatic environment, carotenoids are biosynthesized in the food chain within microalgae or phytoplankton at the primary production level. Such acquisition makes carotenoid ornaments particularly sensitive to environmental factors. Several carotenoids (e.g., lutein, zeaxanthin, anhydrolutein, â-cryptoxanthin) are common in bird plasma. Their use as pigments in aquatic species is well documented, and it appears that their broader functions include a role as an antioxidant and in provitamin A activity, as well as enhancing immune response, reproduction, growth, maturation and photo protection. A commonly used assay for monitoring carotenoid levels is HPLC. The role of parasitism. Endoparasites (incl. coccidians) can directly inhibit the uptake of essential dietary components, including carotenoids, in the gastrointestinal tracts of birds. Interestingly, the rates of coccidian parasitism in Gentoo are of the same magnitude as the occurrence of the yellow spot. McGraw and Hill (2000) tested the effect of endoparasitic infection by intestinal coccidians (Isospora sp.) on the expression of both carotenoid – and melanin-based ornamental coloration in captive male American goldfinches (Carduelis tristis). They found that the carotenoid-based plumage and bill colloration of parasitized males was less saturated than that developed by unparasitized males, but that the brightness and size of melanin-based black caps did not differ between the groups. An investigation on the intestinal coccidian fauna of Pygoscelis penguins from Livingston Island is provided by Golemanski (2002). The results from the quantity of infected birds closely coincide with the percentage of bill colloration penguins. About 1/3 of the penguins investigated were infected, and in the same part of marked birds (Metcheva et al., 2005) a yellow spot appears.

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The role of melanin and UV radiation. Despite studying the sequence variation of MC1R gene (see above) there are no perspective ideas published in the literature, which can explain the variation of the melanin based coloration in birds jet. On the other hand, the complicated biochemical pathway of melanin synthesis gives opportunities to monitor particular steps of this pathway by relatively easy to perform assays. UV radiation exposure stimulates the eumelanin (black-brownish collored) synthesis. To monitor the UV-B radiation levels one can easily use a RIA assay for detecting 25-hydroxyvitamin D3 in Pygoscelys papua blood plasma (Wilske, 1993) and subsequently to correlate the results to the bill colloration variation. Because of ozone depletion, penguins are under the influence of one of the most important environmental factors in Antarctica – a relatively high UV radiation. Jouventin and al. (2005) found UV peaks of reflectance in two large Aptenodytes species, King (A. patagonicus) and Emperor (A. forsteri) penguins. UV reflectance did not occur on the feathers, claws, or skin of these species. UV peaks overlapped with spots of color on the lower beak that appeared orange for human observers, and beak spots differed slightly in location between the two species. Adults of both sexes possessed these UV markings, but they were lacking in juveniles. Measurements of free-ranging King Penguins showed that recently paired birds had higher UV reflectance than courting ones. L-cystein level is another good opportunity to study the bill coloration variation in penguins. Pheomelanin (yellow-redish colored) is the product of nuclephilic oxidation of L-cystein in the presence of DOPA chinone. At decreased levels it incites the eumelanin sythesis (Eller at al., 1994). Therefore the bill color variation might be a result of different ammonts of L-cystein in plasma. CONCLUSION The bill colloration appearance in Gentoo is a result of a single factor variation or of interplay between the factors described above. Further investigations on the heritability of the variation and on environmental and biological variables will elucidate the appearance of the newly described feature – the “yellow spot” on the upper part of the Gentoo’s beak. ACKNOWLEDGEMENT This work was funded through grant INTAS Res. Project, Ref. # 2001 - 0517

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REFERENCES BERROW S. D., R. I. TAYLOR, A. W. A. MURRAY. 1999. Influence of sampling protocol on diet determination of Gentoo penguins Pioscelis papua and Antarctic fur seals Arctocephalus gazelle. Polar Biol., 22, 156-163. DAVIS B. H.1985. Carotenoid metabolism in animals: A biochemist’s view: Pure Appl. Chem., 57, 679-684 . ELLER M., M. YAAR, B. GILCHREST (1994) DNA DAMAGE AND MELANIN SYTHESYS. NATURE, 372: 413-414 GOLEMANSKI, V. 2002. Coccidian parasites (Apicomplexa: Eucoccida) of the penguins from Livingston Island (South Shetland Islands, Antarctica). Bulgarian Antarctic Research, vol. 3, 91-95 GOODWIN T.W. 1984. The Biochemistry of the Carotenoids, 2nd ed., Chapman and Hall, London, pp. 64-96. MATSUNO T., S. HIRAO. 1989. Marine Carotenoids, in “Marine Biogenc Lipids, Fats, and Oils” (ed. by R. G. Ackman), Vol. 1, CRC Press, Florida, pp. 251-388. MCGRAW K. J., G. HILL. 2000. Differential effects of endoparasitism on the expression of carotenoid- and melanin-based rnamental coloration. Proc. R. Soc. Lond,267, 1525 – 1531. METCHEVA, R., P.ZEHTINDJEV, A.SAVOV, E. TRAKIJSKA, Y.YANKOV. 2005. Sex dimorphism established by body weight, beak and flipper length in Gentoo penguins (Pygoscelis papua) from Livingston Island. Acta Zool. Bulg. 57 (1) 65 – 71. METCHEVA R., P. ZEHTINDJIEV, V. BEZRUKOV, A. SAVOV, Y. YANKOV. 2006. BILL COLORATION OF GENTOO (Pygoscelis papua ellsworthii)PART I: A MARKER FOR GENTOO POPULATION STUDIES. Bulg. Antarctic.Res. v.5. MUNDY N., N. BADCOOCK, T. HART, K. SCRIBNER, K. JANSSEN, N. NADEAU. 2004. Conserved Genetic Basis of a Quantitative Plumage Trait Involved in Mate Choice. Science, 303:1870-1873 TELEGEEV G., A. S. DRANITSINA, M. V. DYBKOV, A. S. SAVOV, B.VACHEV, S. S. MALIUTA, V. F. BEZRUKOV, O.GERGIEV. RAPD analysis of gentoo penguins’ populations. Bulg. Antarctic.Res. v.5. WILSKE J. (1993) Plasma concentrations of 25-hydroxyvitamin D3 as an indicator of ultraviolet radiatio. Arct Med Res, 52: 166-169

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© PENSOFT Publishers Bulgarian Antarctic Research YEAST ISOLATED FROM PENGUIN PLUMAGE FROM WESTERN ANTARCTICA STUDY OF 51 Sofia – Moscow Life Sciences, vol. 5: 51-62, 2006

Study of Yeast Isolated from Penguin Plumage from Western Antarctica K. PAVLOVA1, R. METCHEVA2, I. SAVOVA3, V. BEZRUKOV4, Y.YANKOV5, L.WOODWORTH6 1

Department of Microbial Biosynthesis and Biotechnology, Institute of Microbiology, Bulgarian Academy of Sciences, 26, Maritza Blvd. 4002 Plovdiv,

2

Institute of Zoology, Bulgarian Academy of Sciences. 1, Tzar Osvoboditel Boul., 1000 Sofia

3

National Bank for Industrial Microorganisms and Cell Cultures, 125 Tzarigradsko chaussée, Block 2, 1113 Sofia 4

Taras Shevchenko Kiev University 5

The Bulgarian Antarctic Institute 6

VIC 31474; Australia

ABSTRACT Microbiological tests have been applied to the feathers of 55 molting brash-tailed penguins (Gentoo - Pygoscelis papua, Chinscrap - P. antarctica and P. adelie). The samples were collected from different localities in Western Antarctica – from 62 to 650 S. Various percentages of sterile and infected with fungi and yeast samples were determined in localities with and without tourist impact. Eleven strains of yeast have been isolated and classified on the basis of morphology-cultural characteristics, physiology-biochemical properties and sporulation. Preliminary tests were carried out to estimate the biosynthesis of enzymes and polysaccharides in order to select active producers of these biologically active substances. KEY WORDS Antarctica, Pygoscelis papua, Pygoscelis antarctica, Pygoscelis adelie, Yeasts, Feather, Enzymes, Polysaccharides

INTRODUCTION Polar Regions are among the most inhospitable for humans on the Earth. At the beginning of the twenty-first century, the Antarctic Region houses more than 40

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permanent stations where people live and work year a round. From the ecological point of view, the few small ice-free territories around the coastal fringe are the most productive areas of Antarctica. These oases are breeding areas for marine animals which carry ashore nutrients with marine origin. Humans use the oasises for building bases and for tourism. As a result, they input pollutants, microorganisms and disrupt the ecological state of the environment. Ship-based tourism in the Antarctic Peninsula has steadily increased in the last decades. The existing information is insufficient to predict accurately the degree of the physical and microorganisms effects on the biota (Hofman, 2000). As elsewhere in the world, microorganisms are the main components of the Antarctic ecosystem. Yeasts are among them. Wide spread genera of Cryptococcus sp., Candida sp., Rhodotorula sp., Sporobolomyces sp., Trichosporon sp. and other yeasts were isolated from soil, mosses, lichens and different substrates in the dry valleys (Atlas et.al.,1978, Vishniac & Hempling 1979, Vishniac & Beharaeen 1982). Coastal Antarctic locations (Goto et.al.1960) and the region of the Bulgarian base of Livingston Island, Antarctica (Pavlova et al., 2001, 2004) were also among them. Brash-tailed penguins are the most numerous representatives of vertebrates in Antarctica and their feathers are suitable for monitoring the environmental state in these regions. Penguins moult annually, therefore the plumage is a perfect indicator for such type of investigations. It provides an exceptional opportunity to assess whether there exists a microorganisms-contamination trend. There are no data providing information about yeast diversity in feathers, especially in polar birds. The present study deals with the Pygoscelid penguins, most widely distributed (Gentoo – P. papua; Chinstrap – P. antarctica and Adelie – P. adeliae: Del Hoyo et al., 1992). Their feathers are structurally the same and with an identical chemical structure (Murphy et al., 1990). The aim of the present work is to: - study the morphology and physiology of yeast of the Pygoscelid penguin’s molting feathers from different places in Western Antarctica with and without a tourist impact; - identify and investigate the potential of yeasts for the biosynthesis of enzymes and polysaccharides. MATERIALS AND METHODS Molting feathers of adult animals were collected during the summer seasons of 2002, 2003 and 2004 in Western Antarctica: Livingston Island (Caleta Argentina), Cuverville Island, Neko Harbor and Port Lockroy (Yugla point). The samples were collected individually from 26 Gentoo (P. papua), 26 Chinstrap (P. antarctica) and 3

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Adelie (P. adelie) penguins. The molting feathers were carefully removed from the skin at the neck and the upper part of the back of the birds. They are clean and relatively easy to collect. All feathers were collected individually in sterile polyethylene bags. In order to obtain single morphologically different colonies the samples were suspended in 10 ml of sterile water and, after remaining for 3-hours, suitable dilutions from 102 to 106 were made. Each dilution were plated on ME agar (malt extract – 2.0%, agar – 2.0%) and YPD agar (glucose – 2.0%, peptone – 1.0%, yeast extract – 0.3%, agar – 2.0%). The cultivation was carried out at 4°C for 3 to 14 days. The isolated colonies were checked for purity, maintained on malt slant agar and stored at 4°C. The isolates were preserved by lyophilization. The morphology-cultural and physiology-biochemical investigations were carried out using the criteria of Barnett et al. (1990) and Kurtzman and Fell (1998). The exopolysaccharide biosynthesis was determined on basal medium, which contained: sucrose – 4%, (NH4)2SO4 – 0.20, KH2PO4 – 0.1, MgSO4.7H20 – 0.05, NaCI – 0.01, CaCI2.2H2O and yeast extract – 0.1, pH 5.6. The conditions for biosynthesis of polysaccharides from psychrophylic yeasts have been described by Pavlova et al. (2004). The method of obtaining zones of clearness in a solid medium was used. The selection background was as follows: Ca-phytate for phytase activity, starch for amylase and non-fat milk for protease, and pectin for pectolytic activity. RESULTS AND DISCUSSION Microbiological investigations of penguin’s feathers collected from different sites of Western Antarctica show that penguins exhibit various degrees of contamination with fungi and yeasts (Table 1-3). The intensity of microbial growth depends on the composition of the medium – poor on malt extract and rich on nutritive substrates (YPD). The results in Table 1 show that measured on malt agar half of the feather samples of P. papua and P. antarctica from all investigated sites, visited by tourists, and the feathers of P. adelie from Petermann were infected. However, on YPD medium we determined all samples as infected. Yeasts were isolated only from the feathers of P. papua from Cyverville. The degree of microbial contamination of P. papua feathers from Livingston Island, established on malt agar, was considerably smaller (31,6%) than the degree measured on YPD medium, where the contamination was 52,7 % (Table 2). The lowest contamination in the same location was established in the feathers of P. antarctica (Table 3) – infected samples were 11,6% and 19,3% respectively measured on malt agar and YPD medium. To set up the results, in Livingston Island P. antarctica

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K. PAVLOVA, R. METCHEVA, I. SAVOVA, V. BEZRUKOV, Y.YANKOV, L.WOODWORTH

feathers were cleaner than P. papua from microbiological point of view. Comparing the results of the microbial investigation on the penguin feathers (P. papua, P. antarctica, P. adeliae) from Cyverville, Petermann, Port Lockroy (Yugla point) and Neko Harbor with these from Livingston Island (P. papua, P. antarctica) we find out the presence of higher microbial contamination in the human affected places. This indicates a possible disturbance of the ecological balance in such places. The results on morphological and cultural characteristics of the eleven yeast strains are given in Table 4. Nearly 30 % of the stains formed ascospores. Nine percents of spores were formed on 5 % malt agar, acetate agar and test-malt agar. Only one strain produced asexuall- and teliospores on sucrose – yeast agar. Only one of strains reproduces not only asexually but also by teliospores on sucrose-yeast extract agar. Balistospores were not observed in the tested yeast strains. Nine percents of stains formed well developed pseudomycelium on corn agar, potato-dextrose and rise agar. Rudimental pseudomycelium was formed by 18% of the strains. 27% of them did not grow at the temperatures of 30°C, 35°C and 37°C tested. Of the rest of the strains, 45% grow at 35°C, and 28% at 37°C. The cultivation temperature of yeasts isolated from Antarctic soils, mosses, and lichen samples does not exceed 25°C. The cultivation temperature of the yeasts isolated from penTable 1. Microbiological investigation of penguin’s plumage from different localities in Western Antarctica Localities

Species

Cyverville

P. papua

Petermann

P. papua

Petermann

P. adelie

Yougla point Port Lockroy

P. papua

Neko Habour

P. papua

Medium

Sample №

Malt agar

1

sterile

6

infected

2

sterile

3

infected

4

infected

7

sterile

9

sterile

5

infected

10

sterile

8

infected

YPD yeast

fungi

fungi

fungi fungi

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55

guin’s feathers reaches 37°C. Hence, these microorganisms were adapted to the penguin body temperature and they cannot be identified as obligate psychrophylic. The results of the physiolological and biochemical characteristics of the isolated yeasts are shown in Table 5. Most important of them are: none of the tested strains produced acid from glucose; about 72% of them were DBB-positive, and 28% formed ascospores as an exception; urease activity correlated with the DBB test; all cultures possess glucosidase activity-hydrolysis of arbutin. Extra cellular starch-like compounds formed 36% of the strains. Sugars fermentation passed only in two of the investigated strains, which formed 18% of the total and it was too weak and delayed. The results on carbon and nitrogen assimilation show that the utilization of different sources is less delayed and weak in comparison with strains under optimum conditions. It is due to the different conditions of the cultivation. Optimal growth is realized on a sole carbon source medium (sucrose, maltose, melizitose, xylose, lycerol, xylitol, D-glucoronate, lactic acid, succinate and citrate). After preservation by lyophilisation 27% of the yeast strains survived. Taxonomic investigations up to now are the reason to identify the isolates as follows: 55% belong to Cryptococcus sp. Half Table 2. Microbiological investigations on penguin’s plumage from Gentoo (P. papua) from Livingston Island Medium sample No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Malt agar

YPD

sterile sterile sterile sterile sterile yeast sterile sterile sterile sterile fungi sterile fungi fungi fungi sterile sterile sterile fungi

fungi fungi fungi sterile sterile yeast sterile sterile fungi sterile fungi sterile yeast fungi fungi sterile sterile sterile fungi

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K. PAVLOVA, R. METCHEVA, I. SAVOVA, V. BEZRUKOV, Y.YANKOV, L.WOODWORTH

of them were identified as Cryptococcus laurentii and the others as Cryptococcus albidus, Cryptococcus flavus and Cryptococcus antarcticus. Yeasts of Rhodotorula minuta composed 27% of the total amount of strains, 9% belongs to Debaryomyces hansenii and the rest 9% to Leucosporidium scottii. In the cultures identified as Cryptococcus sp., similarities in the morphological and physiological characteristics were observed. The cellular and colonial morphology were almost the same. There are slight differences in the assimilation of carbon and nitrogen sources. They grow at lower temperatures than Debaryomyces hansenii differs in the degree of assimilation of the tested sources. The isolation, identification and study of the metabolic characteristics of Antarctic yeasts were carried out from soils, mosses and lichens. Collections comprise the genTable 3. Microbiological investigations on penguin’s plumage from Chinstrap (P. antarctica) from Livingston Island Medium sample No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Malt agar

YPD

fungi sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile fungi fungi sterile sterile sterile

fungi and bacteria sterile sterile sterile sterile sterile fungi fungi sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile fungi fungi sterile sterile sterile

White, d u ll

Round, singe, double

Reddish to coral

Sort elliptic, oval, single, double

Colour

Shape

Teliospores Balistospores Artrospores Endospores

-

singledouble -

Even

Even

Margins

-

Smooth

Even, smooth

Surface

Acospores

2

1

Characteristics

-

single

Spherical, round, s in g le , double

White, d u ll

Even

Even, smooth

3

-

single

Spherical, oval, single, double

White

Even

Smooth, d u ll

4

-

-

Spores

Sort e l lip t i c , oval, spherical

Cells

Creamy with pink nuance

Even

Smooth, shining

Colony

5

+ -

-

Oval, elliptic, cylindrical

Beige, glossy

Uneven with fiber

Uneven

6

Strains 7

-

-

Oval, short elliptic, simple, double

Creamy with pink nuance

Even

Smooth, shining

Table 4. Morphological and cultural characteristics of yeast strains form Western Antarctica

-

-

Large, oval, spherical, single, double

Creamy with beige nuance

Even

Smooth, d u ll

8

-

-

-

-

Sort elliptic, oval

Pink, glossy

Creamy, Dull, with pink nuance

Oval, elliptic, simple, double

Even

Smooth, shining

10

Even

Smooth, glossy

9

-

-

Spherica l, big, s in g le , double

Creamy to beige

Even

Smooth, shining

11

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STUDY OF YEAST ISOLATED FROM PENGUIN PLUMAGE FROM WESTERN ANTARCTICA 57

Little

+ + -

Little, red

+ + + +

43.88

Sediment

25°C 30°C 35°C 37°C

%

42.00

+

+

Ring

-

-

-

-

-

-

Smooth

Even, smooth

Film

Pseudomyceliu m True mycelium

Surface p

2

1

84.62

+ + Weak -

Little

+

-

-

-

Even, smooth

3

Rudiment al -

Mycel

Smooth, shining

Colony

5

+

+

Uneven

6

Strong

Strong

Islands

77.27

+ + Weak 6.11

Survival

+ -

53.57

+ + -

Growth at different temperature

Little

+ Granular, growth on walls Middle

Growth in a liquid medium

+

-

-

-

Smooth, d u ll

4

Strains

3.43

+ -

Think, growth on walls Little

-

-

Smooth, shining

7

30.00

+ + + +

Weak

Weak

-

-

-

Smooth, d u ll

8

27.50

+ -

Middle

Weak

-

-

-

Smooth, glossy

9

12.8

+ Weak -

Middle

Broken

Weak

Rudi mental -

Smooth, shining

10

62.07

+ + + -

Strong

Think

+

-

-

Smooth, shining

11

58

Characteristics

Table 4. Continued.

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Table 5. Biochemical and physiological characteristics of yeast strains form Western Antarctica Strains Characteristics D-Glucose D-Galactose Sucrose Maltose Lactose L-Sorbose Cellobiose Melibiose Raffinose Melezitose Xylose L-Arabinose D-Arabinose D-Ribose L-Ramnose Salicin Me-á-D-glucoside D-Glucosamine Starch Inulin Ethanol Methanol Mannitol Inositol Erythritol Ribitol Galactitol Xilitol Glicerol Sorbitol D-Glucuronate Lactic acid Succinate Citrate Nitrate Nitrite Cadaverin Ethylamin Fermentation Hydrolysis arbutin DBB

1 + D + + D + + D D + + + D + D + + D D D + D ND + + +

2

3

4

5

6

7

8

9

+ + + + + + + + + + + + + + + + + + + + + + + + + D D + - + + + + + + + + D D D - + + + + + + + + + + + + + - + - + + + + + + + + + + D D + + + + + D D D,W + + + + + + D D + + + + + + D D D + + D,W D D,W D,W D + + + D,W + + D + + + D,W + D + - + - + + + D D,W + D,W + D + + + + + + + + - D W,D + D,W + D,W + D D + - + - + + W D + + + D + + - + + + + + D + - + - + + + D + D + D + + D + - + + + + + + + + + + D + + + D D D D D + + + + + + + + + + + + + + ND ND + + + + + + + + + + + + + + + + + + + + + + + + - + ND ND ND - ND ND ND ND + + + - D D D D + + + D + W - D,W + + + + D + + D + + + + +

W –weak growth; D-delayed; ND-not determined; (-)-negative; (+)-positive

10

11

+ + + + + D + + + + + + D + + W D + + + + + + + + + + + ND + + ND + + +

+ + + + + + + W,D + + + D,W ND D + ND + W + ND D + +

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K. PAVLOVA, R. METCHEVA, I. SAVOVA, V. BEZRUKOV, Y.YANKOV, L.WOODWORTH

Table 5. Continued. Strains Characteristics

1

Acetic acid production Urease Starch formation 50 % glucose 10 % NaCl + 5% glucose Vitamin-free medium 0,1% cyclohexane

2

3

4

+ ND ND ND W - D,W D D,W ND -

5

6

7

+ + ND + ND

+ ND + ND

+ + ND + ND

8

9

10

W + + + + + ND ND ND - + + D ND ND ND

11 + + ND ND ND ND

W –weak growth; D-delayed; ND-not determined; (-)-negative; (+)-positive

era Sporobolomyces, Cryptococcus, Rhodotorula, and Candida. Strain producers of b-glucosidase and protease enzymes were also selected (Pavlova et al. 2002, Zlatanov et al., 2001). In order to investigate a biotechnological application of the selected strains the biological potential and metabolic activity were studied regarding the synthesis of polysaccharides (Table 6) and enzymes (Table 7). Cryptococcus strains accumulate biomass from 4.88 to 5.65 g/l. They also produce a greater quantity of polysaccharides (1.86 – 2.02 g/l) in comparison with Debaryomyces sp. with biomass 3.31 g/l and polysaccharides of 1.28 to 1.46 g/l. Rhodotorula minuta accumulates biomass about 10.97 g/l, but the polysaccharide synthesis was similar to than the other two genera – 1.38 g/l (Table. 6). The strains of Cryptococcus sp. can be used for the investigation of the production of polysaccharides. Considerable zones of clearness in background Ca-phytate were established in the strains of Rhodotorula minuta and Leucosporidium scottii. Therefore they are of imTable 6. Biosynthesis of exopolysaccharides of isolated yeasts form penguin plumage No. samples 1 2 3 4 5 6 7 8 9 10 11

Strains

Final pH

Biomass, g/l

Polysaccharides, g\l

Rhodotorula minuta Debaryomyces hansenii Debaryomyces hansenii Debaryomyces hansenii Cryptococcus laurentii Leucosporidium scottii Cryptococcus laurentii Cryptococcus albidus Cryptococcus laurentii Cryptococcus flavus Cryptococcus antarcticus

1.97 2.49 2.46 2.49 2.15 1.94 2.15 2.08 2.12 2.13 2.03

10.97 3.31 3.31 3.22 5.20 6.92 4.88 5.32 4.89 5.65 6.27

1.38 1.28 1.46. 1.38 1.94 1.15 1.86 trace 2.02 1.86 trace

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Table 7. Biosynthesis of enzymes by Antarctic yeast strains, isolated from penguin plumage No. samples 1 2 3 4 5 6 7 8 9 10 11

Strains

Phytase

Rhodotorula minuta Debaryomyces hansenii Debaryomyces hansenii Debaryomyces hansenii Cryptococcus laurentii Leucosporidium scottii Cryptococcus laurentii Cryptococcus albidus Cryptococcus laurentii Cryptococcus flavus Cryptococcus antarcticus

+++ ++ ± +++ ± + ++ ++ ++

Amylase Protease ± ± +++ ++

+ ++ + ±

Pectinolytic enzymes -

portant interest for further microbiological investigations. Only the strains of Cryptococcus sp. were under the influence of the produced amylase. The strains tested do not synthesize protease and pectolytic enzymes (Table 7). The obtained results enhance the knowledge of the biodiversity of microbial fauna in comparison with the climate and the tourist impact. Penguin plumage is also a good indicator for spots in Antarctica that are frequently visited by tourists. The presence of feathers from Petermann infected (from 50 to 100%) with facultative psychrophylic microorganisms (fungi and yeasts) and relatively sterile samples from Livingston Island (without a tourist impact) constitute evidence of the human impact. New yeast strains were added to the collection of Antarctic microorganisms and their metabolic activity was studied in order to estimate the active producers of biologically active substances. ACKNOWLEDGEMENTS This work was funded through grant INTAS Res. Project, Ref. # 2001 – 0517. REFERENCES: ATLAS R. M., M. E. MENNA, R.E. AMERON 1978. Ecological investigation of yeast in Antarctic soils. Antarct.Res.Ser. 30: 27-34. BARNETT J. A., R.W. PAYNE, D. YARROW 1990. Yeasts: Characteristics and Identification. Cambridge University Press. DELHOYO J, A. ELLIOTT, J. SARGATAL 1992. Handbook of the birds of the world. V.1. Lynx dicions, ICBP, Barcelona.

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GOTO S., J. SUGIYAMA H. LIZUKA 1969. Taxonomic study of Antarctic yeasts. Mycologia 61:748-774. HOFMAN R. J. 2000. Assessment of the Possible Cumulative Environmental Impacts of Commersial Ship-Based Tourism in the Antarctic Peninsula Area. Proceedings of a Workshop La Jolla, California. KURTZMAN C.P., J.W. Fell 1998. The yeasts: a Taxonomic Study. 4th Elsevier Scientific Publisher, Amsterdam (Netherlands) ISBN 0-444-81312-8. MURPHY M. E., J.R. KING, T. G. TARUSCIO 1990. Amino acid composition of feather barbs and rachises in three species of pygoscelid penguins: nutritional implications. The Condor. 92: 913-921. PAVLOVA, K., G. ANGELOVA, I. SAVOVA, D. GRIGOROVA, L. KUPENOV 2002. Studies of Antarctic yeast strains for the production of b-glucosidase. World J.Microbiol.&Biotechnol. 18:569-573. PAVLOVA, K., D. GRIGOROVA, T. HRISTOZOVA, A. ANGELOV 2001 Yeast strains from Livingston Island, Antarctica. Folia Microbiol. 46, 5: 397-401. PAVLOVA K., A. GUSHTEROVA, I. SAVOVA, M. NESTOROVA 2004. Isolation and Taxonomic study of Antarctic yeasts from Livingston Island for exopolysaccharide-producing. Bulgarian Antarctic Research, Life Sciences 4: 27-34. VISHNIAC H. S., S. BAHARAEEN 1982. Five new basidiomycetous yeast species segregated from Cryptococcus vishniacii ement.auct an Antarctic yeast species comprising four new varieties. Int. J. Syst. Bacteriol. 32:437-445. VISHNIAC,H.S.& HEMPLING, 1979. Evidence of an indigenous microbiota yeast in the dry valley of Antarctica. J.Gen.Microbiol. 112: 301-314. ZLATANOV M.,K.PAVLOVA, D.GRIGOROVA.2001. Lipid composition of same strains from the Livingston Island, Antarctica. Folia Microbiol. 46, 5: 401-405.

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© PENSOFT Publishers Bulgarian Antarctic Research A STUDY OF ANTARCTIC YEASTS FOR PROTEASE PRODUCTION 63 Sofia – Moscow Life Sciences, vol. 5: 63-74, 2006

A STUDY OF ANTARCTIC YEASTS FOR PROTEASE PRODUCTION K.PAVLOVA1, A.KOLEVA2, I.SAVOVA3, L.KOLEVA2, I.PISHTIYSKI2, R.METCHEVA4 1

Laboratoty of Applied Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria

2

University of Food Technologies, Department of Biochemistry and Molecular Biology, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria

3

National Bank for Industrial Microorganisms and Cell Cultures, 125 Tzarigradsko Chaussé Blvd., Sofia 1113, Bulgaria 4

Institute of Zoology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

ABSTRACT Yeast strains were isolated from moss and soil samples from the region of the Bulgarian base on Livingston Island, Antarctica, and were screened for protease production. Four strains were selected and identified as Sporobolomyces roseus AL8, Sporobolomyces salmonicolor AL1, Sporobolomyces salmonicolor AL36 and Sporidiobolus salmonicolor AL12 according to their morphology, reproductive behaviour, growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. The strains were examined for protease biosynthesis on different media containing a yeast nitrogen base, yeast extract, albumin, peptone, casein and glucose at pH 4.0 and pH 5.0. The high protease activity (32.5 U/ml) was obtained from S. roseus AL8 when the strain was grown in a medium with 0.1% of yeast nitrogen base, 1.0% of casein and 1.0% of glucose at pH 4.0; and in a medium with 0.3% of yeast extract, 1.0% of peptone, 1.0% of casein and 1.0% of glucose at pH 5.0. The growth and protease production from the yeasts were studied in buffered and unbuffered media. The time course of the growth and protease production of S. roseus AL8 were examined by means of cultivation in unbuffered media at a temperature of 24°C for 120 h. The acid protease had a pH optimum at pH 2.6 and pH 5.5 and a temperature optimum at 60°C. KEY WORDS Antarctica, Livingston Island, protease, taxonomic characteristic, yeasts

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INTRODUCTION In microbial ecosystems of Antarctica, psychrophilic and psychrotrophic organisms play a major role in the biodegradation of organic matter. In the course of their growth, yeasts accumulate metabolites. Yeasts belonging to the Sporobolomyces, Cryptococcus, Torulopsiis, Candida, Trichosporon, Rhodosporidium, and Rhodotorula genera have been isolated from soil, moss, penguin dung, water, etc. from different Antarctic regions (Goto et al., 1969; Pavlova et al., 2001; Ray et al., 1989; Savova, 1999; Vishniac & Kurtzman, 1992). Pigment-formed yeasts are known to be predominant in cold environments like the ones in Antarctica. There is a suggestion that the red pigment contributes towards the change in the membrane or cell wall properties of yeasts so that they could adapt to the specific conditions (Abyzov, 1993; Ray et al., 1989). The nature of extracellular enzymes such as proteases secreted by cold-adapted microorganism would be an interesting subject of research. Earlier studies have indicated that yeasts from the Kluyveromyces, Endomycopsis, Rhodotorula, Candida genera and most Sporobolomyces and Trichosporons genera secreted proteolytic enzymes (Ahearn et al., 1968). In the course of some taxonomic studies on various psychrotrophic microorganisms, Ray et al. (1989) isolated from Antarctic soil a Candida humicola yeast strain, which secreted protease into the culture medium. They found that protease biosynthesis dependents on the medium composition. The protein presence in the medium induced protease production, whereas the presence of amino acids reduced that production. Enzyme secretion during exponential growth at low temperatures was greater than during the growth phase at higher temperatures. Apparently, the secretion of more protease at lower temperatures holds the key for the maximum utilisation of protein substrates of the soil in the Antarctic environment. Similar observations have been reported for Candida albicans by Remold et al., (1968) and Ross et al. (1990). The basidiomycetous Antarctic soil yeasts were not limited to use sugars or the other organic compounds listed in yeast identification handbooks. These yeasts characteristically produced extracellular proteases. Cryptococcus vishniacii used L-aspartate and L-glutamate as sources of carbon and energy (Vishniac, 1985; Vishniac & Baharacen, 1982). Relatively little is known about yeast extracellular acid proteases. Those produced by Candida albicans (Remold et al., 1968), Saccharomyces lipolytica (Yamada & Ogrydziak, 1983) and Rhodotorula glutinis K-24 (Kamada & Murao, 1972) have been purified and characterised in some detail. In this paper, we have presented the results of the taxonomic studies on four Antarctic yeast strains, their ability to synthesise the protease enzyme under different culture conditions and enzyme production from Sporobolomyces roseus AL8.

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MATERIALS AND METHODS Different soil and moss samples, taken from the region of the Bulgarian base on Livingston Island, Antarctica, were analysed for the presence of yeasts. Suitable diluted suspensions were prepared in order to obtain single morphologically different colonies on malt plate agar. The cultivation was carried out at 4°C and 15°C for 3 to 14 days. The isolated colonies were reinoculated several times for purity, maintained on malt slant agar and stored at 4°C. The media for the morphological, cultural, and physiological studies were prepared as prescribed by Kreger van Rij, (1987) and Barnett et al., (1990). The strains were inoculated in a YPD liquid medium for the purpose of determining the cultural characteristics (sediment, ring, film). Observations on cell morphology were made by means of a microscope (Opton 0218, Germany). The assimilation of hydrocarbon was studied using a yeast nitrogen base (YNB, Sigma) with the addition of 1.0% (w/v) of hydrocarbon as a sole carbon source, and incubation at a temperature of 22°C was carried out. Nitrate, ethylamine and cadaverine were used as a sole nitrogen source. The ability of the strains to metabolize certain sugars anaerobically (fermentation) was tested using Durham tubes. Records were taken up to three weeks. The diazonium blue B (DBB) test was used for differentiating ascomycets from basidiomycets yeasts. Yeast strains were tested for their ability to synthesise protease in a medium of malt agar and 10% of sterile milk. Yeasts were plated on this medium in the form of strokes. Cultivation was carried out at 4°C and 20°C. Strain selection was defined on the basis of the zones formed around the strokes. Yeast strains were tested for their protease activity in media with composition as presented in Table 1. Inocula were prepared by growing cells from malt agar in 10 ml Table 1. Composition of the media for protease biosynthesis No. Components,% 1. 2. 3. 4. 5. 6.

Yeast nitrogen base (YNB), Sigma, Aldrich, Deisenhofen, Germany Yeast extract (YE), Difco Laboratories, Detroit, Michigan, USA Albumin, Fluka, Chemie AJ, Buchs, Switzerland Peptone, Fluka, Switzerland Casein, Merck, Darm-stadt, Germany Glucose, Fluka, Switzerland

Media (Number) 1

2

3

4

5

0.1

0.1

0.1

-

-

-

-

0.3

0.3

0.3

0.1

-

-

0.1

-

-

-

0.1

-

1.0

-

1.0

-

-

1.0

1.0

1.0

1.0

1.0

1.0

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K.PAVLOVA, A.KOLEVA, I.SAVOVA, L.KOLEVA, I.PISHTIYSKI, R.METCHEVA

of liquid medium. After 48 h, 2 % (w/v) of the inocula were added into 200 ml Erlenmeyer flasks containing 20 ml of liquid media. The strains were cultivated on a rotary shaker (220 rpm) at 20°C and 24°C for 120 h. The cultures were centrifuged (4000xg for 20 min) and the supernatants were assayed for enzyme activity. The protease activity was determined by Akabori’s method (1956) using casein as a substrate. Briefly, 1 ml of the culture supernatant was added to 5 ml of 0.6 % (w/v) casein at pH 2.6. After incubation at 30°C for 10 min, the reaction was stopped by the addition of 5 ml of 20 % (w/v) trichloroacetic acid. The resulting precipitate was allowed to settle for 20 min and was separated by filtration. A colorimetric reaction was carried out with a 2 ml filtrate, 5 ml of 0.55 M Na2CO3 and 1 ml of Folin Ciocalteu reactive. Intensity of colour was measured at 670 nm. A protease unit was defined by the enzyme amount which degraded the casein amount containing 1 mg tyrosin for 1 min at 30°C. The pH effect on the acid protease activity was tested with in a pH range from 1.6 to 7.5 using casein as a substrate. The temperature effect was examined in the 10°C 70°C temperature range. RESULTS AND DISCUSSION Thirty yeast strains were isolated from samples (soil, moss, and lichen) taken from the region of the Bulgarian Antarctic Base on Livingston Island. About 25 % of the Table 2. Characteristics of the isolated yeast strains Characteristics Shape of colonia Surface Margins Colour Color Cells

AL1 wrinkled, dull dull entired pink to orange ellipsoidal, single, chains

Balistospores Teliospores Pseudomycelium True mycelium Grown at 24°С Grown at 30°С Film Ring Sediment Survival, %

yes no rudimental no + no heavy present 17.30

(-) - negative (+) - positive

Strains AL8 AL12 slightly rough slightly rough semiglossy semidull entired frilly coral red red to orange Ovoidal to ellipsoidal, rare elo-ngate, ovoidal, single, in pairs single, in pairs yes yes no yes no rudimental no no + + thin thin thin heavy light present 95.00 28.46

AL36 wrinkled, dull dull entired red to orange ellipsoidal to elongate, ovoidal yes no no no + thin thin light 68.75

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Table 3. Assimilation tests and other growth characteristics Characteristics D-Glucose D-Galactose Sucrose Maltose Lactose L-Sorbose Cellobiose Trehalose Melibiose Raffinose Melezitose D-Xylose L-Arabinose D-Ribose L-Arabinose D-Glucosamine Salicin Me-α-D-glucoside Starch Inulin Ethanol Methanol Mannitol Myo-Inositol Erythritol Ribitol Galactitol D-Glucuronate Succinate Citrate Nitrate Cadaverine Ethylamine Fermentation Hydrolysis arbutin DBB Acetic acid production Urease Starch formation 50 % glucose 10 % NaCl + 5% glucose Vitamin-free medium 0.1% cyclohexane

AL1 + D + + + + + + + + D, W + + + + + + ND D + D, W + + + + + -

AL8 + + + + + + + + + + D + ND + + + + ND + + + + + + + + + -

Strains AL12 + + + + + + + + + + D + + + + + + + ND + + ND D, W + + + + + -

W–weak growth; D-delayed; ND-not determined; (-)-negative; (+)-positive

AL36 + D + + + + + + + D D, W + + + + + + + ND + + ND + + + + + + -

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colonies were pigment-formed which suggested that the pigments had some protective function and played a significant role for the survival of microorganisms (Abyzov, 1993). When they were dried by means of vacuum sublimation, high values (95 %) of vitality were registered. This phenomenon is not typical of yeasts in general. The protease-producing ability of isolated strains was tested with a medium containing 10% of skim milk. Four strains were selected. The four strains were identified using the yeast classification criteria proposed by Barnett et al., (1990) and Kurtzman & Fell, (1998). The morphological and cultural characteristics and the reproduction type of the strains are presented in Table 2. All strains had the following common characteristics: they were psychrotrophic yeasts, grew well at 18-24°C, did not grow at 30°C, formed orange to red pigments, had the ability to form ballistospores (a sexual reproduction). They are basidiomycetes of the amorphous or teliomorphous type. The carbon sources assimilation and other test characteristics are shown in Table 3. Physiological and biochemical studies were carried out using 16 sugars, 7 alcohols, 3 acids, 2 polysaccharides and 2 glucosides. The results indicated that the strains grew well on media with glucose, sucrose, maltose, cellobiose and starc as carbon source. The other sources were assimilated in a different manner. None of the strains assimilated erythritol and inositol, but all assimilated the nitrates tested. The strains showed a positive reaction in the DBB test, splitting of the arbutin, growth in vitamin-free medium, urea hydrolysis and all were non-fermentative. On the basis of the taxonomic studies the strains investigated were related to the Sporobolomyces salmonicolor AL1 and Sporobolomyces salmonicolor AL36, Sporobolomyces roseus AL8, and Sporidiobolus salmonicolor AL12 species . The four strains were examined for protease biosynthesis in liquid media with pH 4.0 and pH 5.0 at 20°C (Table 4 ). Following yeast cultivation in the medium with pH 4.0, the biomass concentration and protease activity were higher in comparison to those in the medium with pH 5.0. The Sporobolomyces salmonicolor AL1, Sporobolomyces roseus AL8. and Sporobolomyces salmonicolor AL36 strains grew well and actively synthesized the enzyme in medium 2 (YNB + glucose) and medium 5 (YPD + casein) at pH 4.0. The protease was secreted in substantial amounts when an exogenous protein was available and sufficient as the nitrogen source. When casein was present in the media, the production of protease reached the maximum level, which proved that casein induced protease production. The S.roseus AL8 strain produced the highest protease activity of 25,91 U/ ml in medium 2 and 24,14 U/ml in medium 5 at 20°C. On the basis of these experiments, subsequent investigations used a pre-buffered medium (pH 4.0) and an unbuffered medium (pH 5.0) and the strains were cultivated at a temperature of 24°C (Table 5). At that temperature, the S.roseus AL8 strain exhibited a maximum activity of 32.5 U/ ml in medium 2 and 32,0 U/ml in medium 5, with initial pH 4.0. It showed a high activity of 31,1 U/ml in unbuffered medium 5 with pH 5.0 prepared with tap water.

5.1

7.2

7.0

4.4

6.8

1

2

3

4

5

5.9

6.5

6.9

5.2

2

3

4

5

7.18

6.82

7.23

7.36

7.26

5.20

4.20

7.10

4.37

7.22

рН

Final

-

17.37

-

11.82

-

25.09

17.13

6.37

17.81

6.42

U/ml

PА

6.2

5.5

5.7

5.4

5.3

6.2

6.4

5.7

5.8

3.7

g/l

Biomass

7.62

7.22

7.36

7.37

7.14

7.24

7.25

7.13

7.25

6.81

рН

Final

Sporobolomyces roseus AL8

5.2

5.0

6.5

5.7

5.3

g/l

Biomass

19.69

21.18

11.15

17.71

11.82

5.5

5.5

7.0

5.4

5.1

Initial pH 5.0

24.17

24.32

13.37

25.91

11.68

U/ml

PА

Initial pH 4.0

7.41

7.48

7.32

7.28

7.31

7.13

7.04

7.06

6.43

4.17

Н

Finalр

12.59

-

-

-

-

18.14

14.40

-

-

8.44

U/ml

PА

Sporidiobolus salmonicolor AL12

5.8

5.8

7.0

5.2

5.8

6.3

4.6

7.4

5.9

4.5

g/l

Biomass

7.49

7.35

7.07

7.22

7.08

4.14

7.12

6.96

7.12

3.38

рН

Fin

Sporobolomyces AL3

A STUDY OF ANTARCTIC YEASTS FOR PROTEASE PRODUCTION

PA – protease activity

6.1

1

Medium

g/l

Medium

Biomass

Sporobolomyces salmonicolor AL1

Table 4. Growth and production of protease by yeast strains in different media with initial pH 4.0 and pH 5.0, at 200C

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Final рН

7.9

7.0

7.5

Biomass g/l

10.2

8.2

7.4

Strains

Sporobolomyces salmonicolor AL1 Sporobolomyces roseus AL8 Sporobolomyces salmonicolor AL36

Initial pH 4.0

18.1

32.5

PА U/m l 15.3

3.9

4.8

3.6

Biomass g/l

2.6

2.4

2.5

Final рН

Initial pH 5.0

Culture medium 2

9.8

5.9

3.4

PА U/ml

8.4

7.5

9.8

Biomass g/l

7.6

7.4

5.8

Final рН

Initial pH 4.0

9.3

32.0

22.2

PА U/ml

7.3

6.5

7.5

Biomas g/l

Initial p

Culture medium 5

70

Table 5. Effect of the initial pH of the culture medium on the biomass concentration and protease activity of the strains at 240C

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Fig.1. Time course of growth and enzyme production by Sporobolomyces roseus AL8 at 24°C

The time course of the growth and protease biosynthesis by S.roseus AL8 were studied on unbuffered medium 5 with pH 5,0 at 24 °C (Fig.1). In a casein- and peptone-containing unbuffered medium, the protease activity was maximum (32.8 U/ml) at the 72nd h. The protease secretion by S.roseus AL8 was proportional to the biomass in the medium. The protease secretion at 24°C, the optimum temperature for organism growth, was higher than that at 20°C. These results differ from the

Fig. 2. The effect of pH on the activity of the acid protease from Sporobolomyces roseus AL8.

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data reported by Ray et al. (1992) on Candida humicola Antarctic yeast strain. The protease secretion per cell of C.humicola in the log phase at 4°C was higher than that at 22°C. Acid protease expressed its activity in every wide pH range from pH 1.6 to pH 7.5. When casein was used as a substrate, the enzyme exhibited maximum activity at pH 2.6 and a second peak of activity at 5.5, however, its activity was about 40% of that at pH 2.6 (Fig.2). In the presence of casein in the medium, the acid protease produced by the Candida humicola Antarctic yeast strain was also active at two pH values: maximum activity at pH 1.0 and a second peak of activity between 5 and 7. (Ray et al. 1989). Regardless of the fact that the strain was isolated from Antarctic yeasts, at 24oC – the optimum temperature for its development – the strain produced acid protease with a 60°C-temperature optimum for the enzyme action. The enzyme activity at 10°C was only 17% of the maximum, whereas at 65°C it was about 66% (Fig 3). The protease of C.humicola was active at temperatures ranging from 0 to 45°C and exhibited optimum activity at 37°C. The activity at 0°C was about 12 to 15% of the maximum activity (Ray et al. 1992). Since are the first authors we have found to investigate the production and properties of extracellular protease from the Candida humicola psychrotrophic yeast strain, we have discussed the similarities and differences of the acid protease obtained by two different psychrotrophic yeast strains.

Fig. 3. The effect of the temperature on the activity of the acid protease from Sporobolomyces roseus AL8.

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ACKNOWLEDGEMENTS The authors thank Prof. V.Golemansky and Dr. N. Chipev for providing the samples, which were collected on Livingston Island, Antarctica. REFERENCES ABYZOV S.S. 1993. Microorganisms in the Antarctic Ice. In E.I.Friedmann, Antarctic Microbiology, Wiley-Liss New York (US). 265-295 pp. AHEARN D.G., S.P. MEYERS, R.A. NICHLS. 1968. Extracellular proteinases of yeasts and yeast like fungi. Appl. Microbiol. 16: 1370-1374 . AKABORI S. 1956. Methods of Enzyme Research.., Asakaka Booc.CoLtd., 240-244 pp. BARNETT J., R.W. PAYNE, D.YARROW. 1990. Yeasts: Characteristics and Identification, Cambridge University Press. ISBN 0-521 35056-5. GOTO S., J. SUGIYAMA, H. LIZUKA. 1969. A taxonomic study of antarctic yeasts. Mycologia 61: 748-774. KAMADA M., K.ODE, S. MURAO. 1972. The purification of the extracellular acid protease of Rhodotorula glutinis K24 and its general properties. Agric Biol.Chem. 36: 1095-1101. KREGER van Rij N.J.W. 1984. The Yeasts: A Taxsonomic Study. Elsevier science Publishing, Amsterdam (Netherlands). KURTZMAN C.P., J.W. Fell. 1998. The Yeasts: A Taxsonomic Study, 4th and. Elsevier Sientific Publisher, Amsterdam (Netherlands). PAVLOVA K., D. GRIGOROVA, T. HRISTOZOVA, A. ANGELOV. 2001. Yeast srains from the Livingston Island, Antarctica, Folia Microbiologica, 46: 397-401. RAY M., S. SHIVAJI, N. RAO SHYAMALA., P. BHARGOVA. 1989. Yeast strains from the Schirmarcher oasis Anarctica, Polar Biology, 9: 305-309. RAY M., K. UMA DEVI, G. SESHU KUMAR, S. SHIVAJI. 1992. Extracellular Protease from the Antarctic Yeast Candida humicola. Appl.and Environmental. Microbiol., 58: 1918-1923. RENOLD H., H. FASOLD, F. STAIB. 1968. Purification and characterization of a proteolytic activity from Candida albicans. Biochem. Biophys.Acta, 167: 399-406. ROSS I.K., D. F. BERNARDIS, G.W. EMERSON, A.CASSONE, P.A.SULLIVAN. 1990. The secreted aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinase deficient mutant. J.Gen.Microbiol., 136: 687-694. SAVOVA I. 1999. Isolation and taxonomic investigation of yeast strains from the Antarctic. Bulgarian Antarctic Research, Life Sciences 2: 13-18 . VISHNIAK H.S. 1985. Cryptococcus socialis sp. nov. and Cryptococcus consortinus sp. nov Antarctic Basidioblastomycetes. Int.J.System Bacteriol., 35: 119-122. VISHNIAC H.S., S.BAHARAEEN. 1982. Five new Basidioblastomyceteous yeast species segregated from Cryptococcus vishniacii ement an Antarctic yeast species comprising four new varieties Int.J.Syst.Bacteriol., 32: 437-445.

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VISHNIAK H.S., C.P. KURTZMAN. 1992. Cryptococcus antarcticus sp. Nov. and Cryptococcus albidosimilis sp. Nov., basidioblastomycetes from Antarctic soils. International Journal of Systematic Bacteriol., 4: 547-553. YAMADA T., O.M. OGRYDZIAK. 1983. Extracellular acid protease produced by Saccharomyces lipolytica. J.Bacterio., 154: 23-31.

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© PENSOFT Publishers Bulgarian Antarctic Research ... HEAVY METAL CONTENT IN GENTOO PENGUIN FEATHERS 75 Sofia – Moscow Life Sciences, vol. 5: 75-82, 2006

Heavy Metal Content in Gentoo Penguin Feathers at Petermann Island and Livingston Island ALEXANDER V. ANDREEV 1, ALEKSEY A. ANDREEV 1, MARYNA O. KURSA 1,2, ROUMIANA P. METCHEVA3, GENNADI P. MILINEVSKY1,2, SERGEY E. SHNYUKOV1, VALERY R. MOROZENKO1 1 2

National Taras Shevchenko University of Kyiv, Ukraine, [email protected]

National Antarctic Scientific Center of Ukraine, Kyiv, 16 Tarasa Shevchenka Blvd., 01601, Kyiv, Ukraine, [email protected] 3

Institute of Zoology, Bulgarian Academy of Science, 1000 Sofia, Bulgaria,

ABSTRACT Investigations of the heavy metal content in gentoo penguin (Pygoscelis papua) feathers, embryo from dead eggs, and guano by using XRF-analysis at Petermann Island and Livingston Island colonies, Antarctic Peninsula, were undertaken. This is part of an investigation of gentoo penguin populations under environmental change impact. The moulting plumage analysis is a reliable tool for biomonitoring metal pollution because the plumage renews annually. It offers an opportunity for comparison and forecasting environmental changes. The data of the southernmost gentoo population are important as an indicator of the possible changes in the ecological system of the Antarctic Peninsula. The feather samples of 39 gentoo penguins from the back and the chest were collected at Petermann in 2004-2005 season and 30 guano samples for four sites of the colony. The work is based on the XRF multi-element method of analysis with a 1 – 10 mg/kg detection limit for an element with atomic number above 20. The mean content of the Fe, Co, Ni, Cu, Zn, Se, Br, Rb, Sr, Pb elements was determined. The data obtained are the first results of toxicology studies of a gentoo penguin colony at Petermann Island. The results could serve as a database for the study of element migration processes in the Antarctic ecosystem and of heavy metal accumulation in the gentoo penguin feather/guano/embryo. KEY WORDS Pygoscelis papua, heavy metal, feather, guano, XRF-analysis, Petermann Island, Antarctica

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INTRODUCTION The convenient objects for toxicological studies of heavy metals concentration in samples of biological origin to monitor the state of the environment contamination in Antarctica are penguins’ guano, feathers and remains. The further development of investigations in this direction will let us understand the processes of elements migration in the Antarctic ecosystem and to ascertain the genotype-dependant heavy metals accumulation in gentoo. The monitoring on the gentoo population state and their breeding dynamics at Petermann Island (in the vicinity of the Ukrainian Vernadsky Antarctic Station) was established. Long-term data on the most southern population are important to keep vigilant watch on the possible changing of their number as an indicator of the state ecological system of Antarctica. Petermann Island (65°10' S, 64°08' W) is the place of localization of the most southern large population of Pygoscelis papua and the edge of the gentoo population area. The most important values of the site are the medium rate of species diversity, the breeding region of the southernmost colony of gentoo penguins, the convenient area for long-term research. The island position is convenient for studying the climate change influence on the ecosystem and for worldwide research of various areas, including studies in long-term changes. Being under the most unfavorable conditions, the gentoo colony at Petermann could be affected first under the climate change in the Antarctic Peninsula. There exists little information about the level of heavy metals (Pb, Cd, Co, Ni, Cu, Zn) and toxic elements in penguins (Metcheva 2003; Metcheva 2004). Metal distribution in an organism at any time is a result of many extrinsic factors. The moult process is connected with changes of the concentration of many elements in the bodies of birds and with changes of protein and fat contents. Moreover, this process requires a higher energy supply. Bird moulting is important for studying the transportation of elements. Heavy metals are mainly accumulated by birds with food in feathers and inner organs and are expelled with droppings. Element accumulation in feathers takes place during the whole process of feather growth. Particular kinds of feathers can accumulate different amounts of elements (Denneman 1992, Thompson 1998). The moulting plumage is a useful object to monitor metal pollution because the plumage renews annually. It offers an opportunity to estimate annual metal pollution and to study content dynamics. Different kinds of feathers can accumulate different amounts of elements. Therefore, it is possible to use feathers from certain places: the back and the head. Moulting plumage analysis is useful for annual biomonitoring of metal pollution. It may be important to study and predict the environmental changes impact on Antarctic species. A similar study on St. Kliment Ohridski Bulgarian Antarctic Base at Livingston Island (South Shetland Islands) with feather samples of Chinstrap penguins (Pygoscelis antarctica) was carried out by Bulgarian scientists, where the sampling was provided at the end of moult in February 2000 (Metcheva 2002).

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MATERIAL AND METHODS The idea of research based on that particular kinds of feathers and their different parts can accumulate different amounts of elements. The analysis of moulting plumage is a useful biological monitor for metal pollution because the plumage renews annually. It offers an opportunity for comparison and forecasting environmental changes. Collection of gentoo feathers and guano for toxicological studies was undertaken mostly in the moult season at the beginning of March and at the end of March 2005. Feathers from penguins for heavy metals and toxic elements were collected from head and chest of birds. There are feather samples of 39 gentoo penguins from the back and the head collected at Petermann Island in the 2005 season and 30 guano samples for four sites of the colony. The samples were developed using wet air oxidation in nitric acid and hydrogen peroxide with precipitation on spectral pure carbonic dust. The guano was dried at 30 degree Celsius. All feather samples and 30 guano samples were analyzed. The percentage of Fe, Co, Ni, Cu, Zn, Se, Br, Rb, Sr, Pb elements was determined. In addition, an investigation was performed of the heavy elements content in samples of penguin feather and embryo samples from dead eggs from Livingston Islands collected by Bulgarian scientists during the 2004 season. The collection of all biological samples from Petermann Island and Livingston Island was undertaken under a special permission according the Madrid Protocol legislation in Bulgaria and Ukraine.

Fig. 1. Mean element content values and data scattering for different samples and different sampling sites (Fe, Cu, Zn, Br, Sr). The embryo samples were collected at Livingston Island.

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For analyses the energy dispersion X-ray fluorescent method (XRF-ED) with a monochromatic excitation radiation of an X-ray tube with a molybdenum (Mo) anode and a LiF(200) crystal-monochromator tuned to MoKa line under 40 kV voltage and 20 mA current was used. The measurements were made in air without vacuum (Shnyukov 2002). Method description and parameters: the determined elements are Fe – Sr using K-series, Hf – U using L-series; the determination threshold is 2.3 – 0.4 ppm for Kseries and 5 – 0.5 ppm for L-series along the element row accordingly; the measuring line intensity data reproducibility under 1000 s exposition is relative 15% depending on element concentration. The feather investigation was carried out in two regimes: 1) without pre-treatment, 2) using previous wet ashing in order to reach the element concentration. The ashing was provided using nitric acid (aquafortis) with latest hydrogen peroxide addition and evaporation with spectral pure carbon dust assistance. The embryo and guano samples were studied without pre-treatment. The analyzing substance mass for non-ashed feathers was equal to ~1.0 grams, for ashed feathers ~4.0 grams, for guano and embryo samples ~1.0 grams. As standards, biomaterial samples-imitators on the base of the phenol-formaldehyde gum were used.

Fig. 2. Mean element content values and data scattering for different samples and different sampling sites (Se, Co, Ni, Rb, Pb). Embryo samples were collected at Livingston Island.

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As the results of experimenting without a feather sample demonstrate the presence of small amounts of Cu, Zn, Pb in carbon dust after analogous to feather sample treatment by nitric acid and hydrogen peroxide which have to erode the final quantity assessments, the data obtained with the ashed samples were removed from the discussion. RESULTS AND DISCUSSION According to the analysis, presence in samples of the elements Fe, Co, Ni, Cu, Zn, Se, Br, Rb, Sr, Pb was discovered. In several cases the presence of Au, Bi was obtained in ashed feather samples and As in untreated guano samples. The analysis showed the inhomogeneity of the samples of the above-mentioned element content for used weights. The results of the analysis are shown in Figures 1, 2 and Tables 1 and 2. In the figures, the mean values of element concentration are shown by marks, the minimum and maximum values – by a lapse. A difference between groups of samples was found. The content of some elements in Gentoo chicks’ feather samples is different in the different sampling sites. For example, in chick feathers at Petermann Island the Sr content value is much bigger than in adult feather Sr content, which is similar to the adults’ feather Sr value at Livingston Island. The feather samples taken from the head and chest are different on some elements as well.

Table 1. Mean element content in ppm (mg/kg dry weight) in feather and guano samples from Petermann Island. Chick Adult Guano

Fe

Co

Ni

Cu

Zn

Se

Br

Rb

Sr

Pb

102 94.6 305

4.0 5.0 -

0.2 0.3 0.7

29.4 34.9 282

150 88.4 128

6.2 2.6 10.6

54.6 40.5 790

1.7 0.7 -

64.2 16.6 963

2.1 1.1 -

Table 2. Mean element content in ppm (mg/kg dry weight) in feather and embryo samples from Livingston Island. Chick (N=2) Adult Embryo

Fe

Co

Ni

Cu

Zn

Se

Br

Rb

Sr

Pb

75.4

4.4

0.6

32.3

109

9.5

200

3.8

45.9

0.8

59.4 87.4

3.8 3.1

0.6 -

33.4 8.1

99.9 39.6

8.2 5.5

65.4 31.5

1.0 2.1

21.4 12.7

0.6 2.0

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Fig. 3. The strontium (Sr) distribution in Gentoo guano samples (left group – dark guano, right group – more light guano). N – number of samples.

Fig. 4. The correlation of Sr-Zn and Sr-Cu content values in penguin guano samples. Dark marks correspond to dark guano samples, gray marks to all other guano samples, squares correspond to Zn, triangles to Cu.

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Due to analysis the two groups of guano samples can be separated according of guano colour (more dark – more light, which depend on penguin food). The group with a small Sr content (less than 450 mg/kg) is corresponding to dark guano (Fig. 3). For this group the strong correlation between Sr and Zn content and weak correlation between Sr and Cu content are observed (Fig. 4). For other kinds of penguin guano the correlation is missing. CONCLUSION The data of heavy element content in gentoo penguin feather obtained from the southernmost gentoo population are important as an indicator of the possible changes in the ecological system of the Antarctic Peninsula. The data could serve as a starting point for research on the possible changes in the ecological system of the Antarctic Peninsula under climatic and environmental changes. Further investigations will help to understand the elements migration processes in the Antarctic ecosystem. ACKNOWLEDGEMENT Authors are grateful to biologists Dr Leonid Manilo, Dr Mikhail Chesalin, Dr Boris Anninsky, and Dr Vladimir Bezrukov for their assistance in collecting of the samples at Petermann Island. The work was supported by grant INTAS-2001-0517. REFERENCES SHNYUKOV S.E., ANDREEV A.V., SAVENOK S.P., PROSKURKA K.S., MARGULYOV V.M., 2002. Optimal analytical framework for the geochemical data sets creation in environmental pollution study, mineral exploration and petrologic research. – In: Environmental Ecology And Safety of Life Activity, No 5-6, 89-94 (in Russian). METCHEVA R., YURUKOVA L., 2002. Toxic and heavy metals concentration in Chinstrap penguin’s (Pygoscelis antarctica) feathers during molt. - In: Bulgarian Antarctic Research. Life Sciences, 3, 101-105. MECHEVA R., YURUKOVA L., 2004. Major essential and trace elements in Gentoo Penguin (Pygoscelis papua, Forster 1781, Aves: Sphenisciformes) from Livingston Island, the Antarctic. – Acta Zoologica Bulgarica, 56, 3: 331-336. MECHEVA R., YURUKOVA L., 2004. Heavy metals and toxic elements content in Gentoo Penguins (Pygoscelis papua) feathers during moult. - In: Bulgarian Antarctic Research. Life Sciences, 4, 101-106. DENNEMAN W. D., DOUBEN P. E. T., 1993. Trace metals in primary feathers of the Barn Owl (Tyto alba guttatus) in The Netherlands. Environmental Pollution, 82 (3), 301-310

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THOMPSON D. R., BEARHOP S., SPEAKMAN J. R., FURNESS R. W., 1998. Feathers as a means of monitoring mercury in seabirds: insights from stable isotope analysis. Environmental Pollution, 101(2), 193-200.

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© PENSOFT Publishers Antarctic Research OF ELEMENTAL CONTENTS IN PENGUIN FEATHER UBulgarian SING EDXRFA MONITORING 83 Sofia – Moscow Life Sciences, vol. 5: 83-92, 2006

Monitoring of Elemental Contents in Penguin Feather Using EDXRFA E.NIKOLOVA, S.ALEKSANDROVA INSTITUTE FOR NUCLEAR RESEARCH AND NUCLEAR ENERGY, BULGARIAN ACADEMY OF SCIENCES [email protected]

ABSTRACT A three year project for investigation of penguin fear was provided. Three different energy dispersive X-ray spectrometric systems with three kinds of radioactive sources were used in order to cover practically the full periodic system. A sensitive curve for each system was established. Penguin feather from island Livingston and Peterson island was investigated in order to obtain the element contents. The element distribution during this three-year period on Livingston island was found to be stable. A difference between element distribution from Livingston island and Peterson island was found, probably due to the geological status. Down from penguin babies was also measured, in order to compare with the adult exemplars.

I INTRODUCTION Antarctica is still an insufficiently investigated continent and scientists make efforts to change this situation. One of the main points of the investigations is the ecological situation on the southern continent. Practically human technologies are absent there and, therefore, the results obtained in this region can be considered as a basis for the global changes in terms of contamination. There is a very suitable monitor for ecological investigations, namely penguins. These birds live in colonies on the beach and every change in the chemical composition of their feather during a period of one year gives us information on possible impacts. A three-year project for determining elemental contents in penguin feather, using EDXRFA, was carried out.

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The power of this analytical instrument is due to the energy dispersive detector, the simultaneous determination of all elements. Information is obtained rapidly, and no element which is present can be overlooked. For standard excitation the lower detection limits are in the range of µg/g (ppm). This is sufficient for many applications, but for some environmental problems these detection limits are too high II SAMPLE PREPARATION The best procedure for sample preparation is to leave the sample as it is, but in many cases this is unrealistic. In the case of penguin feather we really did not make any interference on the sample. All amounts were put in the sample carrier and pressed in the same way. Nevertheless, the amounts of feathers were not enough to ensure infinitive thick layer. Particular difficulties occur, when very small amounts from the sample are available, as in the case of babies’ down. In this case, an approximation curve intensity versus sample mass was established for the normalization of the characteristic X-ray intensities obtained. Some of the samples were visually dirty and we tried to wash them with distillated water. Because of the grease on the feather, the differences in the results between the dirty and washed sample was insignificant and we decided to measure them in original conditions. The advantage of the measurements of all sample amounts is that you receive information from the whole sample and the reproducibility of the results is quite satisfied. III. INSTRUMENTATION Three spectrometric systems are available in our lab for X-ray analyses. The first one is equipped with a Si(Li) detector with a 12.5 mm Be window and a 180 eV energy resolution at a 5.9 keV Mn-Ka line. The data are acquired with a multichannel analyzer, interfaced to a personal computer that applies specialized software for spectra processing. This system is combined with an exciting head, based on an annular source Am241 with activity 3.7 GBq and three types of secondary targets. For the determination of heavy elements, essential for biology, the system uses the Dy secondary target, that allows detection of elements down to the Z=56. This construction permits the measurement of a great number of elements with a high sensitivity by achieving the desirable experimental conditions of a low background level and at a high-count rate. The second system is equipped specially for light element analysis with a Si(Li) detector with a 12.5 mm Be window and a 160 eV energy resolution for a 5.9 keV

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Mn-Ka line. An exciting head especially designed for low Z elements determination is equipped with three radionuclide sources Fe55. In the case of this construction, the distances source-sample and sample-detector are greatly reduced and optimized, which allows elements with atomic numbers Z=13 to 25 to be analyzed. During the measurements of zoological samples, traces of elements like Cu, Zn, Mn, Pb, very essential for biology, occurred. To acquire a higher sensitivity and a lower detection limit for these elements, another exciting system with a source Pu238 was constructed. Its energy of 13.613 keV is very suitable for exciting elements with Z=22 to 35. A specialized SDD detector with Peltie cooling and an energy resolution of 160 keV is connected to the exciting system .In this way, the full range of Z from 13 to 82 is covered. IV. SPECTRA PROCESSING To obtain results, the measured spectra must be evaluated as to line energies and line intensities, with only the net intensity used for quantification. The correctness with which the peak areas for the analyzed elements are determined is of crucial importance in order to obtain accurate and reliable results for their amounts. Different methods for energy-dispersive X-ray spectra processing have been investigated and now in the software package three different methods for peak area determination are included - namely the total peak area, linear and non-linear least square algorithms, and resolution enhancement procedures. V. CALIBRATION All the spectrometric systems need to be calibrated in order to obtain absolute concentration values from the intensities of the characteristic X-Ray lines. One of the main problem for the EDXRF method are the matrix effects. In the case of biological samples like penguin feather, this problem does not exist because the matrix is organic. That is why we established sensitivity curves for all the systems. As a first step, a set of pure elements was used, covering the energy range for the corresponding exciting source and an equation with a maximum fit to the experimental points was created. In this case, the intensities of the lines used have a good statistics and errors are minimized. The results obtained are in good agreement with the reference concentrations for the standard. The second step was to measure some real standards with similar ones, such as the sample matrix, namely organic. A set from different plant standards – Bowen’s kale, IAEA V10-Hay, orchard leaves, horse kidney, coppepode and fish flash were used

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for the creation of real sensitivity curve. (Figure 1). The estimated curves were of the same type. The IAEA standard V9 was used in order to check the results obtained with the sensitive coefficients. The results obtained are in good agreement with the reference concentrations for the standard. VI. ACCURACY, ERRORS AND DETECTION LIMITS The total error of the analysis consists of many errors, which can be provoked by the measurement of the intensity, reproducibility of the sample preparation and conversion of intensity into concentration. The total relative error of 7 – 10% was estimated during the calibration procedure as a standard deviation error of a previously prepared and analyzed standard with known element concentrations. The detection limits (DL) (the minimum amount that can be analyzed) were determined by the sensitivity and by the background in the X-ray spectrum. The use of three kinds of excitation gives us the opportunity to reach detection limit levels, which are sufficient for environmental control and monitoring purposes. VII. RESULTS Results from three year project are shown in Figure 2 to Figure 11. In Figure 2 to Figure 4, results for elements K, Ca, Cl, S, Fe, Zn, Br, Sr, Rb for feather from adults of the penguin species Pygoscelis Papua are shown. Two penguin colonies from two different islands – Livingston and Peterman are compared. Results from the years 2002 and 2004 are also compared. Practically no significant difference during the three years period was found. The results from different species are also quite similar. Differences in the concentrations between two islands are found. In Figure 5 and Figure 6, results for feather from adults of the penguin species Pygoscelis Antarctica are shown. This species has a colony only on Livingston island and the results are for the years 2002 and 2004. Practically no significant difference for the three-year period was found. In Figure 7 and Figure 8, results for the chemical concentration of baby down is shown. Quite great differences in the concentration of Ca and Sr between babies’ down and adults’ feathers are observed. Also, the presence of the element Ba in the down is observed. In Figure 9 to Figure 11, a comparison between averaged values from different points for Pygoscelis Papua is shown. A significant difference between the concentrations of Rb and Sr from the regions of Livingston and Peterman is observed.

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Figure 2.

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Figure 3.

Figure 4.

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Figure 6.

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Figure 7.

Figure 8.

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Figure 10.

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Figure 11.

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© PENSOFT Publishers APPROACH TO THE ESTIMATION OF THE SURVIVAL Bulgarian Antarctic Research OF PENGUINS A MULTICRITERIA 93 Sofia – Moscow Life Sciences, vol. 5: 93-99, 2006

A Multicriteria approach to the estimation of the survival of penguins BOYKO VACHEV1, ROUMIANA METCHEVA2 1

Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, [email protected] 2

Institute of Zoology, Bulgarian Academy of Sciences, [email protected]

ABSTRACT The main idea of the proposed approach is to use the conventional decision-making AHP (Analitic Hierarhical Process – AHP) methodology for multicriteria estimation of the survival of penguins. The respective model is based on available studies of penguins at the Antarctic bases of this project (identified basic morphological and biological parameters are prioritized and estimated for every marked penguin by use of data from data base). These parameters and studies have to be complemented by results of investigations in different Antarctic regions for several other parameters. The modelling is directed to a complex assessment and respective ranking of penguins by the rate of survival at the individual level. KEY WORDS Pygoscelis papua, Survival, Multi criteria decision making, Analytic Hierarchical Process

INTRODUCTION The idea of the proposed approach is to use the conventional decision-making Analitic Hierarhical Process (AHP) methodology for a multicriteria estimation of penguins’ survival. A multicriteria estimation of penguins’ survival based on the available observation and measurements was made. The approach used (AHP) was successfully applied in the last 15 years when the site selection was made for a repository for different levels of radioactive wastes (Evstatiev et al. 2005, Vachev, 2001, Vachev and Evstatiev, 1994).

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MATERIALS AND METHODS The study was carried out on a Gentoo colony at Livingston Island in the breeding seasons from 2002 to 2005. The penguins were weighed, the bill and flipper were measured and all caught birds were marked (Metcheva et. all, 2005; Williams, 1980). Only marked birds were included in the analyses. Several criteria for estimation of surveillance have been defined. This set includes not all accepted criteria, but some of them that could be measured or estimated. The chosen criteria are from the following three groups: morphological (Body weight – CR 1, Flipper length - CR 2, Bill length – CR 3, Bill color – CR 4); biological (Nest dimension – CR 5 and Nest position – CR 6) and physiological (Body temperature – CR 7). Most of these criteria are well known and accepted (Robertson, Wienecke. 1996) The following model is generated (Fig.1):

Fig. 1. AHP model of penguinssurvival asessement (level 1)

The priorities to the above-mentioned criteria were selected – following the same order (Table 1.): Tab. 1. Criteria priorities Criteria Priority

CR 1 0.730

CR 2 0.110

CR 3 0.110

CR 4 0.010

CR 5 0.034

CR 6 0.001

CR 7 0.005

The criteria were estimated under the 5 level non-linear scales. This non-linearity reflects the fuzziness of importance of the different level of intensity of the presence of criteria (different values). The more important criteria are more convex; the low important criteria

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are more concave. The general expression of the scales is P=F(CR), where P is the degree of preferability of the corresponding specimen according to a given criterion – P ∈ [0,1]: P= ((CRi – CRi min)/(CRi max- CRi min))b, where: CRi are the values of criteria I (or CRi scale levels), and b Î [0,∝] is a coefficient of non-linearity depending on the importance of the corresponding parameter and forming the convex curve for b£1 and a concave curve for b>1. We use the following scales: Tab. 2. Creation of non-Linear Typical Scales

CR 1 CR 2,3 CR 4 CR 5 CR 6 CR 7

0

0.25

0.5

0.75

1

b

Importance

0.000 0.000 0.000 0.000 0.000 0.000

0.933 0.287 0.002 0.000 0.000 0.000

0.966 0.536 0.044 0.016 0.001 0.000

0.986 0.772 0.274 0.178 0.056 0.013

1.000 1.000 1.000 1.000 1.000 1.000

0.05 0.90 4.50 6.00 10.00 15.00

73.0 11.0 3.4 1.0 0.5 0.1

The respective graphical presentation of scales of non-linearity and the resulting AHP model are shown below (Fig. 2 and Fig. 3):

Fig. 2. Non linear scales for criteria assessment

4 4 4 3 5 4 3 3 5 3 4 4 3 3 3 3 3 3 4 4 3 3 3 3 3 5 4 3 3 2

5 5 4 5 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 2 3 3 3 2 3 3 3 3 2

5 5 4 3 4 4 4 4 3 5 3 3 3 3 3 3 3 3 2 2 3 2 2 2 3 1 1 1 1 1

3 3 3 3 3 3 3 3 2 3 2 2 4 3 3 3 3 3 2 2 3 3 2 2 1 3 3 3 3 2

WEIGHT FLIPPER BILL BILCOLOR NESTD NESTP TEMP 0.730 0.110 0.110 0.010 0.034 0.001 0.005 0.9398 1.0000 0.9398 1.0000 0.8896 0.9466 0.8741 0.9301 0.8739 0.9299 0.8637 0.9190 0.8491 0.9035 0.8491 0.9035 0.8479 0.9022 0.8468 0.9010 0.8377 0.8914 0.8377 0.8914 0.8234 0.8761 0.8231 0.8758 0.8231 0.8758 0.8231 0.8758 0.8231 0.8758 0.8231 0.8758 0.8103 0.8622 0.8103 0.8622 0.7957 0.8467 0.7957 0.8467 0.7957 0.8467 0.7957 0.8467 0.7957 0.8467 0.7890 0.8395 0.7787 0.8286 0.7641 0.8131 0.7641 0.8131 0.7127 0.7583

Total 2002 2001 2003 2001 2001 2004 2002 2003 2001 2003 2001 2002 2004 2001 2002 2001 2004 2004 2001 2002 2004 2004 2001 2002 2004 2001 2002 2002 2001 2003

013 013 00-0631-A7B3*(16.02. 2004) 018 028 00-0611-C6A4*(2005) 174 00-0631-8FD5*(01.2005) 018 019 026 026 00-0611-9CC4*(2005) 029 029 174 00-0611-912E*(2005) 00-0611-B17B*(2005) 016 016 00-0611-96FC*(2005) 00-0611-A655*(2005) 119 119 00-0611-9488*(2005) 025 025 023 023 00-0631-BD22*(19.02.2005)

96

005 113 332 276 093 351 014 325 117 303 091 008 342 094 009 097 361 349 116 006 344 348 118 003 370 090 007 004 121 326

/RATINGS/

Table 3. Shortened ranking model table

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Fig. 3. AHP model of penguin survival assessment (level 2)

The next step is the estimation of different specimens under each criteria or building a ranking model. The shortened table (including not all samples but only those that have been encountered at least once) is given above (Table 3). RESULTS AND DISCUSSION

Survival assessment

As a result, we obtain a ranking of all individuals included in the model (all marked animals with available measurements by different criteria). The graphical representation of the result is shown in Fig. 4:

Fig. 4. Ranking graphic based on the AHP ranking model of penguin survival

The ranging area is from 0.75 to 1 and is divided into two areas [1.00,0.88] and [0.87,0.75].

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With a lower priority with respect to survival are 85 specimens, and 12 of them are encountered at least twice, or 14%. In the first area, with a higher rating of survival, 68 specimens are observed, and 18 of them are encountered at least twice, or 26%. These figures give some evidence of the applicability of this set of criteria for the estimation of the level of survival of penguins. (Two of the criteria introduced in the model illustrated above – nest dimensions and position are not applied due to the lack of sufficient data and the specific topography of the penguin site. The third criteria – a bill yellow spot, is disputable, is it only a reflection of some environmental changes or does it have some negative influences related, for example, to the presence of parasites? The last criterion, temperature, is of negligible importance). The rate of observed surviving penguins – approximately 20% (encountered for a second time) appears realistic. CONCLUSIONS On the basis of the discussion here we could make a conclusion about the applicability of the multicriteria approach to the estimation of the survival of penguins. The proposed model, set of criteria and priorities estimations is the first attempt to consider this important population characteristic from the point of view of the decision making theory and by the use of the respective approach. The good interpretability of the results obtained give us assurance that the proposed approach and the method are adequate and that a broadening of the set of the criteria applied and a longer observation (another 3 years) will offer information that is more precise and rich in content. ACKNOWLEDGEMENT This work was funded through grant INTAS Res. Project, Ref. 2001 - 0517 REFERENCES: AINLEY D. G., D. P. DEMASTER. 1980. Survival and mortality in a population of adeli penguin. Ecology, 61 (3), 522-530. EVSTATIEV. D, L. HRISTOSKOV, D. KARASTANEV, K. TODOROV, B. VACHEV. 2005. Report on survey and choice of potential sites for National repository for radioactive wastes, In: Development of the concept for radioactive wastes disposal and for survey and choice of potential sites for National repository for radioactive wastes”.

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METCHEVA, R., P.ZEHTINDJEV, A.SAVOV, E. TRAKIJSKA AND Y.YANKOV. 2005. Sex dimorphism established by body weight, beak and flipper length in Gentoo penguins (Pygoscelis papua) from Livingston Island. Acta Zool. Bulg. 57 (1) 65 – 71 ROBERTSON . G., B. WIENECKE. 1996. The fatter the better. Proceedings of the second int. conf. of the biology of the penguins. Surrei beatty, Sydney. SAATI T. L. 1991 Multicriteria Decision Making: the Analytic Hierarchy Process, Planning, Priority Setting, Resource Allocation, RWS. VACHEV B. I. AND EVSTATIEV D. 1994. Radioactive Wastes Management – AHP Application, Proceedings of the Third Symposium on the Analytic Hierarchy Process, Washington, DC, 585-596. VACHEV B. I. 2001. Radioactive Waste Disposal Site Selection, Proceedings of the International Symposium on the Analytic Hierarchy Process, Berne, Switzerland. WILLIAMS A. J. 1980. Aspects of the breeding biology of the Gentoo penguin Pygoscelis papua. Gefraut 70; 283-295.

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© PENSOFT Publishers Bulgarian Antarctic Research INSTABILITY OF WINTERERS BEFORE AND AFTER ANTARCTIC EXPEDITIONS CHROMOSOMAL 101 Sofia – Moscow Life Sciences, vol. 5: 101-106, 2006

Chromosomal Instability of Winterers Before and After Antarctic Expeditions KATERYNA AFANASIEVA, STANISLAV RUSHKOVSKY, VLADIMIR BEZRUKOV Department of General and Molecular Genetics, Kyiv Taras Shevchenko National University, 64 Volodymyrska Street, Kyiv, 01033, Ukraine ([email protected])

SUMMARY An estimation of parameters of chromosomal instability in human lymphocytes of winterers before and after some Ukrainian Antarctic expeditions (VI - VIII UAE) was carried out. The level of structural chromosome aberrations (metaphases with chromosome aberrations and chromosome aberration) and end-points of chromosomal segregation failure (metaphases with premature centromere division, premature centromere division, total premature centromere division, polyploid mitosis) were selected as parameters of chromosomal instability. The significant increase of the levels of metaphases with chromosome aberrations and chromosome aberration after the expeditions indicates the presence of damaging factors in Antarctica. The end-points of chromosomal segregation failure reflected features of groups of winterers and environmental conditions for each expedition. Key words: chromosomal instability, chromosome aberrations, chromosomal segregation failure, lymphocytes, Ukrainian Antarctic expeditions, winterers INTRODUCTION The environmental impact on the genome of different native Antarctic species is one of the priority studies carried out in Antarctica. These species have lived in Antarctic conditions for millions of years and they are adapted to such an environment. In contrast to native species, these conditions are not ordinary for the human organism, which must adapt to such extreme conditions. The adaptation implies changes in the functioning of all the organs and the physiological system of the organism, including the genome (DECAMPS, 2004). Thus the influence of Antarctic factors should be studied for people living in Antarctica for a long time.

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The main investigations in this area were carried out for some medical and physiological indexes. But there is no research describing the consequences of the influence of Antarctic conditions on the human genome. These studies must determine a presence of any genotoxic factor, its nature and mechanism of action as do all conventional studies in the field of genetic toxicology (ALBERTINI, 2000). The analysis of chromosomal anomalies in human peripheral blood lymphocytes is a universally recognized method for the estimation of the influence of different factors on the human genome (TAWN, 1991). This assay allows the evaluation of the damaging factors according to the level and spectrum of numerical and qualitative chromosomal aberrations. This article offers a comparative analysis of the level and spectrum of chromosomal instability of Ukrainian overwinterers before and after wintering in Antarctica. MATERIALS AND METHODS Blood samples for chromosome assays were obtained from the group of candidates (33 healthy men) for VI - VIII UAE during medical examinations before and after expeditions. Blood was collected by venepuncture. Samples of whole blood (1 ml) were cultivated in 5 ml of IMDM medium. PHA-P (“GENOME”, Donetsk) 5 mg per 1 ml of medium was used for blast-transformation of lymphocytes. Cultures were incubated at 37 °C for 52 h. Colchicine (1 mg/ml, final concentration) was added 1.5 – 2 hours before the end of cultivation. After that cultivation cells were treated with 0.075 M KCl and fixed in a methanol:acetic acid (3:1) mixture. Metaphase plates were prepared by dropping cell suspension onto cool moist slides. Slides were stained by the routine Giemsa technique according to the standard procedure (TAWN, 1991). The level of structural chromosome aberrations (metaphases with chromosome aberrations and chromosome aberration) and end-points of chromosomal segregation failure (metaphases with premature centromere division, premature centromere division, total premature centromere division, polyploid mitosis) were selected as parameters of chromosomal instability. RESULTS AND DISCUSSION Structural chromosome aberrations The average level of metaphases with chromosome aberrations (AM) was 2.25±0.37 % before and 3.01±0.30 % after expeditions. The average rate of chromosome aberration (ChA) was and 2.44±0.41 % before and 3.20±0.34 % after

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expeditions (Figure 1). The values of ChA and AM did not differ significantly (we found only a few metaphases with more than one aberrations). The differences in the levels of investigated end-points of chromosomal instability before and after expeditions were statistically significant (p<0.05). A strong tendency to an increase in the chromosomal aberration frequency was observed for each expedition. This result implies an existence of some Antarctic factors that can induce chromosomal aberrations in human lymphocytes.

Figure 1. The levels of metaphases with chromosome aberrations and chromosome aberration before and after expeditions

It is well known that chemical mutagens induce chromosome aberrations of the chromatid type while ionizing radiation induces the aberrations of the chromosomal type (HSU, 1996). Thus, an analysis of the aberration spectrum allows one to determine the possible nature of the main damaging factor. The predominant contribution in this spectrum was from aberrations of the chromatid type both before and after expeditions. Any alteration of the chromatid to chromosomal aberration types of ratio was not observed after expeditions (Figure 2). Hence, a more detailed analysis of the spectrum was carried out. In general, the spectrum of aberrations was similar both before and after expeditions. The prevalent chromosome aberration was chromatid breaks. The markers of radiation impact (dicentric chromosomes and centric rings) occurred in a few cases (Figure 3). The analysis of the spectrum changes indicates that neither chemical mutagens nor physical mutagens can be considered as essentially more important damaging factors in

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Figure 2. Chromatid to chromosomal aberration types before and after expeditions

Antarctica in comparison with Ukraine. However, growth of AM and ChA was observed after expeditions. This increase may be explained by the presence of nonspecific factors which influence the human genome. Such factors may include longterm isolation, inversion of seasons, achromatization, desynchronoze and other factors called “stress-factors”.

Figure 3. Detailed spectrum of chromosome aberrations before and after expeditions. SF – single fragments, AE – chromatid asymmetrical exchanges, SE – chromatid symmetrical exchanges, DF – double fragments, D – dicentric chromosomes, AR – acentric rings, CR – centric rings, T+I – translocations and invertions.

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End-points of chromosomal segregation failure Some mutagenic factors can damage the genome through a disruption of normal chromosomes segregation during mitosis. The major end-points of such influence are metaphases with premature centromere division (MPCD), premature centromere division (PCD), total premature centromere division (TPCD), polyploid mitosis (PM) (PARRY, 2002). The average rates of these parameters are shown in Table 1. Table 1. The average rates of end-points of chromosomal segregation failure. Before expeditions After expeditions

MPCD, %

PCD, %

TPCD, %

PM, ‰

0.96±0.15 0.89±0.17

1.76±0.39 1.33±0.30

0.33±0.09 0.39±0.06

0.14±0.03 0.13±0.02

The differences in the levels of end-points of chromosomal instability investigated before and after expeditions were statistically insignificant. There are no pronounced tendencies in the changes of the levels of PCD and TPCD for each expedition. The differences in these parameters between the expeditions may reflect some specific features of the groups of winterers as well as environmental conditions for each expedition. Thus, the increase of the levels of metaphases with chromosome aberrations and chromosome aberration after expeditions indicates the presence of damaging factors in Antarctica. The stable tendencies to a chromosomal aberration rise for each expedition suggest uniformity of these factors. We recommend registration of endpoints of chromosomal segregation failure as parameters reflected features of groups of winterers and environmental conditions for each expedition. REFERENCES ALBERTINI R. J. , D. ANDERSON, G.R. DOUGLAS et al. 2000. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. – Mutation Research, 463:111-172. DECAMPS G., ROSNET E., BACHELARD C. 2004. Psychological reactions to stress during a winter-over in Antarctica: definition of the winter-over adaptive troubles. – Abstract Volume. XXVIII SCAR Open Science Conference “Antarctica and the Southern Ocean in the Global System”, 26-28 July, Bremen, Germany. - Bremen: AWS:196-197. HSU T.C., WU X., TRIZNA Z.1996. Mutagen sensitivity in human. A comparison between two nomenclature systems for recording chromatid breaks. – Cancer Genet Cytogenet, 87:127-132 PARRY J.M., AL-OBAIDLY A., AL-WALHAIB M., et al. 2002. Spontaneous and induced aneuploidy, considerations which may influence chromosome malsegregation. - Mutation Research, 504:119-129.

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TAWN E.J., HOLDSWORTH D. 1991. Mutagen induced chromosome damage in human lymphocytes. In: D. E. Rooney and B.H. Czepulkowski (Eds.) . – Human Cytogenetics. Volume II. Malignancy and acquired abnormalities. A practical approach. Irl press, Oxford, New York, Tokyo:189-208.