Anopheline Species Complexes in South and South-East Asia (SEARO Technical Publications)

SEARO Technical Publication No. 57

Anopheline Species Complexes in South and South-East Asia

SEARO Technical Publication No. 57

Anopheline Species Complexes in South and South-East Asia

WHO Library Cataloguing-in-Publication data World Health Organization, Regional Office for South-East Asia. Anopheline Species Complexes in South and South-East Asia. 1. Anopheles 2. Species Specificity 3. Sibling Relations 4. Insect Vectors 5. South-East Asia 6. Asia, Western ISBN

978-92-9022-294-1

NLM Classification No. QX 515

© World Health Organization 2007 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. For rights of reproduction or translation, in part or in toto, of publications issued by the WHO Regional Office for South-East Asia, application should be made to the Regional Office for South-East Asia, World Health House, Indraprastha Estate, New Delhi 110002, India. The designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Printed in India

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Anopheline Species Complexes in South and South-East Asia

Contents Foreword ............................................................................................................... v Acknowledgements ............................................................................................... vi 1. Introduction ..................................................................................................... 1 2. Techniques used in the recognition of Species Complexes ................................ 7 3. Species Complexes ......................................................................................... 17 3.1

The Annularis Complex ........................................................................ 17

3.2

The Barbirostris Complex ...................................................................... 20

3.3

The Culicifacies Complex ..................................................................... 22

3.4

The Dirus Complex .............................................................................. 33

3.5

The Fluviatilis Complex ......................................................................... 41

3.6

The Leucosphyrus Complex .................................................................. 46

3.7

The Maculatus Complex ....................................................................... 48

3.8

The Minimus Complex ......................................................................... 55

3.9

The Philippinensis-Nivipes Complex ..................................................... 62

3.10 The Punctulatus Complex ..................................................................... 65 3.11 The Sinensis Complex ........................................................................... 69 3.12 The Subpictus Complex ........................................................................ 73 3.13 The Sundaicus Complex ....................................................................... 76 3.14. The Anopheles stephensi variants .......................................................... 79 4. Prospects for the future .................................................................................. 84 5. References and select bibliography ................................................................. 87 Anopheline Species Complexes in South and South-East Asia

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Foreword

V

ector-borne diseases continue to be a major health problem in the world. The worsening malaria situation during the 1980s led the World Health Organization (WHO) to declare the control of malaria as a global priority. The World Declaration on Malaria, adopted in Amsterdam in October 1992, committed WHO Member States to the worldwide intensification of control efforts against this disease. Accordingly, a global Malaria Control Strategy was developed which laid emphasis on the following key elements: case management; capacity building for control; containment of epidemics; and basic and applied research. Halting the incidence of malaria is also highlighted as one of the targets to be achieved under the United Nations Millennium Development Goals (MDGs).

It is very important that vector control, as a part of the global as well as the regional malaria control strategy, should succeed. Its success would depend on a systematic review of the available information on vector species and their biology, and of the vector control options and their selective use. Most of the anophelines that are involved in the transmission of malaria in the South and SouthEast Asian countries have been identified as species complexes. Species complexes are of common occurrence among anopheline taxa. More than 30 Anopheles taxa have been identified so far as species complexes and they are important vectors of malaria in different parts of the world. Members of a species complex, commonly known as sibling species, are reproductively-isolated evolutionary units with distinct gene pools and, hence, differ in their biological characteristics.

Differences in the biological characteristics of members of the complexes have an important bearing on malaria transmission dynamics. It is, therefore, imperative to determine sibling species composition and their bionomics as well as their roles in the transmission of malaria. In 1998, WHO published as a technical publication* Anopheline species complexes in South-East Asia authored by Dr Sarala K. Subbarao. This book has received much appreciation both from researchers and programme managers. Since its publication, several papers on species complexes identification tools, especially molecular-based tools, formal designation of members of complexes, and the phylogenetic relationship between members of a complex and also between the complexes have been published. In view of the importance of species complexes in malaria control operations, an updated edition has been prepared to provide the latest information on this important subject. This is part of our commitment to highlight and disseminate the knowledge on species complexes which is so vital to malaria control strategy, especially when target-specific selective and sustainable vector control is urgently needed. In addition to the South-East Asia Region, the present edition covers the work done on the species complexes prevalent in the South Asian countries as well. It presents a clear summary of the work done on anopheline cryptic species, and I am sure it will be very useful for field malaria entomologists, malaria control programme managers and basic researchers working on species complexes.

Samlee Plianbangchang, M.D., Dr.P.H. Regional Director *WHO Technical Publication, SEARO No. 18 (1998).

Anopheline Species Complexes in South and South-East Asia

v

Acknowledgements This edition of Anopheles Species Complexes in South and South-East Asia has been produced by the World Health Organization's South-East Asia Regional Office, Department of Communicable Diseases, Communicable Diseases Control group. The author of the earlier Anopheles Species Complexes in South-East Asia (1998), Dr Sarala K. Subbarao, was commissioned to prepare the revised edition. The issuing of a new edition reflects the fact that new identification tools have been developed and identification of species is critical in control programmes for several complexes. Professor Chris Curtis, Dr Catherine Walton, Professor Nora J. Besanky and Dr Yeya Torre have made very valuable suggestions that have enriched the quality of the monograph. Professor Curtis also provided detailed editorial corrections of the manuscript. Dr K. Raghavendra, Dr Suprabha G. Pulipparacharuvil and Mr O.P. Singh have provided necessary information and help, and Mr U. Sreehari is acknowledged for his help in the preparation of the document. Dr Subbarao also wishes to recognize support given by family members.

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Anopheline Species Complexes in South and South-East Asia

1. Introduction

M

osquitoes are ubiquitous and have a tremendous reproductive potential and great adaptability to different ecological conditions. Adding to their innate ability to adapt, humans are providing them with conditions, which are highly congenial for their multiplication. There are about 4500 mosquito species in different parts of the world, belonging to 34 genera in the family Culicidae, order Diptera, class Insecta and phylum Arthropoda. Some of the mosquito species transmit diseases and consequently form an important target for control in public health programmes. Species belonging to the genus Anopheles transmit malaria. Approximately 424 formally designated anophelines have been identified morphologically, out of which only about 70 species are considered to be the main vectors of malaria in the world. The total number of species has now reached more than 500 because of the identification of biological species within morphologically indistinguishable taxa. Among many Diptera genera, such as Drosophila, Simulium, Anopheles, Aedes, Sciara and Chironomus, the populations within a morphologically defined species do not interbreed. These morphologically-similar, reproductively-isolated species within a taxon are known as cryptic, sibling or isomorphic species, and the taxon as a whole as a species complex. Sibling species are found in other animal groups also. Mayr (1970) gives a detailed account of the groups where sibling species have been found. Most of the anophelines that are implicated in the transmission of malaria in the South and South-

East Asian countries have been identified as species complexes, which include Annularis, Barbirostris, Culicifacies, Dirus, Fluviatilis, Leucosphyrus, Macaulatus, Minimus, Philippinenisis-niyipes, Punctualatus, Sinensis, Subpictus and Sundaicus. Maculipennis was the first complex described in the genus Anopheles. The discovery of this complex resolved the epidemiological paradox that prevailed in the 1930s when there was malaria transmission in Europe and North America. In some areas in southern Europe, there was no malaria in spite of the presence of An. maculipennis, which led to the expression “anophelism without malaria”. Detailed studies on the biological and cytogenetic characters of these populations have now identified eight sibling species in this taxon in Europe. Recognizing the significance of species complexes in malaria epidemiology, Mayr (1970) goes on to state that, “Perhaps the most celebrated case of sibling species is that of the malaria mosquito complex in Europe,” referring to the Maculipennis Complex. So far, about 30 complexes have been described in different regions of the world. The number of sibling species varies in each complex and a total of about 145 species have been identified among these complexes (Table 1). Among the members of the Maculipennis Complex in Europe, An. atroparvus, An. labranchiae, An. messeae, An. sacharovi and An. subalpinus were vectors because they would at least sometimes bite humans, while An. maculipennis sensu stricto, An. melanoon and An. beklemishevi were non-vectors because they were generally entirely

Anopheline Species Complexes in South and South-East Asia

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zoophilic. Some of these species breed in fresh water and others in brackish water. In the Gambiae Complex in Africa, while five sibling species were recognized as vectors with varying levels of efficiency in transmitting malaria, An. quadriannulatus was found to be a non-vector (Coluzzi, 1988). Later, within An. quadriannulatus, two zoophagic sibling species, A and B, were more recently recognized (Hunt, Coetzee and Fetenne, 1998). In India, of the five An. culicifacies sibling species, species A, C, D and E are vectors while species B is a non- vector (Subbarao, Adak and Sharma, 1980; Subbarao et al., 1988, 1992; and Subbarao, Nanda and Raghavendra, 1999). In areas where An. culicifacies A and B are sympatric, DDT spraying in many areas has caused an epidemiological impact on the transmission (Sharma et al., 1986) due to reduction in species A, which is a vector (Subbarao et al., 1988) and is more susceptible to DDT than species B (Subbarao, Vasantha and Sharma, 1988). These are a few examples that demonstrate the differences between sibling species within a species complex and highlight the importance of identifying sibling species in malaria control programmes. The genetic distinctness of each sibling species comes from the definition of the biological species concept whereby each species is an actually interbreeding natural population that is reproductively isolated from other such populations. Reproductive isolation between sibling species is maintained either by pre- or post-mating barriers or both. The post-mating barrier is expressed in the form of non-viability of hybrid progeny at immature stages or hybrid sterility or both. The pre-mating barrier(s) is due to failure in copulation because of physical incompatibilities or behavioural differences in mating procedures. Thus, each sibling species has a specific mate recognition system that is distinctly different from that of the other sibling species in the complex. Population genetic studies involving chromosomal inversions and DNA markers 2

(Besansky et al., 1994, 1997; Garcia et al., 1996) have suggested the possibility of gene flow occurring between members of the Gambiae Complex. Clear evidence for unidirectional introgression leading to gene flow from An. arabiensis to An. gambiae came from a multilocus molecular marker study (Besansky et al., 2003) and a novel population genetic analysis (Donelly et al. 2004). It is intriguing that gene flow is not uniform throughout the genome, i.e. genomes are mosaic with respect to gene flow (Garcia et al., 1996; Besansky et al., 2003). Similarly, introgression was observed between members of the Dirus Complex found in South-East Asia (Walton et al., 2001). The fact that introgressive hybridization occurs between sibling species, this may again raise the issue of whether the sibling species are full biological species or not and whether such introgression would change the biological characterstics of these species and affect vector control strategies. There is, however, so far no evidence that An. gambiae and An. arabiensis have mixed and the characterstic differences of these two species have disappeared, and also very few hybrids were found in nature in spite of their sympatric association over large areas of their distribution (Besansky et al., 2003). Similarly, only a single hybrid of An. culicifacies species A and B was found among several thousands of specimens screened over large geographical areas where these two species are sympatric (Subbarao, unpublished). These observations indicate that introgressive hybridization, even if it occurs between sibling species, is a rare event and pre-mating isolation barriers are strong. Thus, the sibling species should continue to be considered as full biological species and there does not seem to be any likelihood of any of the sibling species changing their biological characters in the near future that should lead to changes in vector control strategies that are being contemplated. Coluzzi (1988) highlights the importance of identification of sibling species by saying that

Anopheline Species Complexes in South and South-East Asia

failure to recognize sibling species of anopheline taxa may result in failure to distinguish between a vector and a non-vector; hence, the assessment of the impact of control measures may be seriously misleading if they are carried out on a morphologically defined taxon which could be a mixture of two or more sibling species. The discovery of sibling species adds a new dimension to vector control. With this background, an effort was made to compile the information available on anopheline species complexes prevalent in South and South-East Asia in a single document. The main objective of this effort was to bring to the notice of researchers, field workers and programme organizers, up-todate information available on species complexes prevalent in South and South-East Asian countries. The first edition of the document was published in 1998. The present document contains the distribution of malaria vectors in South Asia, South-East Asia and neighbouring countries (Figure 1). Most of these anophelines implicated in malaria transmission are species complexes. The species complexes and sibling species discovered in each of the complexes, the formal designations given to sibling species and their prevalence in the South and SouthEast Asian countries are shown in Tables 2a and 2b. The techniques currently being used for the identification of species complexes are described in Chapter 2 and the details of the complexes are given in Chapter 3. The complexes covered in this chapter are: Annularis, Barbirostris, Culicifacies, Dirus, Fluviatilis, Leucosphyrus, Maculatus, Minimus, Philippinensis-nivipes, Sinensis, Subpictus and Sundaicus. An. stephensi, though not yet identified as a species complex, is included because it is an important vector and is a complex of different variants/ecological races. For each of the complexes, information on the types of evidence used for the identification of sibling species, the number of sibling species identified, the techniques that have been developed for the identification of sibling species and the distribution and biological characteristics of the members are presented.

Malaria control strategies are not uniform and at different times and in different areas, programme organizers demand specific strategies to cope with local situations. To meet these challenges, there is a need to generate field data to establish the prevalence of species at the lowest administrative units possible for the implementation of effective control strategies. These aspects are covered in Chapter 4. The references cited are listed by chapter and by complex in Chapter 5. Table 1 : Anopheline species complexes identified so far Complexes

Coustani Gambiae Funestus Marshallii Nili Lungae Punctulatus Annulipes Claviger Maculipennis Quadrimaculatus Albitarsis Crucians Freeborni Nuneztovari Pseudopunctipennis Punctimacularus Oswaldi Annularis Barbirostris Culicifacies Dirus Fluviatilis Gigas Leucosphyrus Lindesayi Maculatus Minimus Philippinensis-nivipes Sinensis Subpictus Sundaicus

No. of species identified 2 7 9* 4 4 3 11 7 2 8 5 5 4 6 2 2 2 2 2 2 3 5 7 4 3 4 4 9 5 3 4 4 4+1**

Distribution in zoogeographical regions Afrotropical Afrotropical Afrotropical Afrotropical Ethiopian Australasian Australasian Palearctic Palearctic Palearctic Nearctic Nearctic Neotropical and Nearctic Neotropical Neotropical Neotropical Neotropical Neotropical Neotropical Oriental Oriental Oriental Oriental Oriental Oriental Oriental Oriental Oriental Oriental Oriental Oriental Oriental Oriental

Source: Information in this table has been compiled from Harbach (2004) and other published and unpublished documents. * The Funestus Group consists of nine species that are morphologically similar at adult stage and, of these, four belonging to the Funestus Subgroup are morphologically indistinguishable at all stages. ** New cytotype

Anopheline Species Complexes in South and South-East Asia

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Table 2a : Species complexes recognized and number of sibling species identified in South Asian countries Anopheles Complexes

Sri Lanka

Annularis (2)

+

Barbirostris (4)

+

Culicifacies (5)

2 B, E

Iran

1 A

Afghanistan

Pakistan

India

Nepal

+

+

+

+

+

+

2 A, B +

+

+

+

2 A, B

5 A, B, C, D, E

1 B

+

+

2 D, E

+

+

1 D

1 A

Dirus (7)2 Fluviatilis (4)

1 T

Bhutan Bangladesh

+

+

4 S, T, U, V

+

+

+

3 B, H, I

2 B, H

4 B, C, H, I

3 B, H, I

+

1 B

+

+

Leucosphyrus (4)2 Maculatus(9)2

1 B

Minimus (5)

+

1 A

Philippinensis/ nivipes (3)

2 n(A), p

+

4 A, B, C, D

+

+

Punctulatus (11) Sinensis (4) Subpictus (4) Sundaicus(4+1)2

4

2 A, B

+

+

1 cytotype D

+

+ +

Anopheline Species Complexes in South and South-East Asia

Table 2b : Species complexes recognized and number of sibling species identified in South-East Asian countries Anopheles Complexes

Myanmar

Thailand

Laos +

Viet Nam

Cambodia

Malaysia

Indonesia

Timor- Philippines Leste

Annularis (2)

+

+

+

+

+

+

+

Barbirostris (4)

+

+

+

+

+

3 A, B, C

+

Culicifacies (5)

+

1 B

1 B

1 B

Dirus (7)2

1 D

5 A, B, C. D, F

1 A

1 A

2 B, F

1 B

Fluviatilis (4)

+ 2 A, b

3 A, B, b 1 B

Leucosphyrus (4)2 Maculatus (9)2

1 A 3 A, B, C

7 A, B, C, G, H, I, K

+

3 A, B, I

1 B

1 B

Minimus (5)

C

4 A, B, C, D

2 A, C

2 A, C

1 A

+

Philippinensis/ nivipes (3)

+

3 n (A, B), p

n, p

n, p

n, p

+

+

+

2 D, J

+

+

Punctulatus (11)

+

2 f, c

Sinensis (4)

+

2 s, sin

Subpictus (4)

+

Sundaicus(4+1)2

+

+

+

+

1 1

+

+

+

+

+

+

1 A

1 A

1 A

1 s. s

3 A, B, C

+

+

( ) No. of sibling species identified in the complex; + Species present but sibling species composition not known; b— balabacensis, n—nivipes (This taxon has two sibling species A and B), p— philippinensis, f—faurauti, c—clowi, s— sineroides, sin—sinensis, nim—nimophilous, s. s.—senso stricto 1

Newly identified sibling species are initially designated either with letters of the English alphabet or occasionally with numbers which are subsequently dropped and are formally designated using binomial nomenclature

2

Sibling species of the following complexes have been given the formal designations:

Dirus Complex dirus (A) cracens (B) scanloni (C) baimaii (D) elegans (E) nemophilous (F)

Leucosphyrus Complex leutens (A) leucosphyrus s.s. (B)

Maculatus Complex sawadwangporni (A) maculatus s.s.(B) dravidicus (C) greeni (D) notonandai (G) willmori (H) pseudowillmori (I) dispar (J)

Anopheline Species Complexes in South and South-East Asia

Sundaicus Complex epiroticus (A) sundaicus sensu stricto (s. s.)

5

6

Anopheline Species Complexes in South and South-East Asia

IRAN

An. minimus

BHUTAN An. dirus An. fluviatilis

An. barbirostris An. dirus An. farauti An. koilensis An. punctulatus An. subpictus An. sundaicus An. aconitus* An. balabacensis* An. bancrofti* An. karwari* An. letifer* An. leucosphyrus* An. ludlowe* An. nigerrimus*

INDONESIA

An. culicifacies An. annularis* An. subpictus*

SRI LANKA

THAILAND

Java

An. balabacensis An. donaldi An. flavirostris An. leucosphyrus An. sundaicus

MALAYSIA East Peninsular Malayasia Malaysia

An. minimus An. aconitus* An. maculatus* An. nivipes* An. philippinensis* An. vagus*

LAOS

Borneo

Sulawesi

An. dirus An. minimus An. sundaicus

PHILIPPINES

BRUNEI

An. dirus An. maculatus An. minimus An. aconitus* An. annularis* An. leucosphyrus* An. philippinensis* An. sundaicus*

SINGAPORE

Sumatra

An. dirus An. minimus

MYANMAR

An. campestris An. letifer An. maculatus An. sundaicus An. nigerrimus*

An. minimus An. aconitus* An. annularis* An. philippinensis* An. sundaicus*

BANGLADESH An. dirus

An. annularis*

An. fluviatilis

NEPAL

An. aconitus An. annularis An. barbirostris An. maculatus An. minimus An. subpictus An. sundaicus

TIMOR-LESTE

An. dirus An. minimus An. sundaicus An. aconitus* An. barbirostris* An. campestris* An. indefinitus* An. jeyporensis* An. maculatus* An. nimpe* An. nivipes* An. philippinensis* An. sinensis* An. subpictus* An. vagus*

VIETNAM

An. dirus An. minimus An. sundaicus An. aconitus* An. barbirostris* An. jeyporensis* An. maculatus* An. nivipes* An. philippinensis* An. subpictus* An. vagus*

CAMBODIA

N

Source: Malaysia — Indra Vythalingam; Cambodia, Laos and Viet Nam – Data from the INCO-DEV Malvecasia project no. IC4-CT-2002-100041-Project Coordinator Professor Marc Coosemans—Sylvia Manguin; Nepal and Bhutan— N. L. Kalra; Timor-Leste— SEA-VBC-82; For all other countries – Kondrashin and Rashid (1987), Rao (1984), and from published and unpublished documents.

Map not to scale

An. culicifacies An. stephensi An. fluviatilis

PAKISTAN

MALDIVES

Primary vectors * Secondary vectors/local importance

An. culicifacies An. dirus An. fluviatilis An. minimus An. stephensi An. sundaicus An. annularis* An. jeyporensis* An. philippinensis* An. varuna*

INDIA

An. culicifacies AFGHANISTAN An. d'thali An. annularis An. fluviatilis An. culicifacies An. pulcherimus An. maculatus An. sachorovi An. stephensi An. stephensi An. superpictus An. maculipennis s.s.

Figure 1: Malaria vectors prevalent in South Asia, South-East Asia and neighbouring countries

2. Techniques used in the recognition of Species Complexes Group(s) of individuals within a species sometimes exhibit distinct differences with reference to resting habitats, preference to feed on a host, the rate of development of resistance to insecticides, susceptibility to acquiring infection, and so on. All these differences may indicate the presence of isomorphic species within a taxonomic species (defined morphologically), but these differences cannot confer species status on populations. Hence, genetic techniques that can demonstrate reproductive isolation within a morphologically similar taxonomic species are needed. Table 3 gives the methods which are available to researchers. Crossing experiments, chromosomal variations and electrophoretic variations at enzyme loci have been extensively used in studies to recognize species complexes. The chromosomal variation and electrophoretic variation at enzyme loci provide evidence for the recognition of species complexes; later the variations are also used in the development of diagnostic techniques/assays to identify sibling species. Cuticular hydrocarbon analysis and molecular approaches are generally used to develop diagnostic assays for the identification of sibling species which have already been recognized by other techniques. Sibling species by definition are species without easily observable morphological differences. A careful examination may sometimes reveal morphological differences that are minute and may be restricted to a particular stage in the life-cycle. The techniques (Table 3) and the principles behind these techniques have been

Table 3: Techniques used in the identification of species complexes z z z

z z z z

z

Morphological variations Crossing experiments Mitotic and meiotic karyotypes – Structural variations – Heterochromatin variations Polytene chromosomes Electrophoretic variations Cuticular hydrocarbon profiles Molecular approaches – DNA or RNA probes Allele specific polymerase chain reaction (ASPCR) – Restriction fragment length polymorphism (RFLP) – Random amplified polymorphic DNA (RAPD) – Sigle strand conformational polymorphism (SSCP) – Heteroduplex analysis (HDA)

described in several papers. Reviews by White, Coluzzi and Zahar (1975), Miles (1981), Green (1985), Coluzzi (1988), Green et al. (1985), Service (1988), Subbarao (1996) and Black and Munstermann (1996, 2004) are a few that describe and discuss these techniques. Readers are also referred to a WHO document prepared by Zahar (1996). This is a review of literature published between 1974 and 1994 on vector bionomics and the epidemiology and control of malaria. The document also includes a compilation of species complexes of the South-East Asia and West Asia regions. Another article recommended is by Harbach (2004). This article is an update of the internal classification of the genus Anopheles, which was earlier reported by the same author in 1994 (Harbach, 1994). The article lists species, species complexes, subgroups, groups and

Anopheline Species Complexes in South and South-East Asia

7

series recognized (formally and informally) so far in the genus. Reviews by Green (1985) and Coluzzi (1988) are strongly recommended for all those who work in this area. Reviews by Besansky, Finnerty and Collins (1992), Hill and Crampton (1994), Collins et al. (2000), Kryzywinski and Besansky (2003) and Black and Munstermann (2004) are also recommended for those who intend to use or develop molecular techniques for the identification of sibling species, and by Philipps et al. (1988) for cuticular hydrocarbon analysis. White (1977) and Service (1988) discussed and described the role of morphological characters in the investigation of species complexes. For the benefit of readers a few salient points are mentioned below.

Morphological variations Morphological characters that are often used to identify adults of anopheline species are largely confined to scale pattern and colour and their distribution. Characters that are used in the description of immature stages are sculpture of eggs, setation and pigmentation of larvae, and the forms of paddles and trumpets as well as chaetotaxy of pupae. Spermatheca and spiracular morphology are also used in the identification of species. In addition to light microscope examination for the specific characters, scanning and transmission electron microscopes are also used to study morphological variations. Morphometrics has proved useful in studying some species complexes when used in conjunction with statistical analyses. For details see White (1977) and Service (1988). White (1977) states that morphological studies should not come too early in the process of detecting anopheline sibling species since it might be misleading to characterize taxa which have not been identified by trustworthy techniques such as cross-breeding experiments or cytological or biochemical characterizations.

8

Crossing experiments The assortative mating observed between sibling species in nature due to pre-mating barrier (s) generally breaks down in the laboratory and different sibling species mate at random and produce hybrid progeny. Genetic differences between sibling species are expressed in the form of non-viability of hybrid progeny at immature stages, hybrid sterility or both. Hybrid males in one or both crosses are sterile and hybrid females are generally fertile. Therefore, hybrid sterility is used as the criterion in designating populations as separate species. Hybrid males exhibit partial development of reproductive organs (the extent of development ranges from atrophied testes and vas deferens to fully developed testes but without sperm; accessory glands and ejaculatory duct are generally normal) and do not produce progeny when crossed. For species which do not mate in laboratory cages, artificial mating methods can be adopted. Thus, laboratory crossing experiments demonstrate post-mating barriers and establish the species status of the isomorphic populations. An. gambiae was first recognized as a species complex from the results observed between two strains which were crossed to study the genetics of resistance to an insecticide (Davidson and Jackson, 1962). It may be noted that though these postmating barriers are studied between members of the complexes, they are not necessarily required to give populations species status. Furthermore, species which exhibit a premating isolating mechanism need not necessarily have any post-mating barrier, as has been observed between species B and C of the Culicifacies Complex (Subbarao, Vasantha and Sharma, 1988). F1 hybrid males of reciprocal crosses between species B and C are fully fertile. Dobzhansky (1970) reports that viable and fertile hybrids may be obtained in experiments between undoubtedly distinct species that maintain complete reproductive isolation in nature. Therefore, the reproductive status of hybrid males is not

Anopheline Species Complexes in South and South-East Asia

always diagnostic in the recognition of species complexes. While studying post-mating barriers, a point to be remembered is that for colonies established from species-specific diagnostic characters such as fixed inversions, enzyme electromorphs of progeny from single female cultures have to be used. A laboratory colony established from natural populations may be a mixture of two or three sympatric sibling species.

Cytogenetic techniques Polytene chromosomes Anopheline females in the semi-gravid stage have the best polytene chromosomes in ovarian nurse cells (Coluzzi, 1968). Larvae at the IV instar stage have polytene chromosomes in salivary glands. For those anopheline species which do not have good ovarian polytenes, larval salivary chromosomes can be used ( but salivary gland polytene chromosomes are not very good in most anophelines). The advantage with adult females is that ovaries can be removed and fixed in modified Carnoy’s fluid (1:3 glacial acetic acid:methanol) and can be used at any time. Another advantage is that the same female can be studied for other parameters such as host preference, presence of sporozoites/sporozoite antigen, susceptibility to insecticides, etc. The recommended references for the preparation of polytene chromosomes are: for ovarian polytene chromosomes from adult females, Green and Hunt (1980), and for salivary gland polytene chromosomes from IV instar larvae, Kanda (1979). Hunt and Coetzee (1986) describe storing of fieldcollected mosquitoes in liquid nitrogen for correlated cytogenetic, electrophoretic and morphological studies. The preparation of polytene chromosomes from adult females is not difficult. Polytene chromosomes are the result of repeated replication of chromosomes at interphase without nuclear division, the process being known as endomitosis.

Chromatids after division remain attached, causing thickening of chromosomes which results in the appearance of long ribbon-like structures with dark and light horizontal portions representing band and interband regions respectively. The dark and light regions represent differential condensation of chromosomes. The banding pattern of each chromosome is specific in a given species; thus, each species differs from others in characteristic banding pattern. Any changes in the pattern can be easily detected. In the polytene chromosome complement, only euchromatic regions are seen and the heterochromatic portions of the chromosomes which are under-replicated are not seen. Therefore, in anophelines, the short arm of the X-chromosome and Ychromosome are not seen in the polytene complement. In some species a definite chromocentre is seen. In such cases, all the chromosome arms are seen attached to the chromocentre by their centromeric ends. Generally, this is not the case with anophelines and chromosome arms are seen separately; occasionally, the two arms of a chromosome are seen attached at the centromeric ends. Homologous chromosomes exhibit high affinity for pairing and, therefore, are seen as a single chromosome. In anopheline cytogenetics literature, two types of designations are seen for the polytene chromosome arms: (i) the two arms of a chromosome are designated as right (R) and left (L) arms; this system is followed by Drosophila cytogeneticists and is adapted by many anopheline cytogeneticists; and (ii) the new nomenclature for arm designation is that suggested by Green and Hunt (1980). In this, each arm is given a separate number—2,3,4 and 5—for autosomal arms and the euchromatic arm of the X-chromosome seen in the polytene complement is designated as X. Taking An. gambiae belonging to the Pyretophorous series as an arbitrary standard, the two arms of chromosome 2, 2R and 2L are referred to as 2 and 3 respectively and

Anopheline Species Complexes in South and South-East Asia

9

those of chromosome 3, 3R and 3L as 4 and 5 respectively in this nomenclature. During evolution in anophelines, wholearm translocations have occurred. This came to notice when banding patterns of different species were compared and studied. In An. culicifacies and An. fluviatilis belonging to the Myzomyia series and An. stephensi, An. annularis and An. maculatus belonging to the Neocellia series, the arm association is 2-5 and 3-4. In the Myzomyia series, for the An. funestus group of species, the arm association is 2-4 and 3-5, indicating another translocation event (Green and Hunt, 1980). With this arm- designation system such events can be incorporated and it is best suited for studies on the cladistic analysis of species belonging to a subgroup, group or series. Researchers who use polytene chromosomes in their work are recommended to read Green and Hunt (1980) for the arguments and justification for the new arm designations. Most of the species complexes identified so far have been by the examination of polytene chromosomes of wild populations. Paracentric inversions are very common in the natural populations of anophelines. The advantage of paracentric inversions in species identification studies is that they act like single gene loci and the alternate arrangements as codominant alleles. There are inversions that are polymorphic within a species (intraspecific) and the three forms of these inversions, the two homozygotes, standard and inverted, and the heterozygotes, are easily recognized on polytene chromosomes, while there are inversions that are fixed and different between species (interspecific). The total absence or significantly deficient proportion of heterozygotes for an inversion in a population indicates reproductive isolation within a taxon; hence, the taxon may be considered as a species complex. Thus, the examination of polytene chromosomes of field-collected adult females provides unequivocal evidence for the existence of different species- specific mate

10

recognition systems (Peterson, 1980). In populations where heterozygotes for an inversion are absent, the inversion is said to be fixed and the two banding patterns on polytene chromosomes, inversion and its standard alternate arrangement become diagnostic tools for the identification of species. The occrrence of a small proportion of heterozygotes can be due to: (i) breakdown of pre-mating barrier(s) between two species (which is rare); and (ii) an inversion which is fixed in one species is polymorphic (floating) in another species and the two species are sympatric (e.g. An. culicifacies species A and D) (Vasantha, Subbarao and Sharma, 1991). In both cases, the number of heterozygotes is far less than the number expected where an inversion is polymorphic with random mating. These situations can be analysed statistically by calculating the expected number based on Hardy-Weinberg equilibrium and applying a Chi-square test. Thus, the population cytogenetic analysis demonstrates and distinguishes intraspecific and interspecific occurrence of inversions. It may be noted that sibling-species having homosequential polytene chromosome banding pattern exists among anophelinespecies complexes, e.g . An. labranchiae and An. atroparvus, two members of the Maculipennis Complex (Coluzzi, 1970) and species B and E of the Culicifacies Complex (Kar et al., 1999). A point to be kept in mind by all those who work with polytene chromosomes is that inversion fixation is an accidental association with speciation. As emphasized by Green and Baimai (1984), where variation occurs in polytene chromosomes due to inversions, it may provide useful markers for speciation and subspeciation events, but in its absence one can say nothing about such events among individuals bearing the same chromosomal rearrangements. In spite of the limitations that homosequential species exist in anophelines and that polytene chromosome complements

Anopheline Species Complexes in South and South-East Asia

can only be examined in semi-gravid adult females or in salivary glands of IV instar larvae, this is the easiest and cheapest tool now available for the recognition of species complexes and for routine use in the identification of members of a complex in entomological studies. This tool is, however, more laborious than the DNA- based tools in large-scale entomological studies. Asynapsis in polytene complements in hybrids is used as one of the criteria in determining species status. The degree of asynapsis may vary, and thus has to be used with caution. In hybrids between members of the Culicifacies Complex, the chromosomes remain synapsed, except in the inversion heterozygote region where a loop or asynapsis is observed (Subbarao et al., 1983). Inversions are designated with lower-case letters of the English alphabet and are specific for each chromosome arm, that is, an inversion a on chromosome arm 2 bears no relationship to a similarly denoted inversion on another arm, that is, inversion a on arm 3. The standard arrangement is designated + before the letter indicating the inversion concerned and these may or may not be written as a superscript, e. g. 2+a or 2+a. Green (1982a) for the Myzomyia Series and Green (1982b) for the Neocellia Series have suggested a unified method for designating inversions for species belonging to these Series, as it provides an efficient means of storing data and its subsequent retrieval. This has been followed for several members belonging to these series (An. fluviatilis, An. culicifacies, An. annularis, An. maculatus, An. philippinensis, etc.). Mitotic and meiotic karyotypes All anophelines studied so far have three pairs of chromosomes—two pairs of autosomes which are either metacentric or submetacentric and a pair of sex chromosomes— which are homomorphic (XX) in females and heteromorphic (XY) in males. X- and Y-

chromosomes have been found as telocentric, acrocentric or subtelocentric, or submetacentric (depending on the position of the centromere in the chromosome) in different species of anophelines. The paper of Levan (1964) is recommended for chromosome nomenclature. The best mitotic chromosomes are found in the neurogonial cells of the brain in early IV instar larvae and meiotic chromosomes in the reproductive organs of newly-emerged adults. The recommended references for the preparation of mitotic and meiotic chromosomes are: Breeland (1961), French, Baker and Kitzmiller (1962) and Baimai (1977). Structural variations due to the position of centromere and quantitative variations in heterochromatin blocks are commonly observed. The variation in autosomes and X-chromosomes, which are found in the homozygous state, demonstrate reproductive isolation between the populations if heterozygotes for the variation are not found or are found in deficient numbers. This is similar to the situations described for paracentric inversions under polytene chromosomes (see above). Unlike the banding pattern due to paracentric inversions, variations at a given position in the chromosome can exist as more than two alternatives. Data have to be generated from single female progeny of wild-caught females or larvae. Structural variations in the Ychromosome, though very common in anophelines, do not by themselves reveal the genetic structure of the population, as the variations in a population give no indication whether they are intra- or inter-specific because the Y-chromosome is inherited from father to son and is found in hemizygous condition. However, one can test the association of the Y-chromosome with other genetic variations, where linkage disequilibrium between Ys and other characters would indicate the presence of different sibling species. Species E in the Culicifacies Complex was identified by

Anopheline Species Complexes in South and South-East Asia

11

associating sporozoite positivity in females with Y-chromosome variants in their sons (Kar et al., 1999). Heterochromatic variants, revealed by special staining techniques using Giemsa, Hoechst, etc., on autosomes and Xchromosomes are distinct and are also diagnostic in the identification of sibling species. Both structural and heterochromatic variants in mitotic karyotypes are not convenient as routine entomological tools for the identification of sibling species in field studies as progeny of wild-caught females have to be examined for the variants. However, these techniques can be used to study the larval ecology of sibling species.

Enzyme electrophoretic variations Enzyme electrophoresis is extensively used in the study of species complexes. The technique involves the detection of the protein bands of an enzyme system with different mobilities as a function of electric charge and molecular structure. On a gel zymogram of an enzyme system, electrophoretic variations in the form of bands with different mobilities represent proteins coded by different alleles (allozymes). These alleles, being codominant, behave like paracentric inversions and the two homozygotes and heterozygotes can be differentiated. Variations at a locus thus enable the detection of the reproductive isolation between populations resulting from positive assortative matings within a population. Because of the simplicity of the procedures for the processing and interpretation of data, this technique permits large-scale sampling of natural populations and is very useful as a diagnostic tool in the routine identification of species. For details on techniques, Ayala et al., (1972), Black and Munstermann (1996, 2004) and Steiner and Joslyn (1979) are recommended. However, one has to remember that unlike inversions, where only one inverted arrangement with reference to a standard arrangement exists, more than two electrophoretic forms at a 12

single locus can exist and in each species more than one allele may be fixed. In An. melanoon and An. sacharovi (two members of the Maculipennis Complex), Hk-190 and Hk1100 alleles at the hexokinase locus are fixed in the former sibling species and Hk-195 and Hk-1 97 alleles in the latter. Therefore, electromorphs found to be diagnostic for each taxon can be used only to identify individuals from populations already sampled (Miles, 1981) as geographical variation within a species can exist. If a single fully diagnostic enzyme system cannot be identified (i.e. without any polymorphism), several enzyme systems which differ in their frequencies of alleles between populations can be identified and used in association, which will reduce the error in identification. For species complexes with several members, enzyme systems that are diagnostic for different members of the same species complex can be identified and used in the form of a biochemical key, as has been developed for the Maculipennis and Gambiae Complexes (for details, see Coluzzi, 1988). Electrophoretic variations at enzyme loci are not only useful for the identification of isomorphic species but can also be used for the correct identification of morphologically identifiable species. An. minimus, An. aconitus and An. varuna are found sympatric and are morphologically very similar. Hence, errors are made in the identifications. For Thailand populations, alleles at the Malate dehydrogenase-1 (Mdh-1) locus were found to be diagnostic (Green et al., 1990). Mdh1100 fixed in An. minimus differentiates it from An. aconitus and An. varuna which have the Mdh-1157 allele. Less than 0.1 per cent An. minimus had the Mdh-1157 allele and a few specimens had the Mdh-1115 allele. Mdh-1115 is diagnostic for An. pampana, but this species is very rare in Thailand (Green et al., 1990). A point to be kept in mind when working with electrophoretic variations is that in order to determine the mobilities of the bands of

Anopheline Species Complexes in South and South-East Asia

samples to be identified, samples of known reference standards should be run on the same gel. Alternatively, non-enzymatic protein standards have to be run along with the samples. For the identification of the Minimus Complex sibling species, human haemoglobin (homozygous for normal haemoglobin) and known laboratory colony material were run on the same gel as reference standards (Green et al., 1990). Mosquitoes collected from the field if not used immediately should be stored frozen (£40°) to retain their enzyme activity prior to electrophoresis.

Cuticular hydrocarbon profiles Cuticular hydrocarbon analysis for siblingspecies identification involves determining species-specific differences in the hydrocarbons contained in the wax layer of insect cuticle. The wax layer lies beneath the outermost cuticular layer. Carlson and Service (1979) were the first to use this technique to identify An. gambiae s.s. and An. arabiensis (two members of the Gambiae Complex). The review by Philipps et al. (1988) is recommended for details on the procedure. This technique has now been used to identify members of several species complexes in mosquitoes and other haematophagous insects. It may be noted that this technique can be employed to identify members of an already recognized species complex but is not recommended to be used to recognize new complexes. The most important criterion for designating different species is reproductive isolation which is not possible with this technique because it is not easy to differentiate between intra- and inter-specifc variations in the hydrocarbon profiles of individual specimens. Furthermore, this tool uses gas liquid chromatography with expensive equipment.

DNA methods Advancements in DNA recombinant technology have facilitated the development of simple and rapid molecular tools for the

identification of sibling species. Several reviews are now available in the literature detailing various techniques and their advantages in the identification of isomorphic species (Besansky, Finnerty and Collins, 1992; Hill and Crampton, 1994; Black and Munsterman, 2004; Collins et al., 2000; and Krzywinski and Besansky, 2003). The first of the DNA methods used to identify species was the use of DNA probes. Clones containing specific DNA segments of the undefined highly repeated component of the genome are identified by differential screening of genomic libraries with homologous and heterologous genomic DNAs. DNA segments from these clones are labelled and used as probes. The paper of Post and Crampton (1988) on DNA probes for the Simulium damnosum Complex and Black and Munstamann (2004) give procedures (with illustrations) used in the isolation of species-specific DNA probes for the identification of sibling species. Initially, radioactive probes were used. Simple nonradioactive probe assays for squash-blot hybridizations have been developed for the identification of members of the Gambiae (Hill et al., 1991), Punctulatus (Cooper, Cooper and Burkot, 1991) and Dirus (Audtho et al., 1995) Complexes. Johnson, Cockburn and Seawright (1992) have improved the procedure to clean up the background in squash-blot hybridizations. Non-radioactive probe methods remove the hazards of radioisotopes and make the assays simple and usable under field conditions. The advantage with DNA probes, as with isozymes, is that species can be identified at all stages of the mosquito life-cycle. And if kits are developed, as they have been for the Gambiae Complex (Hill, Urwin and Crampton, 1992), probes can be used with much more ease in field laboratories. Hill, Urwin and Crampton (1991) have shown that by producing synthetic probes, the cost of each assay can be brought down to between US$ 0.04 and US$ 0.33 depending on the labelling method used.

Anopheline Species Complexes in South and South-East Asia

13

With the improved DNA technologies and reduction in the cost of reagents and equipment, polymerase chain reaction (PCR) assays have become popular for species identification. The PCR assay was developed in 1985 by Saiki et al. (1985). PCR-based methods essentially require variation in neucleotide sequence across species. The major advantage of this technique is that it requires only miniscule amounts of DNA. During the reaction period of 2-3 hours, a particular region of the genome is amplified 1-100 million times, and this can then be simply visualized on agarose gel after electrophoresis and staining. The most commonly used PCR-based methods are: restriction fragment length polymorphism of PCR-amplified product (PCR-RFLP), singlestrand conformational polymorphism (SSCP), Heteroduplex analysis (HDA), and allelespecific PCR (ASPCR). The ASPCR is essentially cheaper and quicker than the other methods and does not involve special treatment following PCR. However, the position of neucleotide variations is critical in designing primers for ASPCR so that all the allele-specific amplicons can be distinguished separately on a gel. Black and Munstermann (2004) show in detail the steps involved in PCR assay with illustrations. Basically, there are two types of PCR strategies: one surveying the genome randomly and the second targeting specific regions of the genome, such as ribosomal DNA (rDNA) or mitochondrial DNA (mtDNA). The randomly amplified polymorphic DNA PCR (RAPD-PCR) belongs to the first category. This technique does not require prior knowledge of the genome, and an added advantage is that commercial kits are available with large number of random decamer primers. Arbitrary regions of the genome are amplified using a single decamer primer. Amplified products by each primer are analysed for differences between the species concerned. This technique was developed by Williams et al. (1990). DNA tools are generally developed once the

14

members of a complex are identified. The taxon An. (Nysorhynchus) albitarsis was, however, identified as a complex of four sibling species by the examination of natural populations from Paraguay, Argentina and Brazil, using RAPD-PCR (Wilkerson et al., 1995). Though RAPD-PCR is simple, this technique has inconsistent reproducibility and therefore has been of limited use for the identification of sibling species. Allele-specific PCR (ASPCR) assays mostly exploit variation in the rDNA cistron. In anophelines this is X-linked (Rai and Black, 1999). It consists of tandem repeated arrays of conserved genes (18S, 5.8S and 28S) punctuated by rapidly evolving non-coding internal transcribed spacers, ITS1 between 18S and 5.8S and ITS2 between 5.8S and 28S. Each gene cluster is separated by intergenic spacers (IGS). Within interbreeding populations the arrays undergo rapid homogenization through concerted evolution, which drives new sequence variations to fixation, leading to speciesspecific differences. For members of many species complexes, differences in ITS2 and variable regions within 28S rDNA gene have been used to develop ASPCR assays for members of the Culicifacies Complex (Curtis and Townson, 1998; Singh et al., 2004a), for the Fluviatilis Complex (Manonmani et al., 2001; Singh et al., 2004b), for the Dirus Complex (Walton et al., 1999) and for several other species complexes prevalent in the Afrotropical, and Neotropical regions. Portions of the genes from mitochondrial genome, COI and COII, are also used to develop diagnostic PCR assays as has been done for members of the Culicifacies Complex (Goswamy et al., 2006). For An. minimus species A and C, Kengue et al. (2001) used the RAPD marker assays of Sucharit and Komalamisra (1997) to develop a robust multiplex ASPCR. This assay distinguished other anophelines, An. aconitus, An. varuna and An. pampanai, which are morphologically very close to An. minimus and are found sympatric with An. minimus.

Anopheline Species Complexes in South and South-East Asia

In PCR-RFLP, the amplified product is cut by certain restriction enzymes which produce a different pattern of digested products when run on agarose gel. A single nucleotide change may alter the restriction enzyme site and thereby a different pattern of bands may be revealed. A PCR-RFLP targeted at ITS2 rDNA was developed by Van Bortel et al. (2000) which distinguishes An. minimus A and C and four related species, An. aconitus, An. pampanai, An. varuna and An. jeyporiensis. Digestion of the PCR-amplified D3 region of 28S rDNA with HPA II endonuclease distinguished An. funestus from An. veneedeni (Koekemoer, Coetzee and Hunt, 1998), and digestion of COII-amplified product with Dde I distinguished species E from species B and C of the Culicifacies Complex (Goswamy et al., 2005). Hiss et al. (1994) developed a technique based on single-strand conformation polymorphism (SSCP) of Orita et al. (1989) as a diagnostic tool. SSCP is a highly sensitive technique and detects point mutations with an efficiency of 99% in products with 100300bp length. As the product length increases, the efficiency is reduced. This technique does not require prior knowledge of DNA sequence data. In this technique the amplified product is denatured to single strands at 95 oC for five minutes and then placed immediately into an ice bath (0-4 oC) so that single-strand duplexes are formed from intrastrand base pairing. Variation in the confirmation of intrastrand duplexes is visualized by a gel retardation assay. Sharpe et al., (1999) developed an SSCP assay for the identification of An. minimus species A and C, An. aconitus and An. varuna, and Koekmoer et al.(1999) used D3 region of rDNA to distinguish four members of the An. funestus group, An. funestus, An. vandeeni, An. rivulorum and An. leessoni. Heteroduplex analysis (HDA) is another technique used in the identification of closelyrelated species. HDA detects the electrophoretic retardation of heteroduplex products (HDPs) formed between the strands

of a probe and a test DNA molecule. The number and type of mismatched nucleotides within a given HDP determine the mobility of the DNA duplex on an electrophoresis gel (Tang and Unnash, 1997). The target DNA and probe DNA are PCR products of chosen loci, which are hybridized and run on a gel. The probe DNA is chosen from a closely related species. The advantage of this technique is that without going through the sequencing of loci, intra- and inter-specific variation can be detected. DNA sequences are also being used to establish the phylogenetic relationship of the species between the subgroups and of those within a subgroup/complex. Portions of genes from mitochondrial genome, COI, COII, Cytochrome b, etc., and from nuclear rDNA regions, D2, D3, ITS1, ITS2, etc., are used (Chen, Butlin and Harbach, 2003; Dusfour et al., 2004 and Garros, Harbach and Manguin, 2005a and b). Sallum et al. (2002) examined the phylogenetic relationship of 32 species of the subfamily Anophelinae, which included species from the genera Anopheles, Bironella and Chagasia. Microsatellite markers are di-, tri- and tetra-nucleotide sequence arrays of variable length found frequently in the genomes. These sequences are identified from genomic libraries using labelled repeat probes. Once the sequences from the positive clones are selected from unique neucleotide sequences flanking the repeated sequences, specific primers for each marker are developed for PCR assays. For An. gambiae s. s., markers were also developed from genome sequence analysis (for this species the entire genome sequence data is now available at http:// www.ensemble.org/Anopheles_gambiae) instead of going through the labour-intensive screening process mentioned above. Microsatellite loci generally have higher mutation rates than other regions of the genome and, therefore, are highly polymorphic. Beacause these are neutral markers, they are preferred for use in population genetic studies. There are a few

Anopheline Species Complexes in South and South-East Asia

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anophelines for which these markers have been developed— An. gambiae s.s. (Zheng et al., 1993, 1997), An. maculatus (Rongnoporut et al., 1996), An. funestus ( Sinkins et al., 2000), An. dirus (Walton et al., 2000a), An. stephensi (Veradi et al., 2002) and An. culicifacies ( Sunil et al., 2004). Microsatellite markers developed for one member can be used for other members of the complex. They can also be used for closely related species as has been done for members of the Leucosphyrus Complex using those developed for An. dirus s.s (Walton et al., 2000a). These markers were initially used for the genetic mapping of refractory gene(s) and morphological markers (Zheng et al., 1993, 1996) and for developing a fine-scale genetic map (Zheng et al., 1997) of An. gambiae. Now they are being used in population genetic analysis and ecological studies in An. gambiae s.s. and also for other members of the Gambiae Complex (Besansky et al., 1997; Kamau et al., 1999 and Donelly and Townson, 2000). Similarly, microsatellites are being used for the population genetic analysis of An. maculatus ( Rongnoparut et al., 1999), An. dirus ( Walton et al., 2000b) and An. culicifacies (Subbarao, unpublished). It is important to highlight the power of simple polytene chromosome analysis using paracentric inversions as a tool in population genetic studies and in establishing phylogentic relationships. This simple tool has brought out the complexities of population structure within the An. gambiae s.s. by identifying five chromosomal forms — Bamako, Mopti, Savanah, Forest and Bissau in West Africa (Coluzzi, Petrarca and DiDeco, 1985). Now, DNA markers together with cytogenetic tools are being used to clarify this complexity

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(Wondji, Simard and Fontenelle, 2002 and Stump et al., 2005). In years to come, some of these forms may be given a species status. Green constructed phylogeny using cladistic analysis based on paracentric inversions seen in ovarian polytene chromosomes for members of the Series Myzomyia (Green, 1982, 1995) and for the Series Neocellia (Green et al., 1985). Programs are now available for calculating gene frequencies, average heterozyosity, per cent polymorphic loci observed and expected heterozygote values based on the Hardy-Weinberg equilibrium, Wright’s fixation indices and genetic distance and identity, and for establishing phylogenetic relationships. Computer programmes can also be used for analysing populations that are polymorphic for many inversions, as has been done for An. gambiae s.s. and An. arabiensis (Garcia et al., 1996) and An. annularis (Atrie et al., 1999). Currently, more than 240 programmes have been listed at http://www.nslijgenetics.org/soft. The BIOSYS-1 program in FORTRAN of Swofford and Selander (1981) and its later vesions were used for the analyses of many of the anopheline species. The most commonly used programmes are—TFPGA, Arlequin, POPGENE, GDA, GENEPOP, GeneStrut, GeneAlEx, etc. MEGA (Molecular Evolutionary Genetic Analysis) is a software for computational molecular evolutionary genetics based on nucleotide/protein sequences. Those interested on population genetic analysis may visit http:// dorakmt.tripod.com/genetics/popgen.html to learn about the basics of population genetics, links to freeware, related literatures and other useful links.

Anopheline Species Complexes in South and South-East Asia

3. Species Complexes

3.1

The Annularis Complex

Anopheles annularis Van Der Wulp 1984 belongs to the subgenus Cellia, Annularis Group in the Neocellia Series. The other members in this Group which are prevalent in South-East Asia are An. nivipes, An. philippinensis, An. pallidus and An. schueffneri (Harrison 1988, Harbach, 2004). An. annularis has a wide distribution in the Oriental region. It is found in Afghanistan, Pakistan, Nepal, India, Bangladesh, Myanmar, Philippines, China and Sri Lanka (Rao 1984; Kondrashin and Rashid, 1987). This species is considered to be an important vector in Nepal and in certain parts in India (Dash et al., 1982; Rao, 1984; Gunasekaran et al., 1989). It is a secondary vector in certain localities though it has a wide distribution and is sometimes found abundantly (Rao, 1984). An. annularis has been incriminated recently in the Thai-Combodia border area (Baker et al., 1987) and is also a vector in Myanmar (Bang, 1985). In Sri Lanka, this species has been found playing a role in the transmission of malaria in a new irrigation development area (Ramasamy et al., 1992).

Evidence for identification of sibling species Two sibling species from Nepal (WHO, 1983) The total absence of heterozygotes for a paracentric inversion and its standard arrangement on the X-chromosome was taken as evidence for the presence of two sibling species in An. annularis in Nepal. No

further reports on details of these species are available. Species A and B (Atrie et al., 1999) Nine inversions – w I , i 1 , j 1 and k 1 on chromosome arm 2; j1 and z on arm 3; h1 and s1 on arm 4; and k on arm 5 – were found polymorphic in six districts in five states in India. The total absence of heterozygotes for inversion j1 and its standard arrangement on chromosome arm 2 in villages in Ghaziabad and Shahjahanpur districts in the state of Uttar Pradesh in India led to the recognition of two sibling species A and B. The deficiency of heterozygotes for three other inversions – 2i1, 2k and 4h – was also observed. However, the partitioning of polymorphic forms of these inversions between the two sibling species characterized by the +j1 and j1 arrangements indicated balanced polymorphisms of the inversions in each of the presumed sibling species. This further supported the conclusion that An. annularis is a species complex and heterozygote deficiencies are due to the difference in the frequencies of these inversions in the two sibling species.

Techniques available for identification of sibling species Polytene chromosomes This technique is based on the difference in the banding pattern of polytene chromosomes due to a paracentric inversion. Species A is characterized by the +j 1 arrangement and species B by the j 1 arrangement on chromosome arm 2. A

Anopheline Species Complexes in South and South-East Asia

17

photomap of polytene chromosomes with the break points for the diagnostic inversion is given by Atrie et al. (1999). The Xchromosome and the autosomal arms 3, 4 and 5 are homosequential in both species.

these two sibling species (Atrie et al., 1999). The assays were developed on the specimens collected from different areas based on the distribution pattern reported for sibling species A and B by Atrie et al. (1999).

Breakpoints of nine polymorphic inversions observed in these species are also marked on the photomap. The photomap is presented in Figure 2. Polytene chromosome maps for An. annularis are also given by Green et al. (1985). The 2+j1 arrangement in species A is homosequential with the one presented by Green et al. (1985). Of the nine inversions observed in India, four inversions - 2w, 3z, 4h 1 and 5k - were found in populations from Taiwan, Philippines, Thailand and Bangladesh (Green et al., 1985).

Distribution and biological characters

The mitotic karyotype of both the species was found to be the same. Both the autosomes and the sex chromosomes were submetacentric (Atrie, 1994). PCR-RFLP Two PCR-RFLP assays, one based on endonuclease restriction sites in the ITS2 sequence and the other based on those in the D3 sequence of rDNA, have been developed by Alam et al. (2006). In the ITS2 sequence, species A showed three restriction sites each for MspI, MyaI and Eco24I enzymes and species B showed restriction sites for MspI, HintI and NruI enzymes. In the D3 sequence, species A had a unique restriction site for Alw26I while species B had the site for KpnI. With the D3 sequence two enzymes are needed for the accurate identification of two sibling species. MspI restriction sites were found in both the species in the ITS2 sequence and fragments differing in lengths were produced in the two species following the digestion. Therefore, ITS2-MspI could be used as a diagnostic system to identify sibling species A and B of the Annularis Complex. However, the two assays have not been correlated with the cytological identification which was the basis for the identification of

18

In the two districts of Shahjahanpur and Ghaziabad in Uttar Pradesh, India, species A and B were found sympatric. In all the other states surveyed, namely, Rajasthan, Haryana, Assam and Orissa, only species A has been found. An. annularis samples from Shahjahanpur district, where both species A and B are prevalent, were found totally zoophagic when blood meals of these species were examined by counter-current electrophoresis (Atrie, 1994). Both the species were found in the riverine, non-riverine and canal-irrigated ecotypes found in the villages in Shahjahanpur. However, in other districts where only species A was found, the same ecotypes were observed, and collections were also made from hilly-forested areas (Atrie et al., 1999). In India, An. annularis is considered a vector only in certain states. Only species A was found in Sundergarh, Orissa state, and Kamrup in Assam state where An. annularis is considered a vector. However, species A was also found in Haryana, Rajasthan and Uttar Pradesh where An. annularis is not considered a vector. Furthermore, species A was totally zoophagic. Thus, the identification of An. annularis as a species complex did not explain why it is a vector only in certain areas in India. An. annularis is a vector of local importance in Nepal and Bangladesh, and a secondary vector in India and Sri Lanka. The status of An. annularis as a vector is not known in Bhutan (Figure1). Recently, in Afghanistan, it was incriminated with a Plasmodium vivax sporozoite rate of 0.58% (Rowland et al., 2002) in villages with river-irrigated rice fields. The distribution of these two sibling species, A and B, has not been studied so far in any of

Anopheline Species Complexes in South and South-East Asia

Figure 2: Photomap of polytene chromosomes of An. annularis species A. The break points of inversions are marked with the letter designations on the right side of chromosome arms. Arrows indicate the centromeric ends of the chromosome arms (Source: Atrie et al., 1999)

2

1 2

3 4

5 6

20 21

8 9

10 11

38 39

30 31

21 22

9 10

5

4 29 30

7 8 2 3

4 5

3

22 23

31 32

23 24

32 33

39 40 40 41 41 42

42 43

11 12

33 34

24 25

43 44 45 46

12 13

4 45 4

X

34 35 25 26

13 14

35 36

26 27 14 15

27 28

36 37

15 16

16 17

17 18

18 19

Anopheline Species Complexes in South and South-East Asia

19

these countries. Studies are required to examine the biological characters of these two species and investigate whether there are more species in this complex.

3.2. The Barbirostris Complex An. barbirostris belongs to the subgenus Anopheles, Barbirostris Subgroup, Barbirostris Group in the Myzorhynchus Series (Harbach, 2004). There are two Subgroups, Barbirostris and Vanus, in this Group. The Barbirostris Subgroup includes barbirostris, campestris, donaldi, franiscoi, hodgkini and pollicaris; and the Vanus Subgroup includes ahomi, barbumbrosus, reidi, manalangi and vanus (Reid, 1968). Anopheles barbirostris is reported from India, Pakistan, Bangladesh, Nepal, Myanmar, Thailand, Malaysia, Laos, Cambodia, Viet Nam, Indonesia, Sri Lanka and South China (Rao, 1984). This species has been suspected as a malaria/filaria vector in Indonesia and Thailand.

Evidence for identification of sibling species Crossing experiments (Choochote, Sucharit and Abeyewickreme, 1983) The evidence showing An. barbirostris to be a species complex came from the progeny of reciprocal crosses carried out between two laboratory strains (Choochote, Sucharit and Abeyewickreme, 1983). Two strains, the Chumphon strain (CHP) established from mosquitoes collected from Bang Luke Canton, Chumphon Province (southern Thailand) and the Chon Buri strain (CHB) from Chan Buri province (central Thailand) were used in this study. The two strains were reared in the laboratory by using the forced mating technique. Both the strains differed in average body weight and number of eggs deposited. The CHP strain always weighed more (female — 20

1.64 + 0.49, male —1.19 + 0.23) and laid 143 + 47.54 eggs/female, while the CHB strain weighed, female — 0.97 + 0.23, male — 0.82 + 0.19, and laid 83.3 + 18.95 eggs/ female. An average of 15–20 mosquitoes of each category were used in this study. In the CHB female x CHP male cross, eggs were laid but none hatched, while in the reciprocal CHP female x CHB male cross there was 60.8 per cent hatching. The two parental strains had more than 80 per cent hatch. In CHP x CHB cross, there was 47.1 per cent of pupation and only 16.7 per cent emerged. When F1 females from this cross were crossed to CHP males, no eggs were laid in spite of 70.4 per cent insemination. F1 males had abnormal genitalia with very short claspers, and atrophied testes and accessory glands. Polytene chromosomes from the salivary glands of the F 1 were homosequential with the maps described by Chowdayya et al. (1970), but exhibited inconsistent asynapsis along the autosomes while the X-chromosomes showed complete synapsis. Based on these results, the authors considered An. barbirostris to be a species complex. Mitotic karyotypes from Thailand and Indonesia (Baimai, Rattanarithikul and Kijchalao, 1995) Four forms of metaphase karyotypes were observed in mosquitoes collected from wild populations. The karyotype consisted of two pairs of autosomes, metacentric chromosome 2 and sub-metacentric chromosome 3 and the X- and Y- chromosomes which differed in size and shape. Three forms, A (X2, X3, Y1), B (X1, X2, X3, Y2) and C (X2, X3, Y3,), were observed in Thailand and one form, D (X2, Y4), in Indonesia. The collections made in Thailand were extensive and covered the entire country. The D karyotype was not found in any of these collections and was restricted to Indonesia. The authors concluded that the X,Y variations found in Thailand could be inter- or intra-specific variations.

Anopheline Species Complexes in South and South-East Asia

Figure 3: Mitotic karyotypes of An. barbirostris found in Indonesia. Plate 1 female, plates 2-4 male of cytological form A; plate 5-6 male and plate 7 female of cytological form B; plate 8 female and plate 9 male of cytological form C; plate 10-11 male and plate 12 female of cytological form D (courtesy of Dr Supratman Sukowati)

Anopheline Species Complexes in South and South-East Asia

21

Mitotic karyotypes from Indonesia (Sukowati, Andris and Sondakh, 2003) Mitotic karyotypes of the progeny of wildcaught An. barbirostris females from their four geographically isolated populations were examined. The mitotic karyotypes differed in X and Y- chromosomes, the variation being in the amount and distribution of constitutive heterochromatin in giemsa-stained preparations. The authors reported that Form A (X1, X2, X3, Y1) is widely distributed in Indonesia, and found sympatric with form B (X1, X2, X3, Y2) and form D (X2, X3, Y4) in Taratara 2, North Sulawesi; Konga, Flores and Tanjung Bunga, Flores, and form A was found sympatric with form B and form C (X2, X3, Y3) only in Boru-Boru, Flores. It may be noted that in Indonesia, Form A included X 1 variation too. Neither form C nor D were found with the X1 chromosome inspite of being sympatric with forms A and B. Y3 and Y4 chromosomes were associated with the C and D forms respectively. Mitotic chromosomes of the four forms found in Indonesia are shown in Figure 3. Thus, there appear to be at least three distinct cytotypes probably representing interspecific variation. All three forms were homosequential in their polytene chromosome banding pattern. S. Sukowati (personal communication) considers these to be three distinct sibling species in this complex. Species A was found highly zoophagic and species B and C were anthropophagic. Species C was found biting during daytime. In the areas where these are found, filariasis due to Wucheraria bancrofti and Brugia malayi and malaria are prevalent. An. barbirostris is considered to be a vector of malaria and filaria. Studies on polytene chromosome difference, allozyme variation and DNA methods need to be initiated to give specific taxonomic status to these populations and establish relationships with the prevalent diseases. Studies have been initiated to develop a PCR assay for the differentiation of these three species (Supratman Sukowati, personal 22

communication). The CHP and CHB strains reported by Chuchote, Sucharit and Abeyewickreme (1983), cytotypes decsribed by Baimai, Rattanarithikul and Kijchalo (1995) and three possible species reported by Sukowati, Andres and Sondakh (2003) have to be correlated with each other.

3.3

The Culicifacies Complex

Anopheles culicifacies Giles belongs to the subgenus Cellia, Culicifacies Subgroup, Funestus Group in the Myzomyia Series (Harbach, 2004). An. culicifacies sensu lato (s. l.) has a wide distribution in India and extends to Ethiopia, Yemen, Iran, Afghanistan and Pakistan in the west, and Bangladesh, Myanmar, Thailand, Cambodia and Viet Nam in the east. It is also found in Nepal and southern China to the north and extends to Sri Lanka in the south (Rao, 1984). Recently, this species was reported from Cambodia (Van Bortel et al., 2002). An. culicifacies is an important vector of malaria in India and Sri Lanka and in the countries west of India. The history of malaria control in these countries concerns mainly the control of An. culicifacies. Significant differences were observed in the bionomics of An. culicifacies in various regions, including differences in seasonal abundance, diurnal activity, man-biting behaviour and vectorial potential (Rao, 1984). As early as 1947, due to such distinct differences in biological characters, it was suggested that the species culicifacies may cover a range of ’biological races’ (Senior White, 1947). An. culicifacies has now been recognized as a complex of five sibling species, provisionally designated as species A, B, C, D and E.

Evidence for identification of sibling species Four sibling species, A, B, C and D in this complex, were identified following the

Anopheline Species Complexes in South and South-East Asia

observation of a total absence or significant deficiency of heterozygotes in natural populations for the alternate arrangements observed in polytene chromosomes due to paracentric inversions. The fifth species, species E, was identified by correlating Ychromosome polymorphism of sons and the sporozoite positivity of mothers. Laboratory colonies established from the progeny of single females with species-specific inversions were used subsequently to study post-zygotic isolation mechanisms between the sibling species.

addition to a and b on the X-chromosome; thus, species B had Xab; 2g1 arrangement (Subbarao et al., 1983). In Gujarat and Madhya Pradesh, two chromosome 2 arrangements, g1+h1 and +g1h1, within Xab populations were seen. In a large sample examined, only four double-inversion heterozygotes (2g 1 +h 1 /+g 1 h 1) were observed; therefore, Xab; 2g1+h1 and Xab; 2+g 1 h 1 were considered as two reproductively isolated populations. The new population with Xab; 2+ g1h1 was designated as species C (Subbarao et al., 1983).

Species A and B (Green and Miles, 1980) Green and Miles (1980) found two distinct polytene X-chromosomes in the An. culicifacies s. l. population in the village Okhla near Delhi, India. One X-chromosome was with two paracentric inversions a and b and the other with a banding pattern that resembled the polytene chromosome photomap described by Saifuddin, Baker and Sakai (1978). No heterozygotes for these inversions were found. The absence of heterozygotes was taken as an evidence of reproductive isolation between the two populations which were considered as two distinct species. The population with the standard arrangement, X+a+b, was designated as species A and that with Xab arrangement as species B. Green and Miles in the same paper (1980), by examining laboratory colonies of An. culicifacies s. l., reported species A and B from Pakistan and species B from Sri Lanka. Following this report, Subbarao, Adak and Sharma (1980) by examining field populations, confirmed the presence of species A and B within An. culicifacies in the villages of Haryana and Uttar Pradesh, both states bordering Delhi.

Species D (Subbarao, Vasantha and Sharma, 1988a; Suguna et al., 1989; Vasantha, Subbarao and Sharma, 1991) In a few populations in northern India, the i1 inversion on chromosome arm 2 was found polymorphic in species A, and it was found fixed in the southern Indian populations of An. culicifacies species A (Subbarao, 1984). The latter population with the X+a+b; 2i1 arrangement was found sympatric with species B. The evidence for reproductive isolation between species A (X+a+b; 2+g 1+h 1) and the population with the X+a+b; 2i1 h1 arrangement came from two locations (inversion i1 includes the g1 inversion region and the distal breakpoint is the same for both the inversions). In northern and central India, a deficiency of heterozygotes was found for i1 inversion in the X+a+b populations (Subbarao, Vasantha and Sharma, 1988a; Vasantha, Subbarao and Sharma, 1991) while in southern India a total absence of heterozygotes was found between species A (X+a+b; 2+g 1+h 1) and the population with the X+a+b; 2i 1 +h 1 arrangement (Suguna et al., 1989). The X+a+b population with the new chromosome 2 arrangement 2i 1+h1 was designated as species D. The explanation for the heterozygotes (+i1/ i1) of i1 inversion observed in northern India is that this inversion is floating in species A with varied frequencies in different populations and is fixed in species D (Vasantha, Subbarao and Sharma, 1991).

Species C (Subbarao et al., 1983) The examination of polytene chromosomes of An. culicifacies from villages around Delhi and in the states of Gujarat and Madhya Pradesh, India, revealed the presence of two fixed inversions on chromosome arm 2. Species B was fixed for the g1 inversion in

Anopheline Species Complexes in South and South-East Asia

23

Species E (Kar et al., 1999) In Rameshwaram island, Tamil Nadu (a state in southern India), An. culicifacies females had the Xab; 2 g1+h1 inversion arrangement on polytene chromosomes, which is diagnostic for species B. However, the biological characters of this population were different than those observed for species B on the mainland. This prompted a detailed examination. Because homosequential species exist in nature, Subbarao et al. (1993) investigated the mitotic karyotype variations. The progeny of the field-collected females revealed the Y-chromosome to be polymorphic with acrocentric and submetacentric types. Because the Ychromosome has patroclinal inheritance, the variation observed could not be used to indicate reproductive isolation. Correlation with sporozoite positivity i.e. total absence of sporozoite positives among females whose sons had acrocentric Y-chromosome and presence of sporozoite positives among mothers whose sons had submetacentric Ychromosome, was taken as evidence for assortative mating between acrocentric and submetacentric populations. The two populations were considered as two sympatric species. The vector population with the submetacentric Y-chromosome was designated as species E and the non-vector population with acrocentric type Ychromosome retained the original designation of species B (Kar et al., 1999). There seem to be at least five specific mate recognition systems operating in An. culicifacies, leading to reproductive isolation and lack of gene flow between the populations; hence, there are at least five sibling species in this complex. Post mating isolation mechanisms (Mahmood, Sakai and Akhtar, 1984; Subbarao, Vasantha and Sharma, 1988b; Kar et al., 1999) In addition to the pre-mating isolation mechanisms, post-mating isolation

24

mechanisms were also observed. From one of the reciprocal crosses between species A and B, Miles (1981) observed hybrid male sterility. In this study, crosses were carried out by the forced copulation technique between the progeny of single females. Hybrid males from the species B female x A male cross were fertile. Mahmood, Sakai and Akhtar (1984) reported fertility of hybrid males varying from 10 per cent to 96 per cent depending on the species B strain used in the cross between B female and A male. Subbarao, Vasantha and Sharma (1988b) observed almost total sterility with an occasional hatch of 1 to 5 per cent in the B female x A male cross, in contrast to 90 per cent hatch in the A female x B male cross. Crosses by these authors were carried out in cloth cages by normal matings. However, in crosses where a low hatch was observed in the B female x A male cross, hybrid males were sterile, having fully developed reproductive organs but without any sperm. In the reciprocal cross (A female x B male), the reproductive organs were partially developed, testes and vas deferens were either totally absent, atrophied or reduced, while the ejaculatory duct and accessory glands were normal (Miles, 1981; Subbarao, Vasantha and Sharma, 1988b). The results from the crosses between species A and C were similar to those between species A and B, except that in the C female x A male cross, no egg hatch had been observed so far (Subbarao, Vasantha and Sharma, 1988b). Reciprocal crosses between species B and C produced fully fertile F1 hybrid males and females, suggesting that there is no postzygotic barrier between B and C. Species E resembled species B and C in the crosses to species A, and there was no post-mating barrier in the crosses with species B and C (Kar et al., 1999).

Techniques available for identification of sibling species The techniques available for the identification of sibling species are summarized in Table 4.

Anopheline Species Complexes in South and South-East Asia

Table 4: Techniques for the identification of An. culicifacies sibling species* Polytene chromosome inversion genotypes

Mitotic karyotypeY-chromosome

LDH enzyme alleles

A

X+a+b; 2+g1+h1; +i1/i1

Submetacentric

Fast

Identified

Yes

Yes

Yes

B

Xab; 2g1+h1

Acrocentric Submetacentric

Slow

Identified

Yes

Yes

Yes

C

Xab; 2+g1h1

Acrocentric Submetacentric

Slow

Identified

as B

as B

Yes

D

X+a+b; 2i1+h1

Submetacentric

Fast

Not done

Not tested

as A

Yes

E

Xab; 2g1+h1

Submetacentric

Slow

Not done

Not tested

Yes

Yes

Species

Polytene chromosomes The use of diagnostic fixed inversions readable on the polytene chromosomes (Subbarao, Vasantha and Sharma, 1988a) has been the only technique available until recently for the identification of sibling species in this complex. This technique has been extensively used to study the biological characters and establish the role of the sibling species in malaria transmission. The problems in using this technique are: (i)

Species B and species E are homosequential, hence cannot be differentiated.

(ii) With reference to species A and D and i1 inversion, one encounters any of the following situations in a population: (a) +i1 and i1 homozygotes without any heterozygotes, suggesting that species A and D are present; (b) + i1, i1 and + i1/ i1 (heterozygotes) in expected proportions, suggesting that only species A is present and i1 is polymorphic; (c) similar to (b) but with a significant deficiency of heterozygotes, suggesting that species A and D are present, and in species A i 1 is polymorphic.

Cuticular hydrocarbon profile

Speciesspecific DNA probes

PCR-RFLP

ASPCR

In a situation such as (c), a population genetic analysis will indicate the presence of species D but individual specimens cannot be identified as species D (Vasantha, Subbarao and Sharma 1991). The photomaps of the polytene chromosome complement of An. culicifacies have been reported by Saifududdin, Baker and Sakai (1978). Chromosome maps with breakpoints of inversions a and b on the Xchromosome are given in Green and Miles (1980), and for those on the X-chromosome and of g1, h1 and i1 on chromosome arm 2, in Subbarao et al. (1983) and Subbarao, Vasantha and Sharma (1988a). The photomaps from the latter paper are reproduced here as Figure 4. In addition to fixed inversions in this complex, six inversions, five on chromosome arm 2 (j1, k1, l1, i1 and o1) and r on arm 3 were found polymorphic in species A, and in species B, l1 on arm 2 and r on arm 3 were seen in natural populations. The breakpoints of all these inversions are mapped on photomaps by Vasantha, Subbarao and Sharma (1991). Mitotic karyotypes Initial studies carried out on limited samples for mitotic karyotypes in sibling species revealed Y-chromosome characters to be

Anopheline Species Complexes in South and South-East Asia

25

diagnostic for the identification. Submetacentric Y-chromosomes in species A and C and acrocentric in species B were observed by Vasantha et al. (1982; 1983). Suguna et al. (1983) also observed corresponding differences in species A and B populations from southern India. Adak et al. (1997) reported both acrocentric and submetacentric Y-chromosome polymorphism in species B and C populations in several areas surveyed. Species E has a submetacentric Y-chromosome (Kar et al., 1999). Thus, the Y-chromosome cannot be used as a diagnostic tool for the identification of members of the complex except for species E. Mitotic karyotypes of specie B and E are given in Figure 5. Electrophoretic variations Out of the nine enzyme systems studied, electrophoretic variation in lactate dehydrogenase (LDH) was found to be diagnostic (Adak et al., 1994). Two electromorphs, fast (F) and slow (S), were represented at the Ldh locus. The frequency of LdhF in species A and D varied between 0.94 and 1.00, as against 0.0 and 0.19 in species B and C. Because of the low-level polymorphism observed within each species, the power of this technique in the identification of sibling species was evaluated by the authors using three indicators: sensitivity, specificity and predictive value. Overall, the probability of correct separation of species pairs by LDH enzyme was determined as 94.6 per cent. In species E, LdhS was found (Kar et al., 1999), thus, this species falls into the same category as species B and C. As Ldh is autosomal and is expressed at all stages of the life-cycle, Ldh allozyme method is useful in the identification of sibling species in certain sympatric associations. Cuticular hydrocarbon profiles Cuticular hydrocarbon profiles were examined in species A, B and C (Milligan et al., 1986). Cuticular wax extracted from single specimens from pure stocks was

26

analysed by gas liquid chromatography. The three species were found to be significantly different in their cuticular hydrocarbon composition by multi-variate analysis of variance. The best separation between the species was obtained using 27 peaks in a discriminant analysis. Using the chromatographic characteristics of these peaks, each specimen analysed was assigned to the group to which its probability of membership was the greatest. With this, the average correct identification was 78 per cent. Analysis of a small number of field samples also exhibited intraspecific variability similar to that observed in laboratory cage samples. DNA probes Three highly repetitive DNA sequences, Rp 36, Rp 217 and Rp 234, were selected from a genomic library of species B (Gunasekera et al., 1995). Radio-labelled fragments of Rp 36, Rp 217 and Rp 234 gave positive signals in dot-blot hybridization assays with sibling species A, B and C. The hybridization signal given by species A was much less than that given by species B and C. Species A can be distinguished from species B and C when single-mosquito extracts are diluted 200-fold and assayed by dot-blot hybridization with any of the three probes, which then give a negative signal for species A. These probes have not been evaluated in different geographical regions. PCR-RFLP The use of restriction endonclease Rsa I for the ITS2 amplicon and Alu I for mitochondrial cytochrome oxidase (CO) subunit II grouped species A and D in one category and B, C and E into another (Goswamy et al., 2005). The COII amplicon digested with Dde 1 distinguishes species E from species B and C. ASPCR Three PCR assays from the rDNA cistron were developed for the identification of sibling species of the Culicifacies Complex. One was developed from the variable D2 domain

Anopheline Species Complexes in South and South-East Asia

(Cornel et al.,unpublished) and the second from the D3 variable domain (Singh et al., 2004) of the 28S rDNA cistron. Both these assays distinguish species A and D from species B, C and E but fail to distinguish species within each of these groups. Primers for both these have been evaluated against field-collected and cytotaxonomicallyidentified specimens. A third assay reported by Curtis and Townson (1998) was developed from the ITS2 region of the 28S rDNA cistron. The assay distinguishes species A from species B. Other species have not been tested with these primers and this method has not been evaluated on field samples. Recently, sequence analysis of ITS2 region revealed that species D is similar to species A and species C and E to species B (Goswami et al., 2005). Two allele-specific PCR assays, AD-PCR and BCE-PCR, were developed from the COII region of mitochondrial DNA to distinguish the species. The strategy in this assay is that once the two groups, i.e. species A and D and species B, C and E are identified either by allele-specific PCR of the D3 or D2 regions or by the PCR-RFLP assays of the ITS2 and COII regions (Goswamy et al., 2005), one can use specific assays developed from the COII region i.e. the AD-PCR assay to distinguish species A and D and the BCE-PCR assay to distinguish species B, C and E (Goswamy et al., 2006). This assay was evaluated on An. culicifacies collected from different areas of India, and simultaneously, identifications were correlated with cytological identification based on species-specific diagnostic inversions. Microsatellite markers About 31 microsatellite markers were developed from the An. culicifacies species A. Some of the markers tested were found polymorphic in species A, B and C. The allele number varied from 2-12 (Sunil et al., 2004)

Distribution and biological characteristics In India all five species of the Culicifacies Complex have been identified. Species A identified in Yemen (Akoh, Beidas and White, 1984) and Iran (Zaim et al., 1993) has been found sympatric with species B in Pakistan (Mahmood, Sakai and Akhtar, 1984). Recently, An. culicifacies has been incriminated as one of the eight species responsible for malaria transmission in Afghanistan (Rowland et al., 2002). A few specimens from Afghanistan were cytotaxonomically identified several years ago as species A. Larger samples from different regions of the country need to be examined for sibling species composition. Extensive surveys carried out in Sri Lanka using polytene chromosomes (Abhayawardena et al., 1996) and DNA probe (de Silva et al., 1998) identified only species B. Surendran et al. (2000) in Sri Lanka found both acrocentric and sub-metacentric Y-chromosome types in An. culicifacies population and by analogy with the situation in Rameshwaram island as proposed by Kar et al. (1999) they assumed that the acrocentric population was species B and sub-metacentric type was species E. Recently, cytologically-identified An. culicifacies sub-metacentric and acrocentric and unidentified sensu lato specimens from Sri Lanka were assayed by the PCR method of Goswami et al. (2006). Specimens belonging to the sub-metacentric and acrocentric categories were identified as species E and B respectively. Among the cytologically-unidentified specimens, both species B and E were also found (Surendran, Sri Lanka, and K. Raghavendra, MRC, India, personal communication). This establishes that in Sri Lanka, species B and E are sympatric as in Rameshwaram island in India. Baimai, Kijchalao and Rattanarithikul (1996)

Anopheline Species Complexes in South and South-East Asia

27

reported species A and B from the Chiang Mai province of Thailand. These identifications were based on mitotic karyotypes described by Vasantha et al. (1982; 1983). Species A was identified as the karyotype which had a submetacentric Y-chromosome and species B as that which had an acrocentric Y-chromosome. Later, it was pointed out that Y-chromosome polymorphism had been observed in species B and also in species C (Adak et al., 1997). Taking into consideration that species B, which is found in the eastern districts of the state of Uttar Pradesh and in the state of Assam in India, the population in Thailand may be species B with Y-chromosome polymorphism. In Cambodia, Van Bortel et al. (2002) reported the presence of species B based on the ITS2 sequence analysis. Following this, the authors also suggest that An. culicifacies from the Sichuan province in China is also species B. This information on the distribuition of sibling species clearly indicates that the distribution of species A extends to countries to the west of India while that of species B to the east of India. In India, where all the five sibling species are prevalent, species B was found almost everywhere throughout the country wherever An. culicifacies was encountered. Species B was found exclusively in some areas, whereas in other areas it was found sympatric with A or C or D or E alone or in combination (Subbarao, Vasantha, Sharma, 1988a; Subbarao, 1991, Kar et al., 1999). Species A and B are sympatric in northern and southern India, with the predominance of species A in the north and species B in the south. However, in the eastern states of north India, species B predominates or is the only species present. Species B and C were predominant in the western and eastern regions, while species D was found in sympatric association with A and B in the north-western region, and with A, B and C in central India and in a few places in the state of Tamil Nadu in the south, where, in Rameshwaram island and also in a few blocks on the mainland in 28

Ramanathapuram district, species B and E were found sympatric. The distribution of species E in other areas is yet to be mapped. A map showing the distribution of sibling species in India is reproduced from Subbarao (1991) as Figure 6. In this map, the recently found species E is also shown. The sibling species were not only found to have definite distribution patterns but, in a given area, the prevalence of a species varied according to the seasonal environmental changes. Species A was found predominant throughout the year. An increase was observed in the proportion of species B in the post-monsoon months in villages around Delhi, where A and B were sympatric (Subbarao et al., 1987). Similar observations were made in the district of Surat, in Gujarat state, where species B and C were found sympatric. Species C of Gujarat behaved like species A did in and around Delhi (Subbarao, unpublished), and also in the district of Sundergarh, Orissa state ( Nanda et al., 2000). Biological variations such as hostspecificity, susceptibility to malarial parasites and response to insecticides have also been noticed between the species. An. culicifacies s.l. is predominantly zoophagic and feeds on man only when the cattle population is low (Rao, 1984). The four species, A, B, C and D, were found to be predominantly zoophagic. However, species A was found with a relatively higher degree of anthropophagy (about 3.5 per cent), compared to species B in several areas (Joshi et al., 1988). Species C and D were also found to have low anthropophagy (<1 per cent). In Rameshwaram island, where species B and E are sympatric, An. culicifacies s.l. was found to be much more anthropophagic than in other areas (Jambulingam et al., 1984, Hema Joshi, personal communication). By analogy, as species B has low anthropophagy, species E can be considered to be contributing to high anthropophagy in Rameshwaram island.

Anopheline Species Complexes in South and South-East Asia

The biting rhythms were distinct among species A, B, C and D (Satyanarayan, 1996). The biting activity of species A, B and C was found all through the night, while no biting activity of species D was observed after midnight. The peak biting activity of species A and B was in the second quarter of the night, between 2200 hours and 2300 hours; while the peak biting activity of species C was found to be in the first quarter of the night, between 1800 and 2100 hrs., in the month of April. However, the peak biting activity of species C shifted to the second quarter of the night in December in one of the study areas. Whether this is true in all areas where species C is prevalent needs to be examined. No seasonal change was observed for species A and B. Though the peak activity of species D was between 2100 hours and 2200 hours, 30 per cent to 40 per cent of this species bite in the first quarter (this is significantly higher than that observed for species A and B). In northern India, where species A and B are sympatric, in the cytologically-identified specimens of species A, sporozoites were found (Subbarao, Adak and Sharma, 1980). Using the two-site immunoradiometric assay technique of Zavala et al. (1982), species A, C and D were found to be vectors of P. vivax and P. falciparum malaria and species B a poor vector if at all (Subbarao et al., 1988b; 1992). The cumulative sporozoite rates for species A, C and D are 0.51 per cent, 0.3 per cent and 0.4 per cent respectively in India (Subbarao and Sharma, 1997). In Pakistan, where species A and B are sympatric, species A was incriminated and is considered the major vector (Mahmood, Sakai and Akhtar, 1984). In the same paper, the authors reported that though species B could not be incriminated under field conditions, in laboratory feeding experiments both species A and B supported sporogony of P. vivax and P. falciparum. Species B used in this study was supplied by the Malaria Research Centre, Delhi, India. This colony was established from An. culicifacies s.l. collected from an area where both A and B are sympatric. The

colony was identified as species B several generations after its establishment. In laboratory feeding experiments on P. vivaxinfected blood (Adak, Kaur and Singh, 1999) and P. vincei petteri and P. yoelii yoelii-infected mice (Kaur, Singh and Adak, 2000), species A had significantly higher oocyst rate compared to species B and C and species B was found the least susceptible. Recently, a strain totally refractory to P. vivax and partially to P. falciparum was isolated from species B (Adak et al., 2006). In this strain malaria parasites are encapsulated very early in the oocyst development leading to the death of the parasites. The low vectorial potential of species B in malaria transmission was also confirmed from our observation where there is no malaria, or its incidence is low, as in the eastern districts of Uttar Pradesh and in northern Bihar in India, where species B is predominant (Subbarao et al., 1988a). The identification of species E, a vector species, now explains and resolves the epidemiological paradox that sporozoitepositive specimens were found in An. culicifacies s. l. in Rameshwaram island (Sebesan et al., 1984), and specimens from the same area were identified as species B by polytene chromosome examination. Similarly, in Sri Lanka, only species B (Green and Miles, 1980; Subbarao 1988; Abhayawardena, Dilrukshi and Wijesuriya, 1996) and several sporozoite-positive specimens were found (Amerasinghe et al., 1991 & 1999). The recent molecular identification of An. culicifacies from Sri Lanka as species B and E (mentioned earlier in this section) also resolved the epidemiological paradox confronted with the cytotaxonomic identifications of this species. In Iran, where only species A is prevalent, it was incriminated for P. vivax sporozoites antigen in May and An. pulcherrimus for P. vivax antigen in the September and October collections by immunoradiometric assay in the same study (Zaim et al., 1993). In Afghanistan, in riverirrigated rice-growing villages, of the eight species incriminated, An. culicifacies had the

Anopheline Species Complexes in South and South-East Asia

29

lowest sporozoite rate of 0.20 per cent (only An. splendidus had lower rate than An. culicifacies) (Rowland et al., 2002). An. culicifacies s.l. is resistant to DDT and HCH in most parts of India, and in a few areas to malathion as well (Subbarao, 1988). Recently, this species was reported to be showing resistance to pyrethroids in Gujarat (Singh et al., 2002) and in Tamil Nadu (Mittal et al., 2002). Species A remains more susceptible to DDT than species B in areas where both A and B are sympatric and DDT has been withdrawn for long periods (Subbarao, Vasantha and Sharma, 1988c). In areas with species B and C sympatric association, species C developed resistance to malathion at a faster rate than did species B (Raghavendra et al., 1991), and species A at a slower rate than species B (Raghavendra et al., 1992). In Gujarat, where An. culicifacies recently developed resistance to pyrethroids, sympatric species B and C exhibited a similar pattern in the resistance levels (Singh et al., 2002). Surendran et al. (2002a & b; 2003) studied the biological variations between Ychromosome polymorphic types. Though the authors refer in their reports to these polymorphic types as species B and E, these studies were carried out before the two forms were correlated either with sporozoite positivity, as has been done by Kar et al. (1999), or with molecular identifications of Goswami et al. (2005 & 2006). As mentioned earlier in this section, the two Y-chromosome polymorphic forms in Sri Lanka have recently been identified as species B and E by molecular assays. Surendran et al. (2002a) reported that species E larvae were found in a wide variety of breeding sites and this species developed oocysts when fed on P. vivax and P. falciparum-infected patients (Surendran et al., 2002b). In species B, oocysts were not developed and it was found

30

breeding in rock pools and sand pools along river margins. These studies need to be extended to more areas, and sporozoite positivity has to be examined in both species B and E in field collections too. Species E was observed to be more resistant to malathion than species B (Surendran et al., 2003). Both species B and E were found equally susceptible to deltamethrin and lambdacyhalothrin. The Sri Lankan research groups are now planning to carry out these studies in detail with field-collected specimens and identifications by molecular methods of Goswami et al. (2005 & 2006) (N. Surendran, personal communication). Taking into consideration the biological differences observed among sibling species, an insecticide spray strategy for the control of An. culicifacies in India has been proposed (Subbarao and Sharma, 1997). Later, Subbarao, Nanda and Raghavendra (1999) stratified the country into seven major divisions based on the prevalence of An. culicifacies sibling species. An. fluviatilis sibling species’ prevalence was also taken into consideration while proposing specific control strategies in each of these divisions. It is considered that the implementation of this strategy will lead to more efficient use of insecticides and this will reduce the expenditure on insecticides and their impact on the environment. In Karnataka state, southern India (Ghosh et al., 2005), selectively-introduced larvivorous fish into tanks and wells brought down the densities of An. culicifacies, the major vector, and consequently malaria incidence in several villages. The concentration on tanks and wells and omission of streams was based on the finding that species A, the vector species, breeds predominantly in wells and tanks and species B, the non-vector species, in streams. This study emphasizes the advantage of sibling species identification and taking account of their biological characters while implementing vector control strategies.

Anopheline Species Complexes in South and South-East Asia

Figure 4: Schematic representation of polytene chromosomes of Anopheles culicifacies sibling species

X chromosome ab

+a +b

a

a

b

a b

b

b

a Chromosome arm 2 h1 i1 +

g1 h1

+ +

1 g1+h

g1

+

h1

i1

+ i1

h1

h1

h1

h1 h1

h1

i1

1 1

g i

h1

h1

g1

g1

g1

g1

g1 i1

1

i

Species A

Species D

Species B

Anopheline Species Complexes in South and South-East Asia

Species C

31

Figure 5: Mitotic karyotypes of Anophles culicifacies; Plates a-c are chromosome plates prepared from larval brain tissue. Plate a–Species B female, plate b–Species E male with submetacentric Y–chromosome; plate c–Species B male with acrocentric Y–chromosome. ( Source : Kar et al., 1999)

Figure 6: Map showing the distribution of members of the Culicifacies Complex in India (Source: Subbarao, 1991)

B B

AB BB AB B A B A AB B

AB AB A B AB AB AB A B A B

A BC D A BD

AB

ABC A BC A

BC

B ABC A B ACD

A BC D A BC D ABCD

A BC D

B

B

A BCB BC

B

B B

BC A BC BC

BC A BC A BC BC BC A BCD BC AB C A BC D BC A BC BC BC BC B C A BCD AB BC BC BC A B C AB C B C BC B AB AB AB AB B A B A BCD B BD

Species A Species B Species C

AB BE

Species D Species E

32

Anopheline Species Complexes in South and South-East Asia

3.4

The Dirus Complex

The Dirus Complex belongs to the subgenus Cellia, Leucosphyrus Group in the Neomyzomyia Series (Harbach, 2004), and occurs in the Oriental region. There has been a considerable interest in the Leucosphyrus Group as three members of this group, Anopheles dirus, An. balabacensis and An. leucosphyrus, are important malaria vectors in South and South-East Asia. However, there has been much confusion, in the literature regarding the taxonomy of this group. Peyton (1989) resolved the confusion and included 20 species and two forms belonging to the Neomyzomyia series in the Leucosphyrus Group based on the following morphological characteristics. In the adult stage they possess a very broad conspicuous white-scaled band covering the apex of the hind tibia and the base of hind tarsomere 1 and speckled legs. The wings have many discrete pale- and darkscaled spots on all veins and with four or more dark spots present on vein Cu-A and terminal abdominal segments always with some scales. Following a detailed morphological study, Sallum, Peyton and Wilkerson (2005) reclassified the species belonging to the Leucosphyrus Group under three Subgroups: Leucosphyrus, Hackeri (earlier referred to as Elegans) and Riparis (new). Leucosphyrus and Dirus are the two Complexes within the Leucosphyrus Subgroup (Table 5). The name, An. balabacensis, has in the past been frequently used in a misleading way until Peyton and Ramalingam (1988) and Peyton (1989) amended the Leucosphyrus Subgroup as follows: (i)

It now includes what was described as An. balabacensis in the South-East Asian mainland but is now referred to as An. dirus (Peyton and Harrison 1979).

(ii) The Balabacensis Complex referred to by Baimai, Harrison and Somchit (1981), Baimai et al. (1984) and Hii (1985) no longer exists.

(iii) An. balabacensis Perlis form is now species B and An. balabacensis Fraser’s Hill form is now species F of the Dirus Complex. (iv) An. balabacensis Taiwan form has been

raised to the species status by Peyton and Harrison (1980) and has been designated as An. takasagoensis Morishita. This is now included as a member in the Dirus Complex.

(v) The mosquitoes now given the name An. balabacensis and included as one of the members of the Leucosphyrus Complex (Table 5) are those belonging to the taxon described from Sabah, east Malaysia. (vi) An. elegans earlier described under the Elegans Subgroup has been redescribed and is now placed in the Dirus Complex (Sallum, Peyton and Wilkerson, 2005) (Table 5). Table 5: Species now (2005) * included in the Leucosphyrus Group Leucosphyrus subgroup 1. An. baisasi 2. Con Son island, Viet Nam Form Leucosphyrus Complex 3. An. balabacensis Baisas 4. An. introlatus Colless 5. An. leutens (leucosphyrus A) 6. An. leucosphyrus s.s. (leucosphyrus B) Dirus Complex 7. An. dirus (species A) 8. An. cracens (species B) 9. An. scanloni (species C) 10. An. baimaii (species D) 11. An. elegans (species E) 12. An. nemophilus (species F) 13. An. takasagoensis Hackeri (elegans) subgroup 1. An. hackeri Edwards 2. An. pujutensis Colless 3. An. sulawasi Waktoedi 4. An. leucosphyrus Sumatra form Riparis subgroup, new subgroup 1. An. cristatus King & Baisas 2. An. macarthuri Colless 3. An. riparis King & Baisas 4. Negros form Source: Peyton (1989) and *Sallum, Peyton and Wilkerson (2005)

Anopheline Species Complexes in South and South-East Asia

33

An. dirus has a wide distribution. It is found in India, Nepal, Bangladesh, Myanmar, Thailand, Indonesia, Malaysia, Viet Nam, Cambodia, south China, and Taiwan. This complex includes seven members recognized and two more suspected (details in the following sections). Peyton and Ramalingam (1988) provided the morphological and geographical descriptions of the Dirus Complex for the first time. They described the morphological characters of this complex which distinguish it from other members of the Leucosphyrus Group. The most significant adult character is: accessory sector pale (ASP) spot on the costa and usually also on the subcosta (an occasional male in some species belonging to the Leucosphyrus Complex exhibits an ASP spot on the costa, but always in less than 6 per cent of specimens in any population). Additional adult characters that serve to characterize this complex are: presector dark (PSD) spot on wing vein R with one or more pale spots on at least one wing (except occasional specimens of An. nemophilous) and hind tarsomere 4 with a distinct basal pale band or dorsal patch.

Evidence for identification of sibling species The cytological (mitotic karyotype) analyses of natural and laboratory colonized populations, crossing experiments between populations and morphological variations observed in natural populations have led to the recognition of members of this complex. Species A and B (Hii, 1985) The unidirectional F1 hybrid male sterility observed between the Bangkok strain identified as An. dirus by Peyton and Harrison (1979) and the Perlis form strain was the first evidence that An. dirus is a complex of two sibling species (Hii, 1985). The Bangkok strain was designated as species A and the Perlis

34

form as species B. A cross between B female and A male produced sterile hybrid males. Mitotic karyotypes of these two strains were described by Baimai, Harrison and Somchit (1981). Species C and D (Baimai et al., 1987) A strain from Kanchanaburi, Thailand, derived from a single female culture and mitotically different from species A and B (Wibow, Baimai and Andre, 1984) was designated as species C as males in one direction in crosses with species A, and in crosses with species B it produced no progeny. Species A female and C male cross produced sterile F1 males. The strain from Ranong and Phangna, Thailand, with a mitotic karyotype different from those of species A, B and C was designated as species D as it produced no progeny in reciprocal crosses with species A, B and C. Natural populations comprising mixtures of the chromosomally identified species A, B, C and D were examined for electrophoretic variations of six enzyme systems to associate chromosomal forms with allozyme genotypes (Green et al., 1992). Data were analysed by GENESTAT of Black and Krafsur (1985). The mean value of FIS for An. dirus sensu lato (sympatric species) was +0.28 (SD 0.02). The partitioning of electromorph data for the chromosomal forms reduced the mean FIS to +0.03 (SD 0.01), suggesting that positive assortative mating is a characteristic of each form (values of FIS range from +1.0 for total absence of heterozygotes in mixtures of two or more homozygotes to -1.0 for total absence of homozygotes). Furthermore, significant deviations observed by chi-square analysis were removed by partitioning. This indicates that different electromorphs at each locus are associated with one or the other of the chromosomal forms, which supports the finding that chromosomal forms are reproductively isolated populations.

Anopheline Species Complexes in South and South-East Asia

Species E (Sawadipanich, Baimai and Harrison, 1990) A colony established from mosquitoes collected from south-western India, and which produced sterile F1 hybrid males in all reciprocal crosses with species A, B, C and D, was designated as species E. The mitotic karyotype was described as different from those of species A and B, and resembling that of species D. Sallum, Peyton and Wilkerson (2005) examined morphologically more than 8000 specimens from seven countries for taxonomic revision of the Leucosphyrus Group. After describing larval, pupal and adult morphological characters of the provisionally designated members of the Dirus Complex, they have given formal designations to sibling species A, B, C, D and E (see Table 5 for the formal designations). Species F (Baimai, Harbach and Kijchalao, 1988) From the mosquitoes collected from the ThaiMalaysia border, an isofemale line selected due to its new cytotype was designated as species F as it produced hybrid males with atrophied testis lobes in crosses with species A, B, C and D and also with An. balabacensis from Sabah. Morphologically, it resembled the Fraser’s Hill form of An. balabacensis recognized by Colless (1956; 1957). The mitotic karyotype resembled that of species B in having acrocentric sex chromosomes (Table 6 and Figure 7) but the heterochromatic short arm is smaller than that of species B. Autosome III is metacentric because of a large block of heterochromatin while this chromosome is submetacentric in species B and also in other species. Peyton and Ramalingam (1988) have formally described species F (Fraser’s hill form) as An. nemophilus after comparing the voucher specimens with the cytotype of species F. This paper has a detailed morphological description of An. nemophilus which is compared with An. dirus (Species A)

and An. introlatus (a member of the An. leucosphyrus complex).

An. takasagoensis Morishita An. balabacensis Taiwan form was elevated to species status by Peyton and Harrison (1980) and designated as An. takasagoensis. The mitotic karyotype of this species was described by Baimai, Harrison and Somchit (1981). At that time it was described as a member of the An. balabacensis complex. This species is not sympatric with other members of the An. dirus complex and is found only in Taiwan (Peyton and Ramalingam, 1988). This species is included in the An. dirus complex because of its possession of diagnostic morphological characters of An. dirus. In addition to taking structural and heterochromatin variations in mitotic karyotypes and hybrid sterility in interspecific crosses as evidence for designating species, the asynapsis observed in polytene chromosomes of F1 progeny of interspecific crosses was also taken into consideration (Hii, 1985; Baimai et al., 1987; Baimai and Green, 1988; Sawadipanich, Baimai and Harrison, 1990). Two suspected new species The ITS2 sequence analysis revealed two types of sequences of species C, from northern Thailand (site 12) and another of species C from southern Thailand (site 14) (Walton et al, 1999). The population genetic analysis using 11 microsatellite markers of northern and southern populations of species C in Thailand, showed significant differences in the genetic structure (Walton et al., 1999 & 2001). Because of the geographical separation of these populations, it is not possible to distinguish the hypothesis of the existence of two species from that of geographical variation within species. From the sequence analysis of amplifications from PCR of the ITS2 region, Walton et al., (1999) report that species D

Anopheline Species Complexes in South and South-East Asia

35

from Thailand (with 493 bps) is different from species D from China as reported by Xu, Xu and Qu (1998). The authors suggest that species D from China may represent yet another species in this complex.

Techniques available for identification of sibling species The techniques available and their diagnostic features for the identification of sibling species are given in Table 6. Additional information, for which there was no space in the table, and the relevant references are given below. Mitotic karyotypes Baimai (1989) gives diagrams of the mitotic karyotypes of all sibling species and their distribution in South-East Asia (this figure is reproduced as Figure 7). All species have two pairs of autosomes, metacentric autosome II and submetacentric autosome III, and X and Y sex chromosomes. Variations in the structure and blocks of heterochromatin in chromosomes are used in the identification. In addition to the variations shown in Table 6, the following features can be used in the diagnosis: (i)

Species B has centromeric heterochromatin in the autosomes (Baimai and Traipakvasin, 1987). Observation of five types of Xchromosome and four types of Ychromosome suggests extensive polymorphism in this species.

(ii) Species F has a block of centromeric heterochromatin in autosome III which makes the chromosome resemble the metacentric type (Baimai, Harbach and Kijchalao, 1988). The short arms of the sex chromosomes are smaller than those of species B. Polytene chromosomes A standard map of polytene chromosomes of speices A has been prepared and has been used in comparative studies of the chromosomes of other species (Baimai, Poopittayasataporn and Kijchalao, 1988). 36

The tips of chromosomes X, 2R and 2L can be used as diagnostic characters for the identification of species. Species B has a fanshaped tip on the X-chromosome, which is quite distinct (Hii, 1985; Baimai, Poopittayasataporn and Kijchalao, 1988). Inversion a on the X-chromosome in species A is polymorphic with the inversion in a minority compared with the standard arrangement. Species D is highly polymorphic with at least a single paracentric inversion in each autosome (break points are given in Baimai, Poopittayasataporn and Kijchalao, 1988). In species D and E hybrids, the Xchromosome was found totally synapsed; therefore, it is inferred that it is also fixed for the a inversion of species D (Sawadipanich, Baimai and Harrison, 1990). The same paper also reports that species E possesses floating inversions in at least two autosomes, but no details are given of these inversions. Though initially mitotic karyotypes were used in the identification of sibling species, Baimai, Poopittayasataporn and Kijchalao (1988) report that polytene chromosomes are now being extensively used, and find this method more convenient. Electrophoretic variations Green et al. (1992) reported that electrophoretic phenotypes were suitable for the identification of sibling species. In this study, two sets of samples were used: (i) chromosomally-identified samples which included species A, B, C and D; and (ii) specimens identified by DNA probes which included sympatric populations of species A and D. Species A and D could be identified with an accuracy of 99 per cent using GCD and AAT enzyme systems. Species B could be identified by slow electromorph(s) of ACP, while in species A, C and D, a fast electromorph was observed. As a marker, human whole blood with normal haemoglobin was used (photographs of gels with a scale were presented to show the actual distances moved by alleles of different enzyme systems and haemoglobin).

Anopheline Species Complexes in South and South-East Asia

Egg morphology under light and scanning electron microscopes (Damron-gphol and Baimai, 1989) The egg size of species A (length 0.524mm x width 0.124 mm) and C (0.548mm x 0.143mm) was intermediate between that of species B (0.570mm x 0.146mm) and species D (0.515mm x 0.144mm). The number of rows of cells formed by tubercles in the outer chorionic membrane between the frill and the float was found to be species-specific (Table 6). Size variation and cells formed by tubercles could be observed under the light microscope. The tubercles that formed aggregates on deck surface were speciesspecific (Table 6) and were examined under the scanning electron microscope. Pupal setae under scanning electron microscope An. dirus Bangkok strain (now identified as species A) and An. balabacensis Perlis form (now identified as species B) were distinguished with 100 per cent accuracy by examining pupal setae under the scanning electronmicroscope (Choochote et al., 1987). An. dirus species A has a stout and simple pupal seta 9-IV on both right and left sides, while in species B it is long and slender and usually has side branches. There is no report on the evaluation of this observation in the field population. DNA probes Panyim, Yasothornsrikul and Baimai (1988) report four species-specific DNA sequences, and Audtho et al. (1995) have developed a simple system for the use of DNA probes, horse-radish peroxidase - labelled DNA probes - and a chemiluminscent detection system. The sensitivity of the system was such that it could detect 1-5 ng of target DNA, which was comparable to 32P labelled probes. This technique successfully identified species A, B, C and D from field collections. RFLP profiles Twenty restriction enzymes were used to study restriction fragment length

polymorphism (Yasothormsrikul, Panyim and Rosenberg, 1988) in species A, B, C and D. Seven enzymes (Ava II, Alu I, Bgl II, Hae III, Hinf 1, Mbob and Sau 3A I) produced unique patterns for each species while other enzymes produced unique patterns for one or two species. Allele-specific PCR (ASPCR) assay Xu, Xu and Qu (1998) reported a PCR assay using three primers, one derived from highly conservative 5.8S coding sequences and two from the internal transcribed spacer (ITS2) region of ribosomal DNA. This assay distinguished species A from species D with 374 base pairs and 663 base pair length amplicons respectively. Walton et al. (1999) reported a multiplex PCR assay with primers designed from ribosomal DNA internal transcribed spacer2 (ITS2) sequences of different species. This assay identifies species A (An. dirus s.s.), B, C, D or F (An. nimophilous) found in Thailand in a single reaction. Under the assay conditions developed, no products were generated with DNA from individuals of An. leucosphyrus, An. macarthuri or An. pujuteasis found in Sarawak. An. hackeri belonging to the Elegans (now Hackeri) Subgroup gave two faint bands of intermediate length between those produced by An. dirus B and C, and thus would not result in a false positive identification. Using this assay, 179 fieldcollected An. dirus s.l. from 15 sites distributed throughout Thailand and Malaysia were identified as one of the five species expected. The results were in agreement with the chromosomal identification of specimens or with known distributions of the species. The fragment size of species D from Thailand in this assay was 306 bases while the sizes for species A, B, C and An. nemophilus were 562, 514, 349 (or 353) and 223 bp respectively. The authors further state that species D from Thailand differs from that described as species D by Xu, Xu and Qu (1998) from China.

Anopheline Species Complexes in South and South-East Asia

37

Manguin et al. (2001) and Huong et al. (2001) also developed PCR assays which distinguished species A, B, C and D of the Dirus Complex. The method of Huong et al. (2001) used a cocktail of four primer sets for identificaton, and the authors report that it is a sensitive method requiring a small amount of DNA. They also report a short method, i.e. homogenizing the mosquito in 1x PCR buffer and use of the supernatant directly for PCR identification. It is a simple and rapid method for the identification of fieldcollected specimens. Microsatellite markers for population analysis Walton et al. (2000a) isolated 11 microsatellite markers from An. dirus species A and tested for polymrophism in species C and D. Walton et al. (2000b) used these markers to study populations of species A, C and D to address the issues of gene flow within each species. All the three species were found well differentiated from each other at the microsatellite loci used. The population genetic analysis using microsatellite markers also showed mtDNA introgression between species A and D (Walton et al., 2001).

Distribution and biological characteristics The association of An. dirus with malaria transmission in forests and forest-fringe areas is well established in Bangladesh (Rosenberg and Maheshwari, 1982), India (Rao, 1984), and Thailand (Rosenberg , Andre and Somachit, 1990). Baimai et al. (1988) report that the distinct differences exhibited by these species suggest some implications for Plasmodium transmission, and therefore recommend the identification of these sibling species during malariometric studies. Approximate information on the distribution of sibling species in Thailand is given below (for details see Baimai 1988, and Baimai et al., 1988). The map from Baimai

38

(1989) is reproduced as Figure 7. For the distinct pattern observed in the distribution of sibling species in Thailand, Baimai et al. (1988) do not suggest any geographical/ topographical reasons. Species A is widespread throughout Thailand except in the south. It occurs exclusively in the central and north-eastern regions. Species B and C have a restricted distribution. Species B is found only in the southern peninsular region of Thailand and extends into peninsular Malaysia. Species B is also reported from Sumatra island, Indonesia. Species C is reported only from Thailand (Kanchanaburi, Nakhon Si Thammarat and Phat-thalung). Species D is commonly found on the north-western side of Thailand. Along the Thai-Myanmar border it is found in sympatric association with species A. It is exclusively found in Myanmar and Bangladesh (Baimai et al., 1988) and north-eastern states of India (Baimai, 1989). The distribution of species D in India was predicted based on its distribution in Thailand and Myanmar. Recently, in four north-eastern states of India (Assam, Arunachal Pradesh, Meghalaya and Nagaland) using Walton et al. (1999) PCR assay, Prakash et al. (2006) confirmed the presence of species D. Species D is reported from Yunan province, southern China, but as mentioned earlier, this may be different from the one reported from Thailand and the other neighbouring countries. Species E is exclusively found in south-western India in the Kyasanur area of Shimoga hills in Karnataka state (Tiwari, Hiriyan and Reuben, 1987; Bhat, 1988). Species F (An. nimophilus, Peyton and Ramalingam, 1988) is found on the Thai-Malaysia border and is also reported from the monsoon forests of the mountain areas of south-eastern, southern and western Thailand and peninsular Malaysia (Rosenberg, Andre and Somachit, 1990). An. takasagoensis is exclusively found in Taiwan. Distinct seasonal variations in relative frequency were observed in sympatric species (Baimai et al., 1988). In areas where species

Anopheline Species Complexes in South and South-East Asia

B and C are sympatric, species B was found in abundance at the beginning of the rainy season (May to October) while species C was abundant at the end of the season. In species A and D sympatric areas, species A is more abundant at the end of the wet season, compared to species D which is abundant in the middle of the wet season. All-night human-bait catches indicated that all the four species bite throughout the night with the peak biting times being strikingly different in the four species studied (for details on study sites and other information, see Baimai et al., 1988). The peak biting activities were: Species A — 2100-2300 hours; Species B — 1900-2100 hours; Species C — 1800-2000 hours; and Species D — 0100-0300 hours. A study was carried out at Mae-tao-kee near Maesod in north-western Thailand (Green et al., 1991) to examine the vectorial potential of the members of the complex. In this area, An. dirus species A and D were sympatric and were found in association with four members of the Maculatus Complex and An. minimus species A. The man-biting rates of species A and D were 1.26 and 0.61 (these were lower than those observed for An. pseudowillmori, a member of the Maculatus Complex, and An. minimus species A), but had the highest sporozoite rates of 6.4 per cent and 2 per cent respectively. The only other species which was found positive for sporozoites was An. pseudowillmori (0.5 per cent) in this study. Baimai (1989), in a review on the members of the Dirus Complex, reported that a study carried out at Mae Sot, Tak province, and Tung Song, Thailand, revealed that species A, C and D were efficient vectors of P. vivax and P. falciparum malaria. (It is not mentioned in the review whether this conclusion was drawn from the epidemiological investigation in the area or whether they were incriminated.) In the same review it is mentioned that there was no evidence to show that species B was a vector under field condition and species F was not known as a vector of human malaria in

Thailand. In Binh Thuan province, southcentral Viet Nam, several An. dirus specimens were found positive for P. falciparum sporozoite antigen by ELISA (Van Bortel et al., 2001). The authors considered An. dirus to be the main vector in the area and suggested that despite the fact that other secondary vectors were found in the area, vector control should target against An. dirus. They further proposed that one round of bednet treatment preceding the transmission season of An. dirus would be sufficient to control malaria in the area. In India, in the north-eastern states, An. dirus D is abundant and implicated epidemiologically in malaria, but there is no information or report to say that species E is involved in malaria transmission. In the state of Assam in the north-eastern part of India, An. dirus (species D) was found to be highly anthropophagic, biting both indoors and outdoors equally (Dutta et al., 1996). In the forest areas of Assam, the vectorial capacity of An. dirus was the highest, 0.779 and 0.649 for P. vivax and P. falciparum respectively, during the hot monsoon season (JuneSeptember) and about 10-fold lower values were seen during the cool-dry season (MarchMay) (Prakash et al., 2001). Prakash et al. (2005) calculated the effective entomological inoculation rate (EEIR) for An. baimaii (formerly An. dirus species D) in a forest-fringe village in Dibrugarh district, Assam state, in different seasons. With an overall sporozoite rate of 1.9 per cent, in the monsoon season (June-September), 0.39 infective bites/ person/night and in the post-monsoon season (October-November), 1.47 infective bites/ person/night were observed for this species. Though biting was observed all night, the maximum infective bites were in the second quarter of the night. As 21 per cent of the total infective bites were recorded before 2100 hours, the authors suggested that appropriate protective measures are needed to supplement the impact of insecticidetreated nets (ITN) against An. baimaii in northeast India. The national malaria control

Anopheline Species Complexes in South and South-East Asia

39

Table 6: Diagnostic characters for the identification of sibling species of the Dirus Complex Species Mitotic appearance of X- and Ychromosomes

Inversion ‘a’ on polytene X- chromosomes

Enzyme systems

DNA probes

Egg morphology Tubercles Rows of cells between frill and float

Aggregates on deck surfaces

RFLP

PCR

An. dirus s.s (Species A)

Telocentric

X+a/a

Gcd 100 Aat 100

pMUA40, 1#5

2.5-3.5 rows broad

Moderately large and widely spaced

?

?

An. cracens (species B)

Acrocentric

X+a

Acp (slow)

pMU-B5

3.5-4.5 rows long and narrow

Same as A and closely spaced

?

?

An. scanloni (species C)

Tel ocentric

X+a

-

pMUC19.2

2-3 rows long and narrow

Large and closely spaced

?

?

An. baimaii (species D)

Telocentric

Xa

2.5-4 rows broad and irregular shaped

Small and closely spaced

?

?

An. elegans (species E)

Telocentric (Y-small rod or dot like)

Xa

-

-

-

-

-

-

An. nemophilus (Species F)

Acrocentric

-

-

-

-

-

-

?

An. takasagoensis

Telocentric

-

-

-

-

-

-

Gcd 128 pMU-D9 Aat 123 or 100

Figure 7: Map showing the distribution and diagrammatic representation of mitotic karyotypes of members of the Dirus and Leucosphyrus Complexes in South-East Asia (Source: Baimai, 1989)

Note: Kampuchea is now known as Cambodia.

40

Anopheline Species Complexes in South and South-East Asia

programme is planning to launch a large-scale ITN programme in the north-eastern states of India. In a study carried out to examine the behavioural heterogeneity of Anopheles species in six different localities in South-East Asia, Trung et al. (2005) found An. dirus s. s. in the central province of China and in Cambodia. This species was found to be highly anthropophagic, exophilic, exophagic and an early biter. Based on these charactersistics the authors state that this species is not suitable for control either by treated nets or by indoor residual insecticide sprays. A detailed review of literature on the distribution and bionomics of all members of this complex is given by Sallum, Peyton and Wilkerson (2005). Prakash et al. (2002) also studied the larval ecology of An. dirus in the rain forest area of Assam. An. dirus-positive shady ground pools showed higher mean values of total alkalinity, hardness and chloride content, while streamside pools showed lower pH and dissolved oxygen and higher total alkalinity and hardness, compared to negative breeding sites of similar type. In Mudon, a coastal area in Mon state in south Myanmar, An. dirus was found breeding in wells, a situation not encountered in other parts of Myanmar (Oo, Storch and Becker, 2002) and from any other country in South-East Asia so far. Shade vegetation and debris on the surface of wellwater were important factors influencing the abundance of larval and pupal density. In Mudon area, the malaria incidence is high, with slide positivity ranging between 9.9 per cent and 34.28 per cent throughout the year (Oo, Storch and Becker, 2003). In Myanmar, Bangladesh and northeastern parts of India, only species D has been reported. An. dirus breeding in wells in southern Myanmar calls for a detailed examination of the population for sibling species composition. Now that effective molecular tools are available, a study should be initiated at the earliest.

Somboon et al. (1999) isolated lines fully refractory and fully susceptible to Plasmodium yoelii nigerriensis (an African rodent malaria parasite) after 17 generations of mass selection. Most of the mosquitoes of the refractory line inhibited the parasite development by encapsulating oocysts, which appeared as melasized spots in the gut. But this line showed normal susceptibility to human malaria parasites, P. falciparum and P. vivax.

3.5

The Fluviatilis Complex

Anopheles fluviatilis James belongs to the subgenus Cellia, the Minimus Subgroup and the Funestus Group in the series Myzomyia (Harbach, 2004). The Fluviatilis and Minimus Complexes, An. flavirostris and An. lessoni, are members in the Minimus Subgroup. An. fluviatilis has a wide distribution in the Oriental region and parts of the West Asia subregion (Rao, 1984). In the Oriental region, it is found in Pakistan, Afghanistan, India, Nepal, Bangladesh, Myanmar, Thailand and south China, and in the West Asia region it is found in Iran, Iraq, eastern and southern Arabia, Oman and Bahrain. It is considered an important vector only in India, Pakistan and Nepal (Rao, 1984). Distinct differences observed in densities, preference to feed on a host and infection rates between the populations led Rao (1984) and several earlier workers, Senior White (1946), Viswanathan (1950), Brooke Worth and Sitaraman (1952), and Bhombore, Sitaraman and Achutan (1956), to suggest that this species may have two biological races. Subbarao et al. (1994) recognized An. fluviatilis as a species complex in India.

Evidence for recognition of sibling species The evidence for the three sibling species came from the absence of heterozygotes for the three polytene chromosome arrangements observed due to two fixed

Anopheline Species Complexes in South and South-East Asia

41

paracentric inversions q 1 and r 1 on chromosome arm 2. Chromosome arms 3, 4 and 5 and X-chromosome were homosequential in all the three species. The three sibling species were designated as species S, T and U (Subbarao et al., 1994). A population differing from the other three sibling species in polytene chromosome arrangement on chromosome arms 2 and 3 was recently recognized in district Hardwar, Uttaranchal state (a new state carved out of the erstwhile Uttar Pradesh state in northern India). In the same area species T and U were found sympatric. The absence of heterozygotes for the two new inversions was taken as evidence to designate this population as a new species (Nanda et al., unpublished). An. fluviatilis now is a comlex comprising four sibling species, S, T, U and new species V. Recently, based on the homology of D3 domain of 28S rDNA, Harbach (2004), Garros, Harbach & Manguin (2005) and Chen et al. (2006) considered An. fluviatilis S conspecific with An. minimus C. Consequently, Harbach (2004) removed An. fluviatilis S from the Fluviatilis Complex in his taxonomic update. Furthermore, all these authors said that the Fluviatilis Complex consists of only two sibling species, species T and U. However, recent analysis of Singh et al. (2006) on the sequences belonging to the internal transcribed spacer 2 (ITS-2) region and D2-D3 domain of ribosomal DNA of An. fluviatilis S and An. minimus C showed that the two species are appreciably different with pair-wise distance (Kimura-2-Paramtetre model) of 3.6 per cent and 0.7 per cent respectively for ITS-2 and 28SD2-D3 loci. Based on this analysis, Singh et al. (2006) concluded that An. fluviatilis S and An. minimus C were not conspecific. Further, from pair-wise and phylogenetic analysis, these authors showed that An. fluviatilis S was more closely related to other members of the Fluviatilis Complex than to An. minimus C.

42

Techniques available for identification of sibling species Ploytene chromosomes Paracentric inversions, q 1 and r 1 , on chromosome arm 2, are diagnostic: +q1+r1 is the standard arrangement diagnostic for species S, q1+r1 for species T and +q1r1 arrangement for species U (Subbarao et al., 1994). Other chromosome arms are homosequential in all three species. A photomap of polytene chromosomes of species S, with inversion break points, is presented by Subbarao et al. (1994). Chromosome arm 2, marked with breakpoints q1 and r1, is shown in Figure 8. Nanda et al. (unpublished) designated new inversions as s1 on chromosome 2 and s on chromosome 3 and the new inversion karyotype 2s1; 3s is diagnostic for species V. Mitotic karyotypes No variations were observed in the mitotic karyotypes of any of these sibling species. ASPCR assays A PCR assay developed from rDNA ITS2 region by Manonmani et al. (2001) was later correlated with cytological identifications (Manonmani et al., 2003). This assay accurately distinguished species S from species T in about 94 per cent of the specimens. Lack of 100 per cent correlation was probably due to the presence of q1 inversion polymorphism. This assay does not distinguish species T from species U (Manonmani, personal communication). An allele-specific PCR assay developed by Singh et al. (2004), based on differences in the D3 variable region of 28S ribosomal rDNA, differentiates all three species S, T and U. This multiplex assay has four primers, two universal primers – D3A forward and D3B reverse and two allele-specific primers – AFS and AFT – which are specific for species S and T respectively. The specimens not reacting to

Anopheline Species Complexes in South and South-East Asia

S- and T-specific primers were regarded as species U. Cytological correlation was done with specimens collected from different parts of India, and in these areas the three species were found in different sympatric associations. PCR identifications were in full agreement with the cytological identifications. In Mandya and Gulbarga districts in Karnataka state (south India), q1 inversion is polymorphic with the karyotypes +q1/+q1, +q1/q1 and q1/q1 in Hardy-Weinberg equilibrium. The question that arose was whether these belonged to species S or T. The allele-specific PCR assay identified all three karyotypes as species T (Singh et al., 2004). Singh et al. (2006), by examining ITS-2 sequences of species X reported by Manonmani et al. (2003) and of species S, conluded that species X was synonymous with species S. The five sibling species of the Culicifacies Complex which are sympatric with An. fluviatilis in many areas did not react with species S or T primers. However, they reacted with the universal primers, giving an amplicon of 380 base pairs like species U. The universal primers may react with other anophelines as well. Therefore, care must be taken to identify mosquitoes morphologically prior to the use of this assay. PCR-RFLP A slight modification of the above assay, by including a restriction enzyme, distinguishes the new species V (O. P. Singh et al., unpublished).

Distribution and biological characteristics Following the discovery of sibling species within An. fluviatilis, studies were carried out to map the distribution of the sibling species and study their bionomics. The biological differences observed are summarized in Table 7. Species S was found either alone or in sympatric association with species T, and species U was more frequently found with species T. In Kamrup district, Assam state, only species U was found but the number of

samples examined were very few. Allopatric populations of T were also observed (Figure 9). From Table 7, it can be seen that species S is distinctly different from species T and U in several biological characters. A longitudinal study was carried out on the bionomics of An. fluviatilis in the foothills of the Shivalik range of the Himalayas in Hardwar and Dehradun districts of Uttaranchal state (which is the newly demarcated state that was a part of the former Uttar Pradesh state in north India) (Sharma et al., 1995). It was found throughout the year with high densities in October/ November and low densities in May to August, densities in peak months being recorded as more than 200 caught per manhour of collection (“man-hour density”). An. fluviatilis breeding in these villages was found in slow-running streams, irrigation channels and subsoil seepage water with grassy margins, preferably under some shade. No breeding was observed in fast-flowing steams or in rice fields. In both the districts, species T and U were sympatric. In Dehradun, which is at an altitude of 640 m, species T was predominant (>95 per cent), while in Hardwar (294.7 m altitude), species U was predominant (>65 per cent). The biological characteristics of the two species were as reported in Table 7. The new species found in a few villages in district Hardwar was sympatric with species U and T (Nanda et al., unpublished). In Nainital district of Uttaranchal state, species T and U were also sympatric (Shukla et al., 1998). In Malkangiri and Koraput districts in Orissa state (in south-eastern part of India), where species S and T are prevalent (Manonmani et al., 2003), the breeding of these species was observed in terraced paddy fields, streams and stream channels (Sahu et al., 1990). Species S was found to be highly anthropophagic (~91 per cent) while species T and U were almost totally zoophagic (Nanda et al., 1996). From the district of Hardwar a small number of species T and U

Anopheline Species Complexes in South and South-East Asia

43

(a total of 189) and 294 specimens of An. fluviatilis s.l. processed by immunoradiometric assay for sporozoite antigen were found not positive (Sharma et al., 1995). In contrast, in the districts of Malkangiri and Koraput where species S is predominant, earlier in 1989 An. fluviatilis s.l. was incriminated for sporozoites (Gunasekaran et al., 1989) and later species S was incriminated (MRC, 1995). In areas where species S has been found, malaria is hyperendemic, and the prevalence of P. falciparum and deaths due to malaria are reported. In Sundergarh, a district in the northwestern part of Orissa, a longitudinal study was carried out to examine the bionomics and to delineate the role of An. fluviatilis and An. culicifacies sibling species in malaria transmission (Nanda et al., 2000). In Birkera block in a village in the forest, mainly An. fluviatilis species S and of the two An. culicifacies sympatric species B and C, species C maintained high transmission. The annual parasite incidence (API) was 269 cases/1000 population with 83.5 per cent P. falciparum. And in a deforested village, only An. culicifacies was found with species B and C sympatric (of these two, only species C is vector). Malaria incidence in this village was relatively low (API 39 cases/1000 population) and P. falciparum cases accounted for 57.9 per cent; P. vivax was the other Plasmodium species in these villages. Another detailed longitudinal epidemiological study is being carried out in the same district in 13 villages, eight in forest and five in the plain areas. This study is being conducted in order to develop a field site for vaccine trial. The observations are similar to the above-mentioned study with 347.9 API in the forest area and 61.0 in the plain area (Sharma et al., 2004a). The contribution of An. fluviatilis species S and An. culicifacies species C to entomological inoculation rate (EIR) in the forest area was 0.395 and 0.009 infective bites per person per night respectively, while in the plain area, only An. culicifacies species B and C were

44

found and the EIR was 0.014 (Sharma et al., 2006). In spite of continuous exposure to insecticides, An. fluviatilis still remains susceptible to all insecticides—DDT, HCH, malathion and pyrethroids in Orissa (Sharma et al., 2004b) and also in other areas where this species was tested in India (K. Raghavendra, personal communication). About 0.5% of An. fluviatilis s.l. from Afghanistan was found infective with both P. falciparum and P. vivax (CSP 210) CSP antigens (Rowland et al., 2002). The villages where this species was incriminated had riverirrigated rice fields. In the mountainous areas of the Hormozgan province, south Iran, An. fluviatilis is considered an efficient vector of malaria. It is exophilic and exophagic and densities start to build up in September and two peaks, one in January and another in May, are observed. Using the PCR assay of Manonmani et al. (2001), An. fluviatilis in the area was identified as sibling species T (Vatandoost et al., 2005). Specimens from Iran were also cytologically identified as species T (H. Vatandoost and N. Nanda, personal communication). Species T in Iran, unlike in India, apprears to be a vector. Recently, Adak et al. (2005) reported that sibling species T from India was susceptible to P. vivax infection in laboratory feeding experiments. This suggests that species T in India is genetically susceptible to Plasmodium infection, but because of its preference to feed on animals and the environmental conditions in addition may be making it a non-vector. Now that well-established cytotaxonomic and molecular methods are available for identification, populations from all the countries where An. fluviatilis is reported need to be examined for sibling species composition and their bionomics need to be studied to find intra- and inter-specific variations, if any.

Anopheline Species Complexes in South and South-East Asia

Table 7: Biological differences and diagnostic characters of An. fluviatilis sibling species observed in India* Inversion karyotypes

ASPCR

S

q1 r1

Yes

T

q1+r1

Yes

U

+q1 r1

Species

Man-hour densities

Feeding preference

Sporozoite positives

Low (1-40)

Anthropophagic (>90%)

Found

High (up to 200)

Almost totally zoophagic (~99%)

Not found

Prefered adult habitat

Ecotype

Human Hilly dwellings forests & foothills Cattle sheds

Foothills & plains

Endemicity

Hyperendemic Hypoendemic

* For details, see "Distribution and biological characteristics" in this section.

Figure 8: Photomap of the polytene chromosomes of An. fluviatilis from ovarian nurse cells. When the blocks are inverted with respect to standard arrangenents (of An. funestus), a dot is placed over the block designation. Break points of paracentric inversions q1 and r1 on chromosome arm 2 are shown (source: Subbarao et al., 1994)

Anopheline Species Complexes in South and South-East Asia

45

Figure 9: Map showing the distribution of members of the Fluviatilis Complex in India (Source: The map is courtesy of Dr Nutan Nanda, NIMR, Delhi)

Kangra Kulu Hardwar

Sonapur

Nainital Allahabad

Alwar

Jabalpur Purulia

Kheda

Koenjahar Mayurbhanj Sambalpur

Bahraich

Phulbani Sundergarh

Gulbarga

Balangir Kalahandi Koraput

Tumkur

Malkangiri

Kolar Mandya

Species S Species T Species U

3.6

The Leucosphyrus Complex

The Leucosphyrus Complex consists of Anopheles balabacensis Baisas, 1936, An. introlatus Colless, 1957, and An.leutens (leucosphyrus A) and An. leucosphyrus s. s. (leucosphyrus B) (see Table 5).

46

Evidence for recognition of sibling species Two isomorphic species, An. leucosphyrus A and B, were identified following the cytological examination of mitotic karyotypes and reproductive isolation observed in crosses between allopatric populations (Baimai, Harbach and Sukowati, 1988). The three allopatric populations of An. leucosphyrus were collected on human bait from two islands of Indonesia: (i) Sumatra (Bukit Baru,

Anopheline Species Complexes in South and South-East Asia

near Muarabungo, Bungo Tebo regency, Jambi province); (ii) South Kalimantan (Salaman, near Kintap, Tanah Laut regency); and (iii) Southern Thailand (Pedang Besar, Songkla province). There was no evidence of reproductive isolation between the Thai and South Kalimantan populations, as the F1 progeny were fertile from both reciprocal crosses. In contrast, from both reciprocal crosses between the Sumatra population and the populations from South Kalimantan and Thailand, F1 males were sterile, indicating reproductive isolation. Based on these results, the authors concluded that An. leucosphyrus includes two allopatric species, the one inhabiting Borneo, west Malaysia, and southern Thailand designated as species A, and the population confined to the island of Sumatra designated as species B. Species B is An. leucosphyrus Doenitz, the nominotypical member of the widely distributed Leucosphyrus Group. Baimai, Harbach and Sukowati (1988) presented photographs and diagrams of the mitotic karyotype of species A and B (see Figure 7). The differences between the two species were due to major blocks of heterochromatin. In species A, the X- and Ychromosomes of specimens from South Kalimantan possess a very small segment of extra heterochromatin at the centromeric region, which gives the chromosomes a subtelocentric configuration. In the Thai population, because of the absence of this block, the sex chromosomes appear telocentric (as shown in Figure 7). The Sumatra population of species B possesses submetacentric X- and Y-chromosomes. The short arm of the X and the whole of the Ychromosome is heterochromatic. The short arm of the X has a secondary constriction in the middle which is not found in any of the members of the Dirus Complex. On the X-chromosome of both the species, the presence of a distal block of heterochromatin is very conspicuous and

appears to be a character which distinguishes these two species from An. balabacensis. This diagnostic character also distinguishes the above-mentioned species A and B from the members of the Dirus Complex. Two types of Xs due to size variation in the long arm of the X were observed in each species. A conspicuous pericentric heterochromatic segment was seen in both the autosomes. In the Sumatra population (species B), this was more prominent in autosome III. Hii (1985) reported, for the first time, that mosquitoes from Sabah, which he referred to as An. balabacensi s. s., were distinct from An. dirus A and An. dirus B (Perlis form). An. balabacensis s.s.produced sterile hybrid males (partially-developed reproductive organs) when crossed with An. dirus A and B. Peyton (1989) has assigned An. balabacensis for the first time to the Leucosphyrus Complex (see Table 5) becuase it is morphologically closely similar to An. leucosphyrus. A diagram of the mitotic karyotype of An. balabacensis is presented by Baimai, Harbach and Kijchalao (1988) (see Figure 7). This species has acrocentric X- and Y-chromosomes. The Y-chromosome is totally heterochromatic and autosome III has a large block of heterochromatin. The authors also report that An. balabacensis, in crosses with species F of An. dirus, produced F1 males with atrophied testes without sperm, and the polytene chromosomes were totally asynapsed in the F1 progeny. No information on the fourth species, An. introlatus, has been found. Formal designations Sallum, Peyton and Wilkerson (2005), after morphological examination of several thousand specimens belonging to this complex, formally designated An. leucosphyrus species A as An. leutens and species B as An. leucosphyrus s. s.

Anopheline Species Complexes in South and South-East Asia

47

Techniques available for identification of sibling species Structural variations in mitotic karyotypes and the banding pattern associated with herochromatin variations are the only available methods for the identification of species.

Distribution and biological characters An. leucosphyrus species A is widely distributed in southern Thailand, west Malaysia, and Sarawak, east Malaysia, and Kalimantan in Indonesia, while species B is probably confined to the island of Sumatra, Indonesia (Baimai 1988; Baimai, Harbach and Sukowati, 1988). An. balabacensis is confined to the locality from which the type specimen came in Balbac island and neighbouring areas, i.e. Polawan island, Sabah and north-east Kalimanatan (Peyton and Harrison, 1979; Hii, 1985; Peyton, 1989). All species of this complex breed in shaded temporary pools in forests. An. balabacensis was found positive for P. falciparum sporozoite antigen by IRMA along with another species An. flavirostris on Banggi island, Sabah, Malaysia (Hii et al., 1988). Based on the human landing rate and sporozoite positives, the EIR/Year was calculated as 160. The vectorial capacity calculated for March was 1.44-7.44 and for November 9.97-19.7. On Banggi island, malaria is holoendemic. In south Kalimantan where An. balabacensis and An. leucosphyrus species A are sympatric, these two species together comprised 97.7% of the total anophelines collected (Harbach, Baimai and Sukowati, 1987). Large numbers of these specimens were collected from within the village than in the forest. The P. falciparum sporozoite antigen detection rate was 1.0% for An. leucosphyrus species A and 1.3% for An. balabacensis. After more than 50 years of effective management of malaria, in the subdistricts of Menoreh Hills and Dieng Plateu,

48

Java, Indonesia, a sharp increase in malaria occurred in the year 2000. Two important vectors, An. maculatus and An. balabacensis, which favour forested hill sides in Java, were considered responsible for the transmission (Barcus et al., 2002). An. leutens (species A) is considered highly anthropophilic and an important vector of human malaria, both in villages and forest areas of Sarawak, east Malaysia (Chang et al., 1995). This species is also considered a vector of Wuchereria bancrofti to humans in Sarawak (from Sallum, Peyton and Wilkerson, 2005). There is no report on the incrimination of An. leucosphyrus species B.

3.7

The Maculatus Complex

Anopheles maculatus Theobald belongs to the subgenus Cellia and the Maculatus Group in the Neocellia Series (Harbach, 2004). This species is recognized as an important vector of human malaria parasites in Thailand, Indonesia and peninsular Malaysia. To facilitate the understanding of the Maculatus Complex, information on eight nominal forms described in the literature (Christophers, 1931; Rattanarithikul and Green, 1986) is given below. Christophers regarded maculatus as a single species based on studies of the morphological variation in adults and recognized two vectorial forms: one with reduced abdominal scaling, the nominotypical form (maculatus), and the other with heavy abdominal scaling, var. willmori. He considered pseudowillmori, dravidacus and hanabusai as synonyms of the nominotypical form and considered dudgeonii, indicus and maculopsa as synonyms for var. willmori. Rattanarithikul and Green (1986) further state that in spite of extensive studies by Puri (1931), Christophers (1933), Crawford (1938), Reid, Wattal and Peters (1966), and Reid (1968), the morphological concept and formal

Anopheline Species Complexes in South and South-East Asia

taxonomy of this group have remained unchanged. The cytotaxonomy and morphological studies together have now unequivocally identified eight biological species, and the DNA analysis has recently identified a new species corresponding to the chromosomal form K in this complex. Nine biological species are described and discussed below.

Evidence for recognition of sibling species All the rearrangements in An. maculatus are referred to the An. stephensi map (Green, 1982) which serves as an arbitrary standard. (This is in contrast to other species complexes where chromosome maps prepared for an unspecified member of the complex or one of the sibling species are used as standard). The first evidence that An. maculatus is a species complex came from two largely independent studies on chromosomes– polytene and mitotic chromosomes from natural populations of An. maculatus. Within An. maculatus polytene chromosomes, no variation has been seen in chromosome arms 3, 4 or 5 while fixed and floating inversions were seen on the X and arm 2. A total of 18 paracentric inversions were observed and these inversions were observed in six distinct chromosomal forms in Thailand. These chromosomal forms were found in different sympatric associations in different areas. The six chromosomal forms were given the species status based on the population genetic data. The absence of heterozygotes for the alternate arrangements of the inversions in a given area was taken as evidence for the reproductive isolation. Species A, B and C (Green and Baimai, 1984, Green et al., 1985) In addition to the differences in paracentric inversions (as mentioned above), heterochromatin variation in X- and Ychromosomes was also observed (Green and Baimai, 1984). Three types of X-

chromosomes (X1, X2 and X3) and four types of Y-chromosomes were observed. No heterozygotes were found of X1 either with X2 or X3. And X1 was considered indicative of species A in contrast to X2 and X3 in species B, which were found in heterozygous condition. Y-chromosome polymorphic forms were not found associated with any particular species. The three species, A, B and C, were found sympatric in three localities, two near Kanchanaburi, west of Bangkok, and one in northern Thailand, near Chiang Mai and no heterozygotes were found for the diagnostic inversions (Green and Baimai, 1984). Two allopatric populations that are close to species B but differ from it in the frequency of two paracentric inversions on Xchromosome and four on chromosome arm 2 were identified as the E and F forms. The observation of heterozygotes for all the inversions that distinguish between species B and the E form in Petchaburi (near Kanchanaburi, Thailand) suggested that the form E is a chromosomal race of species B (Green and Baimai, 1984). The authors, however, caution that the sample size was small, hence the conclusions drawn are not final. Furthermore, F1 progeny from both reciprocal crosses between the B and E forms were fertile, supporting the concept that the form E may be a chromosomal race of species B. The status of form F could not be confirmed as B/E and F are separated by the Chao Phraya river basin from which An. maculatus s.l. is absent (Green et al., 1985), and larger samples of the F form are required. Cuticular hydrocarbon analysis (Kittayapong et al., 1990) differentiated species B from E form. There is also indirect evidence from cuticular hydrocarbon analysis that the two forms coexist in peninsular Malaysia (Kittayapong et al., 1993). Microsatellite marker analysis suggested that there was restricted gene flow between the

Anopheline Species Complexes in South and South-East Asia

49

northern populations of An. maculatus o o extending from latitude 11 to 16 N and the o o southern populations from latitude 7 to 6 N (Rongnoparut et al., 1999). Race B (species o B) extends northwards from latitude 13 N, while race E extends southwards from latitude o 12 N into peninsular Malaysia (Green et al., 1985). Based on microsatellite data analysis, the authors suggest that the southern populations may become distinct species (details in the next section). Rongnoparut et al. (1999) report that unpublished data of R. Rattanaritikul shows that inversion karyotypes show little indication of hybridization between B and E. Green et al. (1985) noted that B and E forms are either two distinct sibling species or they represent geographical variation within An. maculatus. Keeping in view that distinct cuticular hydrocarbon profiles were observed for the two forms, Harbach (2004) and Walton et al. (2005) suggest that they may be distinct species. Species G (Green and Baimai, 1984) A population fixed for inversions x and y on chromosome arm 2, and for d and e on Xchromosome, was found along with species A and B in a locality near Petchaburi in the northern part of peninsular Thailand. No heterozygotes were observed for these inversions and the inversions were unique for the population. This population was designated as species G. The results from reciprocal crosses between species A, B, C and G produced sterile F1 males and fertile females, which provided support for the designation of separate species based on cytogenetic evidence. Species D and J (Rattanarithikul and Harbach, 1990) An. maculatus form D was identified on the basis of chromosomal differences noted during the comparative cytological examination of populations from the Philippines, Thailand and Malaysia (Green et 50

al., 1985). Another population was collected from Subic Bay, to the west of Manila (Philippines), and the cytological examination of this population revealed that this was different from form D collected from the north-east of Manila. These two cytological forms, though very different, could not be designated as separate species because the two forms were identified from allopatric populations. Rattanarithikul and Harbach (1990) correlated distinctive morphological traits to each of the cytotypes. The observation of two morphotypes from single localities in the Philippines led them to state that there is reproductive isolation between An. maculatus form D and the new (J) cytotype, and, hence, these are distinct species. An. maculatus form D described in Green et al. (1985) was given the formal name An. greeni, and the new cytotype, J form, the name An. dispar (Rattanarithikul and Harbach, 1990). This is the first case where the morphological and cytological differences taken together demonstrated reproductive isolation. Species H and I (Green, Rattanarithikul and Charoensub, 1992) An. pseudowillmori and An. willmori were described morphologically by Rattanarithikul and Green (1986) and they were also identified as distinct cytological forms (I and H respectively) (Green, Rattanarithikul and Charoensub, 1992). The evidence for their species status came from the cytotaxonomic examination of populations from four localities in north-western Thailand. In these localities, An. willmori, cytologically designated as form H, and An. pseudowillmori as form I, were found in sympatric association with species A and B, with a total absence of heterozygotes for the inversions fixed in them. Enzyme polymorphism at 6-Pgd (6phosphogluconate dehydrogenase) also supported the specific status of An. pseudowillmori. Three allozymes or electromorphs were found associated with

Anopheline Species Complexes in South and South-East Asia

three species: An. pseudowillmori — 6Pgd70, An. maculatus (species B) — 6Pgd100, and An. sawadwongporni (species A) — 6-Pgd130, with a total absence of heterozygotes. In Thailand, six species (A, B, C, G, H and I) were found, while forms D and J were confined to the Philippines. The six forms in Thailand were found in the following different sympatric associations (Rattanarithikul and Green, 1986; Green, Rattanarithikul and Charoensub, 1992): A, B and C —

from several widely separated localities in western Thailand;

A, B and I —

from Mae Sariang, Mae Hong Son province;

B, H and I —

from Doi Inthanon, Chiang Mai province;

B and H —

from Mae Sa, Chiang;

A, B, C and G — in a single locality near Phet Chong , Nikhon Ratchasma province;

they have a unique sequence and is 3.7 per cent divergent from the next closely related taxon An. sawadwongporni in the group (Walton et al., 2007). The authors consider that this corresponds to the chromosomal form K reported by Baimai (1989). Based on the negligible intra-specific variations observed in the specimens analyzed, the authors designated the chromosomal form K as a new species in the complex. The biological species that were provisionally designated with the letters of the English alphabet were formally recognized by studying the morphological variations in progeny broods from wild-caught females that were identified by chromosomal rearrangements observed in their ovarian polytene chromosomes. Papers by Rattanarithikul and Green (1986) and Rattanarithikul and Harbach (1990) are strongly recommended for details regarding the formal recognition of these species. The provisional letter designations and their species names are given in Table 8. Species K does not have a formal designation yet.

A, B and G —

in one locality near Phet Buri in the northern part of peninsular Thailand;

Techniques available for identification of sibling species

A and F —

in a single locality near Nokhorn Nayak, northeast of Bangkok;

A, B, H and I —

in four localities in northwestern Thailand.

Polytene chromosomes The members of the Maculatus Complex can be identified unequivocally from each other and from An. stephensi (which is used as an arbitrary standard) by examining polytene chromosomes for paracentric inversions on autosomes. Sixteen inversions on chromosome arm 2, one on arm 3 and one on arm 5 are unique to this complex and hence diagnostic. Though differences in the banding pattern in the X-chromosomes are reported, the homologies have not been worked out well. The diagnostic inversions given in Table 8 are from Green, Rattanarithikul and Charoensub (1992). This paper gives the photomap of An. stephensi chromosome arm 2 with breakpoints of the inversions marked, and the diagnostic inversions summarized in a figure. For

In all these localities no heterozygotes were seen for the fixed inversions identified for these forms, which strongly supports the species status given to these cytologically distinct populations, and establishes the presence of six species specific mate recognition systems in the An. maculatus Complex in Thailand. New species, putative species K (Walton et al., 2007) The ITS2 sequence analysis of the specimens collected from eastern Thailand revealed that

Anopheline Species Complexes in South and South-East Asia

51

photomaps of chromosome arms 3, 4 and 5, the readers are referred to Green (1982). Morphological key A morphological key for the identification of all eight members of the Maculatus Complex is provided by Rattanarithikul and Green (1986). Upatham et al. (1988) found a small percentage of errors in the identification of species A, F form of species B and species B, using a morphological key (identifications were confirmed by cytological examination). Electrophoretic variations/Cuticular hydrocarbon profiles 6-Pgd allelic variations (Table 8) can also be used to distinguish An. sawadwongporni (species A), An. maculatus (species B) and An. pseudowillmori (species I) (Green, Rattanarithikul and Charoensub, 1992). The two forms of species B, E and F could be distinguished by gas-liquid chromatographic analysis of cuticular lipids in association with a multivariate principal component analysis (Kittayapong et al., 1990). PCR-RFLP An. greeni and An. dispar occur sympatrically in the Philippinnes. In order to develop a simple and reliable method of identification, Torres, Foley and Saul (2000) carried out an analysis of two regions of rDNA, ITS2 and D3 domain of the 28S gene of An. maculatus s.l. specimens from localities throughout the Philippines. Two distinct sequence groups were observed, one corresponding to An. greeni and the other to An. dispar. Digestion of the ITS2 amplicon with Hae II restriction enzyme yielded distinct fragments, and the two species could be identified with ease and accuracy. PCR assay Based on interspecific variation in the ITS2 region, a diagnostic PCR assay that distinguishes five members of the complex found in China, An. sawadwongporni, An. maculatus, An. willmori, An. dravidacus and

52

An. pseudowillmori, was developed (Ma, Li and Xu, 2006). Another PCR-based diagnostic assay has been developed to facilitate field research in northern Thailand, which distinguishes An. maculatus, An. dravidacus, An. pseudowillmori, An. sawadwongporni and species K (Walton et al. 2007). Microsatellite markers About 23 microsatellite markers were identified from An. maculatus s.s. (species B) (Rangnoparut et al., 1996). Seven of these microsatellites were used to study the genetic variation in eight widely dispersed localities in the western and peninsular Thailand (Rangnoparut et al., 1999). The data suggested that the populations could be grouped into two clusters: one including the upper and lower northern populations (extending from latitude 11o to 16o N) and the other including the southern population (extending from latitude 7o to 6o N). Among the populations, within each cluster, extensive gene flow was observed, while restricted gene flow was observed between the northern and southern populations. Geographical or genetic barriers could be limiting the gene flow between these populations.

Distribution and biological characters The distribution of the species given below is from Rattanarithikul and Green (1986), Baimai (1989), Ma, Li and Xu (2006) and Walton et al (2007): An. sawadwongporni (species A) — Myanmar, China, Cambodia, Thailand and Viet Nam. It is found at low and high altitudes in association with all other members of the group An. maculatus s.s. (species B) — Bangladesh, Myanmar, China, India, Indonesia, Cambodia, Malaysia, Nepal, Pakistan, Sri Lanka, Taiwan, Thailand and Viet Nam An. dravidicus (species C) — Myanmar (Kaley valley), India, China and Thailand An. notanandai (species G)—Thailand

Anopheline Species Complexes in South and South-East Asia

An. willmori (species H) — India, Nepal, Pakistan, China and Thailand (Chiang Mai) An. pseudowillmori (species I)— China, India, Nepal, Thailand and Viet Nam Species K — Thailand An. greeni (species D) and An. dispar (species J) are indigenous to the Philippines. The distribution of members of this complex in Thailand is given in Baimai (1989) and the map is reproduced in this document as Figure 10. An. greeni is widely distributed both in the low land and hilly areas of the Philippines (Rattanarithikul and Harbach, 1990). An. dispar appears to be more common, particularly at higher elevations than An. greeni (Rattanarithikul and Harbach, 1990). No sporozoite-positive specimens were found in An. greeni. However, Rattanrithikul and Harbach consider that this may be the same species which Ejercito (1934) found infected with oocysts and sporozoites of P. falciparum in the Bulacan province of Luzon island, Philippines. The first study conducted on the bionomics and vector potential of members of the Maculatus Complex was by Upatham et al. (1988). An. maculatus s.l. were collected from a village in the Pakchong district, Nakhon Ratchasima province, central Thailand, and another village in the Sadao district, Songkhla province, southern Thailand. In the central Thailand, species A, B (form F) and C, and in southern Thailand, only form E of species B, were identified. In Pakchong, An. maculatus species A was the most dominant species, followed by species B (form F) and species C, which was rare. The densities of species A and species B (form F) were high between July and November, with their peaks in October. The biting activities of both the species occurred throughout the night, with a major peak

during the first quarter of the night in all seasons. In the Sadao district, only An. maculatus species B (form E) was detected with peak densities between February and June. The biting activities of this species varied according to the season. The prevalence of mosquitoes was influenced by monthly rainfall, relative humidity and air temperature. All species identified in the study were found to be predominantly zoophagic and preferred to bite humans outdoors rather than indoors. The life expectancy recorded was the highest for species B (E form) — 0.7 days to 21.2 days; the maximum recorded for species A was 6.6 days and for the F form 8.1 days. No sporozoite-positive glands were found in any species (out of 4472 dissected) but 0.23 per cent oocyst-positives were found. In both the areas in the same study, An. dirus were found positive for P. falciparum sporozoites. This indicates that species A, B (the E and F forms) and C do not play any role in the transmission of malaria in Thailand. The E form was incriminated as the primary vector of human malaria in the peninsular Malaysia. Another study was carried out to determine the vector potential of the members of the Maculatus Complex at MaeTao-Kee near Maesod in north-western Thailand (Green et al., 1991). Four species, An. maculatus, An. dravidacus, An. sawadpongpornii and An. pseudowillmori, were found sympatric. One specimen of An. pseudowillmori was found infected with P. vivax and one with P. falciparum sporozoites (0.5 per cent sporozoite rate). These specimens were collected on human baits. The man-biting rate of An. pseudowillmori was 4.41/night while that of An. maculatus and An. sawadwongporni were 0.89/night and 0.41/night respectively. The man-biting rate of An. pseudowillmori was almost equivalent to that of An. minimus species A collected during the same study. No sporozoite-positives were found in An.

Anopheline Species Complexes in South and South-East Asia

53

minimus, but An. dirus A and D in the same area had the sporozoite rates of 6.4 per cent and 2 per cent respectively. These results clearly suggest that An. pseudowillmori is capable of maintaining a low-grade transmission in the absence of efficient vectors like An. dirus A and D. According to Green, Rattanarithikul and Charoensub (1992), An. pseudowillmori is at the limit of its distribution in Thailand, and they therefore suggest that its role in malaria may be different in Myanmar and north-east India, which are the centre of its distribution. Sporozoite-positive mosquitoes were earlier reported from Assam state in north-east India, Myanmar and Nepal (Rao, 1984). Two studies by Upatham et al. (1988) and Green et al. (1991) have shown that An. sawadpongpornii (species A), An. maculatus s.s. (species B) and An. dravidacus (species C) may not be playing a role in the transmission of malaria in Thailand. However, it should be noted that An. maculatus s.s. in penninsular Malaysia

(chromosomal form E at present assigned to species B) is a major vector of malaria (Green et al., 1991). An. willmori from the higher altitudes in Nepal is considered responsible for malaria transmission (Pradhan et al., 1970). Since An. willmori is found at high altitudes in Thailand, Green, Rattanarithikul and Charoensub (1992) suggest that its role should be studied. In river-irrigated, ricegrowing districts of eastern Afghanistan, An. maculatus s.l. was incriminated for malaria transmission during a study carried out from May 1995 to December 1996 (Rowland et al., 2002). One specimen was found positive for P. vivax CSP210 from the 30 tested. After more than 50 years of effective management of malaria, in sub-districts of Menoreh Hills and Dieng Plateu, Java, Indonesia, a sharp increase in malaria occurred in the year 2000. Two important vectors, An. maculatus and An. balabacensis, which favour forested hill sides in Java, were considered responsible for the transmission (Barcus et al., 2002).

Table 8: Diagnostic inversion genotypes and other methods available for the identification of Maculatus Complex members

Species name

An.sawadwongporni Rattanarithikul & Green 1986 An. maculatus s.s. Theobald 1901 An. dravidicus Christophers 1924

Cytotaxonomic designation

Diagnostic inversion genotypes on arm 2*

6-Pgd electromorphs

PCRRFLP

A

pt1u1v1w1

130

-

?

B,E,F C

j x1y1z1

100 -

-

-

? -

? ? -

An. greeni Rattanarithikul & Harbach

D

q

An. notanandai Rattanarithikul & Green 1986 An. willmori James 1903

G

xy

An. pseudowillmori Theobald 1910 An. dispar Rattanarithikul & Harbach 1990 Putative species K

ITS2-based PCR assay

-

H

-

-

-

?

I J

o1p1q1 r1

70 -

?

? ?**

* These inversions are used to distinguish An. maculatus sibling species from An. stephensi. In addition to these, in all species of the Maculatus Complex, inversions a on arm 3, x on arm 4, and c and d on arm 5 are fixed, an exception is An. pseudowillmori in which the +x arrangement on arm 4 is seen as in An. stephensi. An. willmori is distinguished from An. stephensi only by 3a, 4x and 5cd. ** ITS2 sequence of this species was different from other species but the ITS2 assay developed by Ma, Li and Xu (2006) was not tested on this species.

54

Anopheline Species Complexes in South and South-East Asia

Figure 10: Map showing the distribution of members of the Maculatus Complex in Thailand (Source: Baimai and Green (1988))

95

o

97

o

99

o

101

103

o

105

o

o

VIETNAM

20

o

MYANMAR

LAOS

18o

16o THAILAND

14o COMBODIA

12

o

10

o

GULF OF THAILAND

8o

6o MALAYSIA

3.8

The Minimus Complex

Anopheles minimus Theobald belongs to the subgenus Cellia, the Minimus Subgroup, the Funestus Group in the Myzomyia Series (Harbach, 2004). This species has a wide distribution in the Oriental region, and throughout the range of its distribution it is considered an important vector of malaria. It is found in India, Nepal, Sri Lanka, Bangladesh, Myanmar, Thailand, Malaysia, Indonesia, south China, Hong Kong, Taiwan and the Ryuku Archipelago, Japan (Rao, 1984). Harrison (1980) reported 12 morphological variants in An. minimus

populations from Thailand. Suthas et al. (1986a) observed significant differences in parous rates between females fed on bovids and those on humans. Also, a significant tendency was observed in females to return to the type of host upon which they were first caught (Suthas et al., 1986b). To explain the genetic heterogeneity observed in An. minimus populations in Thailand, the authors suggest the possibility that the taxon comprises morphologically cryptic species. Yuan (1987) reported two morphological forms, A and B, from the hilly regions of China. Sucharit et al. (1988) identified three

Anopheline Species Complexes in South and South-East Asia

55

morphologically variant forms in the Thai population: Typical minimus form (M), Varuna form (V) and Pampanai form (P). Among the morphological differences described, the characters that are distinct in the three forms are: M form - wing with presector pale spot (PSP) on costa; V form

- wing with completely dark prehumeral and humeral bands on costa;

P form

- with two pale spots, presectoral (PSP) and humeral (HP), on costa.

An. minimus has now been recognized as a complex of 5 sibling species, A, B, C, D and E. Though species B (Sucharit et al., 1988) and species D (Baimai, 1989) have been reported, no further information is available on these species.

Evidence for recognition of sibling species Species A, B and C (Sucharit et al., 1988) Sucharit et al. (1988) examined wild populations in Thailand for electrophoretic variations in seven enzyme systems in relation to the morphological forms. The enzyme systems studied were: esterases (EST), malic enzyme (ME), leucine aminopeptidase (LA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), xanthine dehydrogenase (XDH) and aldehyde oxidase (ALDOX). Of these, ALDOX and MDH were monomorphic. Although for such studies more enzyme systems are required to draw reliable conclusions, a clear-cut relationship was nevertheless seen. In the Pu Toei population, the P form was predominant (frequency 0.947), in other populations the M form was predominant (frequency 0.963 to 1.00), and in Sattahip, the M form frequency was 0.794, and that of the V form was 0.206. This was the highest frequency recorded for the V form; in other populations the frequency ranged from 0-0.033. Furthermore, the authors reported that,

56

among the five alleles observed at the Est-2 locus, Est-298 was predominant (frequency 0.833) in the Pu Toei population, while Est2 100 allele was common in all other populations (frequency 0.357 to 0.604). Based on these results, the authors, for the first time, concluded that An. minimus is a species complex consisting of three species. Typical An. minimus (M form) with predominant Est-2100 or 102 electromorph was designated as species A, the P form predominant in Pu Toei with Est-2 98 electromorph was designated as species C, and the V form found in low frequency in Thailand was designated as species B. Specis A and C (Green et al., 1990) The population genetic evidence demonstrating a lack of gene flow within An. minimus in Thailand came from the study of wild populations for electrophoretic variations in six enzyme systems by Green et al. (1990). Sympatric occurrence of two electromorphs, 134 and 100, at the Octanol dehydrogenase (Odh) locus, and the absence of heterozygotes in two localities, was taken as evidence that An. minimus is a complex comprising two sibling species. This conclusion was further supported by the relative deficiency of heterozygotes observed at both the Manose phosphate isomerase (Mpi) and the Glycerol dehydrogenase (Gcd) loci, and the disappearance of the deficiency when the genotypes of these loci are classified into the two Odh classes. In the other enzyme systems—phosphoglucomutase (Pgm), hydroxyacid dehydrogenase (Had), lactate dehydrogenase (Ldh) and malate dehydrogenase (Mdh)—studied, the relative frequencies of alleles were different in the two populations. The authors ruled out possibilities such as immigration/emigration and close linkage between loci which could cause such associations. The evidence for this was based on genetic analyses which established linkage relationship of these loci (Thanaphum et al., 1990) and consistent absence of heterozygotes over a four-year

Anopheline Species Complexes in South and South-East Asia

study period at Ban Phu Rat. Following the nomenclature given by Sucharit et al. (1988) to sibling species in this taxon, Green et al. (1990) equated Odh100 to species A and Odh134 to species C. Van Bortel (1999) studied An. minimus populations from Viet Nam using electrophoretic variations in 14 enzyme systems. Significantly high positive Fis values at the Odh locus gave a clear indication of non-random mating within this species, and the deficiency of heterozygotes indicated reproductive isolation between two sympatric populations, species A and C, as in Thailand. Species E (Somboon et al., 2001) An. minimus specimens collected from Ishigahi island, Ryuku Archipelago, Japan, were identified as species E by Somboon et al. (2001). Based on morphological characters and results from genetic crosses, the population from Ishigahi island was recognized as a new species. 99.5% of the specimens from Ishigahi island resembled species A in having a pale spot on the costa, but differed with species A and C in having a pale fringe spot at the tip of vein A (a character rarely found in An. minimus populations from other countries). In scanning micrographs, cibarial armamature showed cone filaments differing in shape from those of species A and C. In a cross between ISG males (strain established from An. minimus collected from Ishigahi island) and species A (CM strain) females, the hybrid males were sterile due to atrophied testes or presence of abnormal spermatozoa. The hybrid females, when back crossed to the parental males, laid very few eggs and there was low hatch. No F1 progeny were produced from the reciprocal cross. Metaphase karyotype of ISG differed from CM strain (species A) in having different polymorphic Y-chromosomes. Polytene chromosomes of F1 hybrids exhibited no asynapsis. These data conclusively suggested that species A and E are distinct species. Recently, Somboon et al., (2005), based on results from genetic crosses between species

C laboratory colony and provisionally designated species E ISG strain, showed that species C and E are distinct species. In both the crosses, F1 males were sterile and polytene chromosomes of F1 hybrid larvae exhibited partial asynapsis. In all the chromosome preparations an inversion heterozygote in 3R chromosomal arm was observed. Suspected new species (Sharpe, Harbach and Butlin, 2000; Somboon et al., 2001) From the DNA sequences at a mitochondrial locus (Cytochrome Oxidase II) and three nuclear loci (ITS2 and D3 regions of rDNA and Guanylate cyclase), Sharpe, Harbach and Butlin (2000) confirmed the presence of species A and C within An. minimus from Thailand and also reported the possible presence of another species. The specimen suspected to be a new species (specimen no. 157) morphologically resembled species C (humeral pale spots on both wings), and had unique D3 and ITS2 sequences. But the COII sequence was identical to that of some specimens belonging to species C; thus, it resembled species C more than species A. Since only one specimen was observed, a species status was not assigned to this specimen. The authors also reported a personal communication from C. A. Green which stated that allozyme data from the same locality supported the presence of a new species. Somboon et al. (2001) observed variations in D3 sequences in the specimens from Viet Nam. Sequences from two of the four specimens were similar to those of species A and C, while two sequences differed from each other and also from those of species A and C. These sequences also differed from that of species E. The authors argue that novel sequences might represent new species because each specimen is homozygous for a particular sequence, and suggest that this represents reproductive isolation. Based on this evidence the authors consider that four species are present in Viet Nam. Van Bortel and Coosemans (2003)

Anopheline Species Complexes in South and South-East Asia

57

argue that this evidence is unconvincing. According to them, the two novel sequences probably are due to intra-specific variation. And this has been insufficiently ruled out by Somboon et al. (2001) and considered that the sequences represent new species.

Techniques available for identification of sibling species Mitotic karyotypes Sucharit et al. (1988) briefly described the sex chromosomes and a fluorescent band in the X-chromosome of the M and P forms. Baimai, Kijchalao and Rattanarithikul (1996) described mitotic karyotypes of species A and C from Thailand. Species A can be distinguished from species C, as species C has prominent pericentric heterochromatin in the autosomes and the short arm of the X. Furthermore, two types of X-chromosomes differing in the length of the long arm in species A and a third type of X in species C were described. Both microphotographs and karyotype diagrams are given in Baimai, Kijchalao and Rattanarithikul (1996). Polytene chromosomes Kanda et al. (1984) reported photmaps and line-drawing maps of salivary gland polytene chromosomes of An. minimus. The authors concluded that the polytene chromosomes of ISG strain and of two strains isolated from Kanchanburi, Thailand, have homosequential banding pattern as no asynapsis was observed in F1 progeny from the crosses between the strains. Although the species status of the two strains from Kanchanburi is not known, the ISG strain has recently been identified by Somboon et al. (2001) as species E. Somboon et al. (2005) reported that species A and species E have homosequential banding pattern, while species C differs from species E by a fixed inversion in chromosome arm 3L and one in arm 3R, and partial asynapsis was observed on chromosome arms 2R and 3L in F1 hybrid larvae.

58

Diagnostic Electrophoretic variations Alleles at the Odh locus, allele 100 in species A and 134 in species C, are diagnostic for the Thai populations (Green et al., 1990) (Table 9). In the absence of known reference standards, human AA haemoglobin has to be run on each gel to estimate the mobility of the bands of unknown specimens. The relative mobility of haemoglobin AA was more useful in the TEB (Tris-boric acid EDTA buffer system) gels than in the TC (Tris-citric acid buffer system) gels. Relative mobilities of a single human Had electromorph and three intense bands of human Ldh were more useful on TC gels. Green et al. (1990) also reported Mdh-1 electromorphs to be diagnostic for distinguishing An. minimus s.l., An. aconitus and An. pampanai. Van Bortel et al. (1999), following the Green et al. (1990) technique, could distinguish An. minimus s. l. from the closely related species An. aconitus and An. jeyporiensis at the Odh locus in northern Viet Nam. The authors further reported that in Viet Nam An. minimus A was monomorphic for Odh100 allele but in contrast to Green et al. (1990) finding in Thailand, An. minimus C in Viet Nam was polymorphic for the Odh locus. However, Van Bortel et al. (1999) in Viet Nam could not separate unambiguously An. minimus s.l. from An. aconitus using Mdh-1 variation. Est-2 alleles reported by Sucharit et al. (1988) should be tested for their specificity against diagnostic Odh alleles. Yuan (1987) also reported that their A form and B form exhibit distinct esterase banding patterns. Molecular assays Now there are molecular assays available which not only distinguish species A and C (Table 9) but also other sympatric anophelines found with An. minimus. Sucharit and Komalamisra (1997) used RAPD-PCR to distinguish species A and C. Fifteen different commercially available primers (Operon oligonucleotide kit M from Operon Technologies, Inc.) were used. Six primers were found diagnostic on the basis of the

Anopheline Species Complexes in South and South-East Asia

presence or absence of amplified fragments in species A and C. However, these markers have not been validated on the field samples. Sharpe et al. (1999) developed an allelespecific amplification assay (ASPCR) from the D3 variable region of the 28S rDNA gene, which distinguished species A and C. A single strand confirmation polymorphism (SSCP) assay of the D3 amplified region was also developed, which distinguished species A from C and also An. aconitus and An. varuna. Van Bortel et al. (2000) developed a PCRRFLP technique which distinguished species A and C and An. pampanai Buttiker and Beales, An. aconitus, An. varuna and An. jeyporiensis, which are sympatric. ITS2 rDNA amplification followed by digestion with BisZ l enzyme accurately distinguished six species belonging to the Myzomyia Series (Van Bortel et al., 2000). The technique was evaluated on specimens collected from Viet Nam, Thailand, Cambodia and Laos. Garros et al. (2004b) extended the above PCR-RFLP assay by including more species from the Funestus Group and An. gambiae as outgroup in the evaluation. The assay distinguished An. minmus A and C, hybrids between A and C and 11 other species. In an effort to provide a simple and robust technique for the identification of members of this complex, Kengue et al. (2001) developed a multiplex PCR method based on RAPD markers. From the sequences of the RAPD markers, sequence characterized amplified regions (SCARs) specific primers of 20-24mer were designed. In the assay, one

primer set for distinguishing species A and C and one each for An. pampanai, An. varuna and An. aconitus were combined. The method was validated on a large number of specimens collected from Viet Nam, Thailand, Cambodia and Laos. In northern Viet Nam and north-western Thailand where species A and C are sympatric, <1% of hybrids were observed by the isoenzyme technique (Van Bortel et al., 1999), the PCRRFLP method (Van Bortel et al., 2000) and by the multiplex PCR of Kengue et al. (2001). This suggests that all techniques are equally sensitive and specific. Studies correlating the morphological characters with the electrophoretic variation by Van Bortel et al. (1999) and with molecular diagnostic markers by Sharpe (1999) and Chen, Harbach and Butlin (2002) have established that the morphological characters mentioned by Sucharit et al. (1988) are unreliable in distinguishing species A from species C. Furthermore, it has been clearly shown that form A and form B described in China by Yuan (1987) should not be equated to species, and these forms have no taxonomic status. The An. minimus described by Harrison (1980) has been identified as species A. Another multiplex PCR assay has been developed for the identification of An. minimus and four other anopheline species belonging to the Minimus Group (Phuc et al., 2003). Allele- specific primers were developed from the rDNA ITS2 region. Species A and C and An. varuna, An. aconitus,

Table 9: Techniques1 available for the identification of sibling species of An. minimus

Species A C E

SEM

Mitotic karyotypes

Electrophoretic

RAPD-

Variation

PCR

PCRRFLP

SSCP

ASPCR2

Yes Yes Yes

Yes Yes -

Odh100 Odh134 -

Yes Yes -

Yes Yes -

Yes Yes -

Yes Yes -

1 Details in the text. 2 Several PCR assays are available which not only distinguish species A and C, but also distinguish other sympatric anophelines (details in the text).

Anopheline Species Complexes in South and South-East Asia

59

An. pampanai and An. jeyporiensis could be easily differentiated by this assay. The authors claim that their assay results can be interpreted more readily than previous assays. Garros et al. (2004a) developed a multiplex allele-specific PCR which not only distinguishes members of the Minimus Group, An. minimus A and C, An. aconitus, An. varuna, An. leesoni and An. pampanai, but also four members of the Funestus Group, An. funestus, An. vandeeni, An. rivulorum and An. parensis, in a single step. The assay was developed based on the variation seen in the ITS2 region.

Distribution and biological characters Baimai and Green (1988) give a map showing the distribution of the sibling species of An. minimus in Thailand (see Figure 11). Species A is widespread in Thailand, in the ThaiMyanmar border areas species C is present, and in one locality species D is also seen. Species A has now been reported from Viet Nam, Laos and Cambodia and is found sympatric with species C in Viet Nam (Van Bortel et al., 2000, 2001; Kengne et al., 2001; Garros et al., 2005), Laos (S. Manguin, personal communication) and China (Zhou et al., 2002; Chen, Harbach and Butlin, 2002). Though Sucharit et al. (1988) reported species B from Thailand and China, recent molecular studies identified only species A and C from these countries. Both species A and C from Thailand were reported to be predominantly zoophilic, feeding on humans more outdoors than indoors. At MaeTao-Kee near Masod in northwestern Thailand where members of the Maculatus, Dirus and Minimus Complexes were prevalent, An. pseudowillmori and species A and D of the Dirus Complex were found positive for sporozoites, but no sporozoite-positive An. minimus species A were found, even though the man-biting rate/ night (4.41) of this species was almost equivalent to that of An. pseudowillmori (Green et al., 1991). Furthermore, species A

60

and D of the An. dirus complex had manbiting rates of only 1.26 and 0.61. Green et al. (1991) suggest that An. minimus species A in this area is not as efficient as members of the Dirus Complex in transmitting malaria. An. minimus populations in India are highly anthropophagic (Rao, 1984). In Sonapur, Assam state, this species has been found resting indoors, highly anthropophagic and with sporozoite rates ranging between 2.3 and 3.35 (Wazihullah, Jana and Sharma, 1992). It is from this area, based on Odh electromorphs An.minimus specimens were identified as species A (T. Adak, personal communication). Recently, from four northeastern states of India–Assam, Arunachal Pradesh, Meghalaya and Nagaland An.minimus specimens were identified as species A using Phuc et al. (2003) PCR assay (Prakash et al., 2006). In Orissa state, An. minimus species A was collected from hilltop villages in Singhbhum hills of Koenjhar district after 42 years of its disappearance following DDT spraying (Jambulingam et al., 2005). These findings suggest that there is only one member of the Minimus Complex, species A in India. The introduction of insecticidetreated bednets in Assam, reduced the slide positivity rate considerably (Jana-Kara et al., 1995). The An. minimus species A from Assam appears to be different from that found in Thailand with respect to host-feeding preference and sporozoite positivity. In northern Viet Nam, Van Bortel et al. (1999) observed species C to be more exophagic and zoophilic than species A. Species A was found to be highly endophagic and five times more abundant in indoor collections than species C. This behavioural difference is considered significant, because cattle are kept inside the houses. These observations suggest that An. minimus C may not be a vector of importance in Viet Nam. In China this species is considered a vector (Chen, Harbach and Butlin, 2002). Van Bortel et al. (2001), using the PCR-RFLP method developed by Van Bortel et al. (2000) for the Minimus Group, correctly identified a

Anopheline Species Complexes in South and South-East Asia

majority of the specimens from a village in south-central Viet Nam as An. varuna, which is predominantly a zoophagic species. Earlier, following morphological identifications, a majority of the specimens from this area were wrongly identified as An. minimus s. l. Because it is considered an important vector, the control strategies in the area were focused against two vectors, An. minimus and An. dirus. Now, it has been established that An.

dirus is the main vector in the area, several specimens of An. dirus being found sporozoite-positive by ELISA (Van Bortel et al., 2001). A study by Van Bortel et al. (2003) found species A breeding along the banks of slowmoving rural streams, while in the suburban areas of Hanoi it was found breeding in water tanks. This demonstrates the adaptability of

Figure 11: Map showing the distribution of members of the Minimus Complex in Thailand (Source: Baimai and Green (1988))

VIETNAM

MYANMAR

LAOS

THAILAND

COMBODIA

GULF OF THAILAND

MALAYSIA

Anopheline Species Complexes in South and South-East Asia

61

this species to different breeding environments. In a recent study carried out by Van Bortel et al. (2004), intra-specific behavioural differences among the populations of species A collected from six different localities were observed. This species was found more anthropophagic in central Viet Nam, Laos and Cambodia where cattle were scarce, than in northern Viet Nam where cattle were in good numbers. A similar observation that the anthropophagy of species A was dependent on cattle was made by Trung et al. (2005). These authors observed that species A is a late biter and it bites throughout the night. These observations led Trung et al. (2005) to suggest that insecticidetreated bednets was a suitable strategy for the control of this species. Garros et al. (2005) reported that with the introduction of permethrin-treated bednets from 1999 to 2001 in central Viet Nam, species A population was drastically reduced, and during the same priod species C which was found in low numbers in the area, increased significantly. In this study, molecular identifications were done with AS multiplex assay developed by Garros et al. (2004a).

3.9

The PhilippinensisNivipes Complex

An. philippinensis and An. nivipes belong to the Annularis Group of mosquitoes in the Neocellia series. (Harbach, 2004) An. philippinensis and An. nivipes are morphologically very similar to the only diagnostic wing character being: presector dark mark on vein 1 does not reach as far back as the distal end of the humeral dark mark on the costa in An. philippinensis, and the presector dark mark overlaps the humeral dark mark on the costa in An. nivipes. However, this character is equivocal (Reid, 1968). Because of the overlap in the morphological characters, An. nivipes was considered as a synonym for An. 62

philippinensis. Reid (1967) reported that there is little or no overlap in distinguishing these two species if the characters on the paddle at the pupal stage are used, and he elevated nivipes to species level. An. philippinensis is reported to be distributed from India to the Philippines, and northward to China. Because of the overlapping adult wing characters with An. nivipes, accurate distributions of these two species cannot be defined. Klein et al. (1984) reported that malaria workers in Thailand refused to accept An. nivipes as a separate species from An. philippinensis because of the overlap in the adult morphological characters. In Bangladesh, An. philippinensis is considered to be the principal vector of malaria in the vast plains of its central, western and northern regions. After the withdrawal of the large-scale DDT spraying in the late 1970s, An. philippinensis reappeared in some of the areas (Elias et al., 1987). In India, An. philippinensis was found abundantly in West Bengal, Assam and the neighbouring states (Rao, 1984). For the first time, Nagpal and Sharma (1987) have reported An. nivipes, along with An. philippinensis, from Assam and Meghalaya states. In Thailand An. philippinensis/nivipes are considered secondary vectors of malaria.

Evidence for recognition of sibling species An. philippinensis and An. nivipes (Klein et al., 1984) Two laboratory colonies were established, based on the distinguishing characters on the paddle at the pupal stage (Klein et al., 1982). The genetic incompatibility, observed in reciprocal crosses between An. philippinensis and An. nivipes laboratory colonies in the form of hybrid mortality at egg, larval and pupal stages, and hybrid sterility in the surviving males, established that these two are distinct species and justified the decision of Reid (1967) to raise An. nivipes from the synonym to species level (Klein et al., 1984).

Anopheline Species Complexes in South and South-East Asia

An. nivipes species A and B (Green et al., 1985) Two allopatric populations fixed for the alternative banding pattern on the Xchromosome due to a paracentric inversion, b, were observed in Thailand (Green et al., 1985), which suggested that there was another species in this complex. An. nivipes with X+b is designated as species A, and Xb as species B.

Techniques available for identification of sibling species Morphological variations The differences between An. nivipes and An. philippinensis are clear at the pupal stage on the paddle (Reid, 1967). In An. nivipes, the paddle has a longer refractile border with distinctly numerous teeth and shorter paddle hair. In An. philippinensis, the paddle has less than ten fringe teeth on the refractile border and has longer paddle hair, the unstraightened length being about one third that of the paddle. Polytene chromosomes By examining polytene chromosomes of An. nivipes and An. philippinensis colonies (Klein et al., 1982), two autosomal inversions were identified that could unequivocally distinguish An. nivipes from An. philippinensis at the adult stage by Green et al. (1985). The two inversions were t on the chromosome arm 2, and l on arm 5. The X-chromosome and chromosome arms 3 and 4 are homosequential in An. philippinensis and An. nivipes. The b inversion on the X-chromosome differentiates An. nivipes A from B. The diagnostic inversion genotypes of the species are: An. philippinensis

— X+b; 2+t; 5+l

An. nivipes species A — X+b; 2t; 5l An. nivipes species B — Xb; 2t; 5l The photomaps of the polytene chromosomes of An. philippiensis with the

break-points of inversions found in An. nivipes were presented by Green et al. (1985), and the photomaps of An. nivipes are shown in Figure 12. Molecular Assays There are now two assays, an allele-specific PCR assay of Walton et al., (2007) and a PCRRFLP assay of Alam et al., (2007), developed to distinguish members of the An. annularis Group which can be used to accurately identify An. nivipes and An. philippinensis. The allele specific PCR assay was developed based on differences seen in ITS2 sequences of five species—An. annularis, An. nivipes, An. philippinensis, An. pallidus and An. schueffneri—of the An. annularis Group, which are morphologically very close (Walton et al. al., 2006). While the An. schueffneri is restricted to Java and Sumatra islands of Indonesia, An. pallidus to Sri Lanka, India and Myanmar, the other species are widespread in the region. The authors report that the assay developed will be a rapid and reliable epidemiology tool, which should work from northeastern India through Myanmar and Thialand to Laos and Cambodia. Prakash et al. (2006) examined ITS2 sequences of morphologically confirmed An. nivipes and An. philippinensis specimens from Assam and Nagaland states in the northeast India to assess the applicability of Walton et al. (2006) PCR assay. The sequences of An. phillipinensis shared 99.2% and An. nivipes shared 99.3% respective similarity to those from the Thialand. The PCR-RFLP assay was developed based on endonuclease restriction sites in the D3 sequence to differentiate four members in the An. annularis Group—An. annularis, An. nivipes, An. philippinensis, and An. pallidus— (Alam et al., 2007). Restriction digestion of D3 fragment individually with SmaI, ApaI and NcoI produced distinct pattern of all four species on 3. 5 per cent agarose-gel. D3-SmaI system can be used to distinguish An. nivipes from An. philippinensis.

Anopheline Species Complexes in South and South-East Asia

63

Distribution and biological characters Subbarao, Vasantha and Sharma (1988) and Subbarao et al. (2000) carried out extensive cytotaxonomic examinations of specimens collected from five states in north-east India following the report of Green et al. (1985).

Collections were made from Kamrup district in Assam, Umbling in Meghalaya, Bhalapur in Arunachal Pradesh, Imphal in Manipur and Dimapur in Nagaland. All the specimens were found to be An. nivipes A. (All these specimens had been examined for the

Figure 12: Photomap of polytene chromosome of An nivipes. The break points for inversions b,t and l on chromosome arms, X,2 and 5 respectively are marked. C indicates the centromere end of each chromosome arm (Source: Subbarao et al., 2000). b

X+

2t

3

4

5l

b

b l c

t

l

t c

c

c

c

64

Anopheline Species Complexes in South and South-East Asia

characters on the wing which distinguish An. nivipes and An. philippinensis). Some of these areas were the same as those surveyed by Nagpal and Sharma (1987) who reported the presence of both the species based on their wing characters. Thus, the wing character was not found diagnostic for Indian populations as concluded by Reid (1967) for the Thai populations. However, the presence of An. philippinensis cannot be ruled out in these five states. Prakash et al. (2000) reported An. nivipes and An. philippinensis, based on larval and pupal diagnostic characters, from the Changlang district of Arunachal Pradesh and recently (2004) from Dibrugarh district in Assam, and also from the neighbouring states, Arunachal Pradesh and Meghalaya. In Dibrugarh, all the nine females from humanlanding collections were identified as An. philippinensis, while all the 16 females from the resting collections made near cattle sheds were An. nivipes. An. philippinensis, once dominant in the deltaic regions of West Bengal and responsible for the high incidence of malaria in this area, is almost absent today (Rao, 1984). The earlier dissection records showed sporozoite-positive specimens from West Bengal, but not from Assam, in spite of a large number of dissections (Krishnan, 1961). However, An. philippinensis, identified based on its wing character, was incriminated as a vector of malaria in Burnihat, which is now in Meghalaya state (Rajgopal, 1976). The cytotaxonomic examination (Subbarao et al., 2000) has revealed only An. nivipes in Meghalaya, and from pupal paddle characters recently by Prakash et al. (2004). Among An. philippinensis-nivipes s. l. collected from CDC light traps placed in human dwellings in districts Dibrugarh and Jorhat, Assam state, specimens positive for P. vivax and P. falciparum sporozoite antigens were found by ELISA (Prakash et al., 2005). Extensive cytotaxonomic (Subbarao et al., 2000) or molecular identifications (Prakash et al., 2006) along with ELISA tests are needed to establish unequivocally the relative role of An.

philippinensis and An. nivipes in malaria transmission in the north-eastern states of India. Following the discovery of the diagnostic inversions, the Thai populations were examined for the presence of two species. An. nivipes was found to be more common, but An. philippinensis was found to be more widespread than was earlier thought (Baimai et al., 1984). In the light of the findings from India, the populations from Bangladesh need to be examined.

3.10 The Punctulatus Complex Anopheles punctulatus belongs to the subgenus Cellia, and the Punctulatus Group in the Neomyzomyia Series. This taxon has a wide distribution from the Moluccas in the west through New Guinea and the Solomon Islands to Vanuatu in the east, and as far south as northern Australia. Rozeboom and Knight (1946) identified four species in the Punctulatus Group, based on the morphological characters of the proboscis (Bryan, 1973a; Foley et al., 1993). (i)

An. farauti Laveran — totally dark proboscis, except for a pale subapical ring (based on this character, An. moluccensis from the Moluccas and Irian Jaya, Indonesia, was synonymized with An. farauti).

(ii) An. punctulatus Doenitz — with the apical third to half of the proboscis pale, the remainder dark. (iii) An. koliensis Owen — white scaling on the proboscis variable but more often restricted to a small discrete patch on the apical third of the proboscis. (iv) An. clowi (Rozeboom and Knight) — proboscis is similar to that of An. koliensis, and the legs are yellowish instead of having dark areas on at least some or all of the fore- and mid-tarsomers. This

Anopheline Species Complexes in South and South-East Asia

65

species was described from Irian Jaya, Indonesia (Rozeboom and Knight, 1946). This species was rediscovered by Cooper et al. (2000) in Papua New Guinea. Marked differences observed in the An. farauti s.l. populations in peak biting times and breeding habitats suggested the presence of more than one species within this taxon. Cross-mating experiments and cyto- and biochemical-taxonomic studies have identified 11 species so far in the Punctulatus Complex. In the South-East Asia region, members of this complex occur only in Indonesia. Kondrashin and Rashid (1987) report An. punctulatus, An. koliensis and An. farauti as important vectors in Irian Jaya/ Maluku islands.

Evidence for identification of sibling species An. farauti No. 1 and An. farauti No. 2 An. farauti (Bryan, 1970; 1973a and b) from two colonies established from (1) Rabraul, New Britain, Papua New Guinea, and (2) Innisfail, northern Queensland, Australia, were crossed. Both female and male progeny from the reciprocal crosses were sterile. This was taken as evidence for the existence of two species within An. farauti; the colony from Papua New Guinea was designated as An. farauti No. 1 and the colony from Australia as An. faruati No. 2. Bryan and Coluzzi (1971) observed two paracentric inversions, one on the left arm and another on the right arm of chromosome arm 2 in An. farauti No. 2. In hybrids between An. farauti No. 1 and An. farauti No. 2, inversion heterozygotes were observed in these regions. This suggests that the two populations differ by fixed paracentric inversions. An. koliensis (Bryan, 1973a and b) Doubts about whether An. koliensis is a hybrid between An. punctulatus and An. farauti (because of the variable pale scales on the proboscis) were removed by cross-mating experiments carried out by Bryan (1973a and

66

b). There was either reduced hatchability or total sterility in crosses between An. koliensis and An. farauti and An. punctulatus. The adults which emerged had reduced or partially developed reproductive organs. These results proved that An. koliensis is a separate species. An. farauti No. 3 (Mahon and Miethke, 1982) A population found sympatric with An. farauti No. 1 and An. farauti No. 2 in Australia produced sterile progeny in reciprocal crosses with An. farauti No. 1 and No. 2. A fixed paracentric inversion was found in this population, and the population was considered a new species and was designated as An. farauti No. 3. An. farauti No. 4, No. 5 and No. 6 Bryan et al. (1990) studied variations at 14 enzyme loci in the populations from Papua New Guinea and the colony material of An. farauti No. 1, No. 2 and No. 3. The phenogram revealed nine clusters for which more than 20 per cent loci were fixed for different alleles. The authors considered that there may be nine species in this complex, as so many fixed differences could not be maintained within a population which shares the same gene pool. Subsequently, Foley et al. (1993) analysed cellulose acetate gels of populations from 19 localities in Papua New Guinea for electrophoretic variations at 35 loci. Based on fixed allele differences, six species were identified. Of these, An. punctulatus, An. koliensis and An. farauti No. 1 had already been reported; three were new species, which were provisionally designated as An. farauti No. 4, No. 5 and No. 6. The population designated as An. farauti No. 4 from Gonoa produced sterile F1 progeny when crossed with An. farauti No. 1, and asynapsis in polytene chromosomes of F1 progeny (Bryan et al., 1990).

Anopheline Species Complexes in South and South-East Asia

An. farauti No. 7 (Foley, Meek and Bryan, 1994) The specimens collected either as larvae or adults from 33 localities in the Solomon Islands and six localities in Vanuatu were analysed for electrophoretic variations. The same enzyme systems that were used for the Papua New Guinea population (Foley et al., 1993) were used in this study, except that β-galactosidase was added and the Ak-1, Ao, Fdp-1 and Mpi loci were excluded because of unreliable staining. Enzyme analysis revealed the three already identified sibling species, An. farauti No. 1 and No. 2 and An. punctulatus, and a population which did not correspond with any of the earlier identified species was found in two localities on northern Guadalcanal, Solomon Islands. Fifteen specimens belonging to the new population were examined and all had dark probosces as described for An. farauti s. l. This group of mosuqitoes was considered as a new species and was designated as An. farauti No. 7. An. sp. near punctulatus (Foley, Cooper and Bryan, 1995) Within the specimens identified morphologically as An. punctulatus, two genetically distinct groups were found when examined for enzyme electrophoretic variations and analysed for genetic distance. The new group differed from An. punctulatus populations with 34-39 per cent fixed genetic differences and Nei’s D ranging between 0.5 to 0.61. The maximum genetic divergence recorded within An. punctulatus collected from widely separated localities in Papua New Guinea was 18 per cent (Foley et al., 1993) and 6 per cent for specimens collected in the Solomon Islands (Foley, Meek and Bryan, 1994). This was taken as evidence to show that in the western province of Papua New Guinea there is a population conspecific with An. punctulatus as described earlier and a population that was not described earlier, i.e. a new species designated as An. sp. near punctulatus.

Formal names Schmidt et al. (2001) studied the morphological variations and identified markers for three species. An. farauti No. 1, No. 2 and No. 3 in the adult female, IV instar in larvae and pupae. On this basis formal names were given: An. farauti No. 1 (conspecific with An. farauti sensu stricto as described by Foley and Bryan, 1993, and Foley, Meek and Bryan, 1994) designated as An. farauti Laveran. An. farauti No. 2 designated as An. hinesorum Schmidt sp. n. An. farauti No. 3 designated as An. torresiensis Schmidt sp. n.

Techniques available for identification of sibling species Crossing experiments An. punctulatus, An. koliensis and An. farauti No. 1, No. 2, No. 3 and No. 4 produced progeny in reduced numbers, and with reduced reproductive organs in reciprocal crosses between the above-mentioned species (Bryan, 1973a and b; Mahon and Miethke, 1982; Foley et al., 1993). Polytene chromosomes Polytene chromosomes from IV instar larval salivary glands were used for the identifications, since polytene chromosomes from ovarian nurse cells were not readable in contrast to those in other anopheline species. Bryan and Coluzzi (1971) identified paracentric inversions diagnostic for An. farauti No. 1, No. 2 and No. 3. Salinity tolerance Test (STT) An. farauti No. 1 showed a higher tolerance of salinity than An. farauti No. 2 and No. 3 (Sweeney, 1987). Tests carried out with 03.4 per cent salinity showed complete mortality of An. farauti No. 2 and An. farauti No. 3 at 1.7 per cent. At the same concentration, An. farauti No. 1 from field

Anopheline Species Complexes in South and South-East Asia

67

and Rabaul colony showed about 92 per cent survival, while Port Moresby colony showed 73 per cent survival. An. farauti No. 7 was thought to be an obligatory fresh-water species which would not tolerate salinity. However, Foley and Bryan (2000) found An. farauti No. 7 could survive the STT and thus could not be efficiently differentiated from An. farauti s.s. which is a vector on Guadalcanal, Solomon Islands. The authors, therefore, recommend alternatives to the STT for the vector species on Guadalcanal. Biochemical taxonomy and electrophoretic keys Bryan et al. (1990) and Foley et al. (1993) analysed populations from Papua New Guinea for electrophoretic variations and developed electrophoretic keys. Foley and Bryan (1993) give the keys for six species, An. farauti No. 1, No. 4, No. 5 and No. 6, An. punctulatus and An. kolinesis, from Papua New Guinea, and for An. farauti No. 2 and No. 3 from Australia. Alternative keys are given depending on the availability of different species for standards. Foley, Meek and Bryan (1994) presented a key for distinguishing An. farauti s.s., An. farauti No. 2 and No. 7, and An. punctulatus which are prevalent in the Solomon Islands and Vanuatu. These species can be distinguished by staining for the enzyme LDH only. The authors also discuss combinations of standards and diagnostic enzymes required to identify the species. Specimens of new species An. sp. near punctulatus were found homozygous for a unique allele at Pep D-1 (Foley, Cooper and Bryan, 1995). The authors provisionally reported this allele to be diagnostic for this species based on the small number of specimens examined. Additional alleles that discriminate An. punctulatus and An. sp. near punctulatus are at the Hk, Me-1 and Hbd II loci.

68

DNA probes Three DNA probes, pAf1, pAf2 and pAf3, were selected by screening partial genomic libraries with radio-labelled total genomic DNA from each species (Booth, Mahon and Sriprakash, 1991). pAf1 hybridized with both An. farauti No. 1 and No. 2, pAf2 hybridized strongly to An. farauti No. 3, less to An. farauti No. 1 and faintly to An. farauti No. 2, suggesting that there is cross-hybridization. Hartas et al. (1992) sequenced these probes and identified oligonucleotides of 25-26 base pairs specific for each probe. Radio-labelled oligonucleotides, oAf1, oAf2 and oAf2, hybridized respectively to An. farauti No. 1, No. 2 and No. 3. These oligonucleotide probes identified both dot-blots and squashblots of larvae, pupae and adults very efficiently. Cooper, Cooper and Burkot (1991) independently identified three DNA probes specific for An. farauti No. 1, No. 2 and No. 3. Total genomic libraries of the three species were screened by hybridizing with radiolabelled homologous and heterologous total genomic DNA of species. Three probes, 1/1, 9/2 and 5/3, specific for An. farauti No. 1, No. 2 and No. 3 respectively, were identified. Probes labelled with P32 or horseradish peroxidase with enhanced chemiluminescence detection system or biotinylated streptavidin-conjugated alkaline phosphatase with the chromogenic detection system were all found equally sensitive in identifying the species both in dot-blots and squash-blots. Beebe et al. (1994) made DNA probes for the identification of five members of the Punctulatus Complex namely, An. punctulatus, An. koliensis and An. farauti No. 4, No. 5 and No. 6 by differentially screening genomic libraries with P32-labelled homologous and heterologous DNA. The sizes of the probes that were selected ranged between 273 and 630 bp. The probes identified specimens from DNA dot-blots and

Anopheline Species Complexes in South and South-East Asia

squash-blots. These probes are sensitive enough to identify fragments of mosquitoes. Beebe et al. (1996) developed two DNA probes specific for An. farauti No. 7 and An. sp. near punctulatus, by screening genomic libraries. A probe which hybridizes to all members of the Punctulatus Complex has also been developed. These probes have been field-tested. All members of this complex can be identified with DNA probes. PCR-RFLP Beebe and Saul (1995) differentiated 10 species, An. farauti No. 1 to No. 7, An. koliensis, An. punctulatus and An. sp near punctulatus, by restriction enzyme digestion of rDNA ITS2 product (750bp amplicon) with Msp-I. rDNA-ITS2 sequence analysis Beebe et al. (1999) analysed sequence variations in the rDNA ITS2 region among 10 species of this complex. The length of ITS2 ranged from 549 to 565bp. Sequence variation among the 10 species studied ranged from 2.3 per cent to 2.4 per cent mainly due to indels of simple sequences.

Distribution and biological characters Information on the distribution of members of this complex in Papua New Guina, Solomon Islands and Australia is avilable from recent investigations using allozymes (Bryan, Reardan and Spark, 1990; Foley et al., 1993 and Foley, Meek and Bryan, 1994). From Irian Jaya Islands of Indonesia, An. punctulatus, An. koliensis and An. farauti s.l. have been reported (Kondrashin and Rashid, 1987), and An. clowi (from an earlier report). An. farauti No. 2 and No. 7 were found to be zoophilic while An. farauti No.1 (now An. farauti Levaran) was highly anthropophilic. Differences in anthropophily were observed on north Guadalcanal in the Solomon Islands by Foley, Meek and Bryan

(1994). In Gonoa, Papua New Guinea, An. farauti No. 4 and An. koliensis were caught on opposum, pig and human bait, while An. punctulatus was caught on human and pig baits (Foley et al., 1993). On human bait four out of six An. farauti No. 4 were caught before 2000 hours, suggesting early biting activity of this species. Now that eleven members have been identified in this complex and techniques are available for their identification, studies should be undertaken to map their distribution and to establish their role in malaria transmission.

3.11 The Sinensis Complex Anopheles sinensis Wiedman, 1828 belongs to the subgenus Anopheles, the Hyrcanus Group in the Myzorhynchus Series (Harbach, 2004). An. sinensis is an important vector of malaria throughout the Republic of Korea (Chow, 1970). This species is considered to be the main vector responsible for malaria in central China. Malaria in this area is unstable and outbreaks have regularly been reported since the 1950s. As recently as in 1995, Honan province in China experienced an outbreak of P. vivax (Sleigh et al., 1998). This species is also considered to be a vector in Japan, Indonesia and Thailand (O’Connor 1980, Kanda et al., 1981). However, in Thailand, it is not considered an important vector.

Evidence for recognition of sibling species Sibling species in China— An. sinensis and An. lesteri syn. anthropophagus Two morphological variants based on the size of the deck of the egg were reported in the 1950s (cited in Reid, 1953). Later, it was found that the narrow deck egg variety was more anthropophilic, endophagic and endophilic than the wider deck variety which fed outdoors and predominantly on cattle. Because the narrow deck egg variety resembled An. lesteri, which also has a narrow

Anopheline Species Complexes in South and South-East Asia

69

deck, it was initially considered as a subspecies of An. lesteri and was called An. lesteri anthropophagus. Later, Ma (1981) (as mentioned in Gao, Beebe and Cooper, 2004) raised anthropophagus to the species status and designated it as An. anthropophagus based on its morphology, distribution and vectorial capacity. The wider deck variety is designated as An. sinensis. An. anthropophagus and An. sinensis are isomorphic species at the adult stage. Wilkerson et al. (2003), however, reported that An. anthrophagus and An. lesteri are identical in ITS2 sequences and are, therefore, synonymous. This was based on the examination of An. lesteri from the type locality in Laguna province from the Phillippines and An. anthropophagus from Jhangsu Province, China. Ma and Xu (2005) supported this conclusion and said that An. anthrophagus is a junior synonym of An. lesteri. Wilkerson et al. (2003) clarified the status of An. lesteri present in China by stating that it is another unknown species and is also found in Korea. This is supported by Gao, Beebe and Cooper (2004) observation that it is different from An. anthrophagus and An. sinensis based on ITS2 sequences. Sibling species in Japan — An. sinensis, An. engarensis and An. sineroides Successful colonization of An. sinensis using an induced copulation method devised by Oguma and Kanda (1976) led to crossing studies. Strains collected from Koniya, Kanoya, Karurizawa, Yomogita, Yakumo and Engaru ranging from north to south in Japan were used in the crosses (Oguma, 1978). In two crosses, Engaru with Kanoyo and Engaru with Yakumo, F1 males were sterile and females were fertile. In the other crosses (of the 36 possible crosses, 19 were carried out) where Engaru was not involved, the progeny were fertile. Therefore, it was concluded that the Engaru strain is distinct and was given species status, while all the other strains were considered to belong to one species, An. sinensis. The Engaru strain was also found to

70

be different physiologically from An. sinensis, in the mean frequencies of the clasper movements during induced copulation (Kanda and Oguma, 1978). Based on this evidence the Engaru strain was considered a distinct sibling species, morphologically similar to An. sinensis and designated as An. engarensis. Kanda et al. (1981) reported from the polytene chromosomal examination of about 2000 specimens from Engaru, where both the species exist sympatrically, that no heterozygotes for 2RB inversion were found. The absence of heterozygotes further supported the view that there are two distinct species in the area, and also suggested that there is pre-mating isolation in addition to the post-mating barrier already reported (Oguma, 1978). An. sineroides Yamada 1924 has a restricted distribution, being found from Kokkido to Kyushu in Japan, in China and in Korea. An. sineroides and An. sinensis are morphologically similar, hence hybridization experiments were conducted to establish the taxonomic status (Kanda and Oguma, 1977). In crosses between the strains of An. sinensis and An. sineroides collected from Japan, heavy mortality in F1 larvae and pupae, and asynapsis in polytene chromosomes of F1 progeny were observed. Based on these results, the authors concluded that An. sinensis and An. sineroides are two distinct sibling species. The X-chromosome of An. sineroides was found similar to that of An. koreicus rather than to that of An. sinensis (Oguma and Kanda, 1970). Cytotypes of An. sinensis in Thailand and Republic of Korea Two forms designated as form A and form B were identified within An. sinensis, based on structural difference in the Y-chromosome (Baimai, Rattanarithikul and Kijchalao, 1993). Form A has a telocentric or acrocentric Ychromosome (short arm being very small) and form B has a sub-metacentric Y-chromosome.

Anopheline Species Complexes in South and South-East Asia

Choochote et al. (1998) carried out crossing experiments between form A and form B using induced copulation. In all crosses viable progeny were observed and no genetic incompatibility was observed in crosses with the F1 progeny. Polytene chromosomes of 4th instar larvae of F1 progeny showed complete synapsis. In another study (Min et al., 2002), form A from Thailand was crossed with form B isolines from Thailand and Korea. In the reciprocal crosses, and corresponding back crosses, no genetic incompatibility was observed affecting hatchability, viability of immature stages, sex ratio or adult emergence. Thus, both cytotypes, form A and form B, were considered as intra-specific Ychromosome variations within An. sinensis. This was further supported from sequence analysis of ITS2 and COII regions of form A and B. The sequences were near identical with a sequence variability of 0.0-0.6% (Min et al., 2002). Thus, it appears that An. sinensis is a complex of at least four species, An. sinensis s.s., An. lesteri (syn. anthropophagus), An. sineroides and An. engarensis.

Techniques for identification of sibling species Morphological variations At the adult stage all four species, An. sinensis, An. engarensis, An. sineroides and An. lesteri syn. anthropophagus, are isomorphic. The size of the deck of the egg varies between the two species, i.e. it is narrow in An. lesteri syn. anthropophagus and is wider in An. sinensis. An. lesteri (an earlier designation and needs formal redesignation following Wilkerson et al., 2003) also has a narrow deck and thus resembles An.lesteri syn. anthropophagus. Both An. lesteri and An. yatsushiroensis (another species in the Hyrcanus Complex which is sympatric with these species) can be distinguished morphologically at the adult stage from An. sinensis and An. lesteri syn. anthropophagus.

Molecular methods Restriction fragment length differences of genomic repetitive DNA (Li et al., 1991 from Abstract)

High molecular weight DNA of five species, An. sinensis, An. lesteri syn. anthropophagus, An. liangshanensis, An. crawfordi and An. xiaokuanus, belonging to the Hyrcanus Group, was cut with three restriction endonucleases. All three enzymes, Bgl II, Hae III and Pst I, produced diagnostic fragments discernable on agarose gel for all species. Thus, all five species could be distinguished by this method. An allele-specific PCR assay (Ma Qu and Xu, 1998 cited in Gao, Beebe and Cooper, 2004)

This assay was developed from ribosomal DNA to differentiate An. sinensis from An. lesteri syn. anthropophagus. With this assay only 78.9% of field-collected samples could be identified, i.e. many remained unidentified because of failure to produce PCR products. According to the authors, this was because unidentified samples belonged to another yet unknown cryptic species. Another possible reason as pointed out by Gao, Beebe and Cooper (2004) could be due to intra-specific sequence variation within the primer region which inhibited annealing. PCR-RFLP assay (Gao, Beebe and Cooper, 2004)

Universal primers from the rDNA ITS2 region were used to amplify, and the amplicons were digested with Hind I and Rsa I restriction enzymes. After digestion the patterns on acrylamide gel were distinct with both the enzymes. To validate the assay, field-collected specimens from seven areas in China and An. sinensis specimens from an established laboratory colony were used. All the specimens identified morphologically based on egg deck size as An.lesteri syn. anthropophagus were also identified by the PCR as the same species except for 4 of 8 specimens from Guangdong province which were identified as An. sinensis. The sequence analysis also confirmed that these specimens

Anopheline Species Complexes in South and South-East Asia

71

were An. sinensis. An. sinensis from Liaoning province mostly had narrow decks typical of An.lesteri syn. anthropophagus, with a few with wide decks typical of An. sinensis. But all the specimens were identified as An. lesteri syn. anthropophagus by molecular assay. This suggests that the egg deck size is not reliable to distinguish these two species as it was thought earlier.

Distribution and biological characters An. sinensis forms, A (XY 1) and B (XY 2), identified in Thailand by Baimai, Rattanaritikul and Kijchalao (1993) were also reported to be found in Taiwan (China) and China. Recently, Ma and Xu (2005), based on ITS2 sequences reported that in China only B form haplotype was found. Throughout the Republic of Korea, An. sinensis has been considered for a long time to be an important vector. Recently, Coleman et al. (2002) collected anopheline mosquitoes from 29 locations distributed throughout the country. Three species, An. sinensis/lesteri (as these two species cannot be identified accurately, no effort was made to separate them), An. yatsushiroensis Miyazaki and An. sineroides Yamada, were found. 89.7% were An. sinensis/lesteri, and of these two pools, one of 10, and another of nine (screened by ELISA) were found positive for P. vivax (CSP 247). For An. sinensis, the anthropophilic index (AI) and average probability of daily survival rate respectively were 0.7 per cent and 0.89 in the Guonggi–do (Ree et al., 2001) and 0.8 per cent and 0.79 per cent in the Paju (Lee et al., 2001) areas of the Republic of Korea. In both publications the investigators suggest that in spite of their tendency to zoophily and with moderate survival rate, this species is responsible for malaria transmission because of high population densities. In Thailand, An. sinensis is not considered an important vector. In a laboratory-feeding experiment, two cytologically different An. sinensis strains (form A-XY1, and form B-XY2) were found to be totally refractory to P. falciparum with none 72

developing oocysts or sporozoites while both exhibited low susceptibility to P. vivax with 0.00– 0.571 per cent and 0.00-.588 per cent oocyst and sporozoite rates respectively. Both the strains were refractory to P. yoelii when fed directly on infected rodents (Rongstrriyam et al., 1998). Malaria is endemic in central China but it is unstable and with regular occurrence of outbreaks (Li et al., 1995). Gao, Beebe and Cooper (2004) report An.lesteri syn. anthropophagus to be widespread throughout central China and it occurs sympatrically with An. sinensis. However, they report that the distribution of An.lesteri syn. anthropophagus is patchy compared to An. sinensis. An. sinensis is predominantly a cattle-feeder in China. Throughout central China, An.lesteri syn. anthropophagus is considered a primary vector and An. sinensis a secondary vector. Molecular identifications (Gao, Beebe and Cooper, 2004) have extended the distribution of An. lesteri syn. anthropophagus northward by 1200 km (420 N, 1200 E). This region is north of the Korean peninsula. A small sample examined using molecular techniques by Ma et al. (2000) cited in Gao, Beebe and Cooper (2004) identified only An. sinensis, An. lesteri and An. yatsushiroensis. Along the North/South Korea border, Stricman et al. (2000) reported that, based on morphological identifications, An. sinensis was the most abundant mosquito biting humans, constituting more than 80 per cent of the anopheline human-landing collections. These mosquitoes were collected during an outbreak of P. vivax malaria. Now that a molecular assay is available (Gao, Beebe and Cooper, 2004) for accurate identification of An.lesteri syn. anthropophagus and An. sinensis, large samples from the Republic of Korea are to be identified to establish whether only An. sinensis is prevalent as is being reported. This may resolve the anomalies reported in the feeding behaviour of the mosquito hitherto described as An. sinensis.

Anopheline Species Complexes in South and South-East Asia

3.12 The Subpictus Complex Anopheles subpictus Grassi belongs to the subgenus Cellia, and the Pyretophorus Series (Harbach, 2004). An. subpictus is very widely distributed and found in abundance in the Oriental region. It is found to the west of India in Pakistan, Afghanistan, and Iran, and in the east as far as Papua New Guinea and the Marina island, Malaysia. It is also found in Sri Lanka in the south and China in the north. In India, it is found throughout the mainland and in Lakshadweep islands but not in the Andaman and Nicobar islands (Rao, 1984). In Indonesia, it is considered as a secondary vector (Van Hell, 1952; Sundararaman, Soeroto and Siran, 1957). In India, sporozoite-positive specimens were reported from a coastal village in the state of Tamil Nadu (Panicker et al., 1981) and in the district of Bastar in Madhya Pradesh (Kulkarni, 1983). Amerasinghe et al. (1991; 1992) reported this species to be a vector in Sri Lanka. Based on the morphological differences in the eggs of An. subpictus, Reid (1966) suggested that this might be a species complex, and Suguna (1982) for the first time provided clear evidence that this taxon is a complex of two sibling species, A and B. Later, this taxon was found to be a complex of four sibling species, A, B, C and D (Suguna et al., 1994).

Evidence for recognition of sibling species Differences in egg morphology and in the banding pattern of polytene X-chromosome due to a paracentric inversion were the basis for the identification of species A and B. Species A with the X+a karyotype (standard arrangement) was predominant in inland villages while in the coastal villages species B with the Xa inversion arrangement was found

together with species A in the Union Territory of Pondicherry in India (Suguna,1982). The four sibling species, provisionally designated as A, B, C and D, were identified by examining polytene chromosomes from the ovaries of adult females collected from the field and those from the salivary glands of larvae collected from breeding sites. Two inversions on the X-chromosome, a, a small inversion towards the tip of the chromosome, and b, an inversion in the middle of the chromosome (this same inversion was designated as a in the earlier publication of Suguna, 1982), and their combinations +a+b, ab, a+b and +ab were found with a total absence of heterozygotes in the natural populations examined. This was taken as an evidence for the recognition of four species (Suguna et al., 1994).

Techniques available for identification of sibling species Inversions on polytene X-chromosome are diagnostic characters in the identification of sibling species. The four inversion arrangements observed were: species A – X+a+b, species B – Xab, species C – Xa+b and species D – X+ab. A photomap of polytene chromosomes complemented with break-points marked for inversions a and b are given in Figure 13. Reuben and Suguna (1983) reported morphological differences in eggs, larvae, pupae and adults between sibling species A and B. Now that species C and D have also been reported within the fresh waterbreeding populations in Pondicherry (Suguna et al., 1994), the differences reported by Suguna (1982), and Reuben and Suguna (1983) between species A and B, are not discussed here as they are no longer valid for the identification of all the sibling species. Differences in egg, larval, pupal and adult morphological characters among the four species were also observed (Suguna et al., 1994) and these are summarized in Table 10.

Anopheline Species Complexes in South and South-East Asia

73

Distribution and biological characters An. subpictus population from villages around Delhi, India, were identified as species A (Subbarao, Vasantha and Sharma, 1988) by examining the ovarian polytene chromosomes of adult females collected from the field, following the report of Suguna (1982). The ridge number on the egg float ranged between 21 to 30, and this did not correspond with the characters of species A which has, on an average, 33 ridges (Suguna, 1982), but it corresponds to those of species

C of Suguna et al. (1994). Atrie (1994) reported that in the Delhi population, the apical pale band in adult females was longer than the sub-apical dark band, which is the characteristic feature of species A (Table 10). Thus, it was felt that there was a need for a detailed study to correctly identify the specific taxonomic status of the Delhi population. Recently, Singh et al. (2004) conducted a detailed study on An. subpictus collected from Sonepat district, Haryana (a state neighbouring Delhi). In this study, fieldcollected adult females, and from their

Figure 13: Polytene chromosome complement of An. subpictus species A. Break-points of inversions, a and b, reported by Suguna et al. (1994) are shown on the X-chromosome. (Source: Subbarao, Vasantha and Sharma, 1988)

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Anopheline Species Complexes in South and South-East Asia

Table 10: Morphological, biological and cytological differences among An. subpictus sibling species (Source: Suguna et al. (1994) Larva

Pupa

Species

Inversions on Xchromosome

Mean ridge No. (range)

Egg Frill

Seta 4M

Seta 7-1

A

+a+b

35 (31-36)

Opaque

2-branched (rarely 3)

Simple; as long as hairs 6&9

Longer than sub apical dark band

Paddy fields (0.0054-0.2636), Riverine pools (0.0247-0.7827) and Back waters (0.5574-5.3554)

B

ab

18 (16-20)

Transp arent

2-branched (rarely 3)

Branched 4-5; shorter than hairs 6&9

Shorter than sub apical dark band

Back waters (0.5574-5.3554)

C

a+b

27 (25-29)

Semitransparent

3-branched (rarely 2)

Branched 2; shorter, but longer than in sp. B

Equal to sub apical dark band

Same as A

D

+ab

22 (21-24)

Semitransparent

3-branched

Branched 3; shorter

isofemale lines, eggs, larvae, pupae and adults, were examined for morphological characters following Suguna et al. (1994). Species A, C and D were found in almost equal proportions, and no variation was observed in the proportion of the three sibling species from field-collected adults and isofemale lines. An. subpictus breeding in the villages was found in the river-bed pools. Thus, in northern India, all three fresh waterbreeding species are sympatric. In Sri Lanka, both species A and B were identified (Abhayawardana, Wijesuriya and Dilrukshi, 1996) based on species-specific diagnostic inversion genotypes reported by Suguna (1982). In Sri Lanka, species A was found to be more endophilic and seasonally more abundant than species B (Abhayawardana, Wijesuriya and Dilrukshi, 1996). No studies have been reported so far on the biological characteristics of these four sibling species in India. Detection of sporozoite-positive specimens in coastal

Adult Length of apical pale band on female palpi

Breeding habitats (Salinity range %)

villages of Pondicherry by Panicker et al. (1981) suggests that species B may be a vector. Data of Kulkarni (1983) in Bastar district, Madhya Pradesh state in India, and Amerasinghe et al. (1991 and 1992) in an irrigation development area of the Mahaweli project in Sri Lanka suggested that fresh waterbreeding sibling species may also be playing a role in malaria transmission. Fresh waterbreeding An. subpictus from Delhi, when fed on Plasmodium vivax-infected blood developed oocysts and sporozoites (Nanda et al., 1987). This suggests that fresh water breeding An. subpictus is genetically susceptible to plasmodial infection. Generally, the densities of this species are relatively high. The species may not be playing a role in malaria transmission probably due to high zoophagy and poor longevity, which are relevant for the species to be a vector in the field. Resistance to malathion and fenitrothion was observed in An. subpictus predominant

Anopheline Species Complexes in South and South-East Asia

75

in inland regions, while in coastal areas resistance to permethrin was observed in this species (Kelley-Hope et al., 2005). Abhayawardana, Wijesuriya and Dilrukshi (1996) reported species A’s predominance in inland regions and that of species B in coastal areas. Following this, Kelley-Hope et al. (2005) suggested that different resistant mechanisms may prevail in the two sibling species, with species A being more resistant to organophosphates and species B to pyrethroids, with little evidence of introgression between the two sibling species. Now that four sibling species are identified, the distribution of the sibling species should be mapped in all the areas wherever An. subpictus is prevalent and is considered a vector, as in Indonesia and Sri Lanka, and the biological characters of the sibling species should be studied.

3.13 The Sundaicus Complex Anopheles sundaicus belongs to the subgenus Cellia and the Ludlowae Group in the Pyretophorus Series (Harbach, 2004). An. sundaicus is widely distributed in the Oriental region. The distribution extends from India, and eastward to China through Bangladesh, Myanmar, Viet Nam, Cambodia, Thailand, Malaysia, Singapore, Philippines and Indonesia (Rao, 1984). In India it has now disappeared from the mainland (Rao, 1984; Dash et al., 2000), except for a small focus in the Kutch area of Gujarat state (Singh, Nagpal and Sharma, 1985) and is now found abundantly and widely in the Andaman and Nicobar islands (Rao, 1984). This species is considered an important vector throughout the areas of its distribution. An. sundaicus is generally a salt-water breeder. The major breeding places are swamps and pits along bunds (embankments) containing brackish water (Sundararaman, Soeroto and Siran, 1957). Rao (1984) reports that the most suitable breeding places are

76

those in which sea water and fresh water mingle, but it was also found adapted to fresh water in some areas. In Vishakapatnam, Andhra Pradesh state, India, it was found exclusively in fresh water-breeding places such as tanks, ponds and borrow pits. In Viet Nam and Indonesia, shrimp/fish ponds found along the coast or those irrigated by inland sea water were reported as favourable breeding sites for this species (Collins et al., 1979). Its populations were found both indoors and outdoors in Malaysia (Reid, 1968), and in Car Nicobar, Andaman and Nicobar Islands, India (Kumari and Sharma, 1994). In Indonesia, in brackish water-breeding areas, this species is exophilic except in Kampund Laut, Cilacap, where it is endophilic (Kirnowardoyo and Gambiro, 1987). The preference to feed on man varies in this species. In Vishakapatnam area, India, the anthropophilic index was found to be 5.6 per cent (Rao, 1984). In Car Nicobar Island (Andaman and Nicobar islands), India, this species was found to be predominantly zoophagic, with an anthropophilic index of 2.5 per cent, but in human dwellings it was 18 per cent (Kumari et al., 1993). However, in Indonesia, it is primarily anthropophagic except in some places like Purworejo (Central Java) and Kulon Progo (Yogyakarta), where the human blood index was only 1.3 per cent (Kirnowardoyo, 1988). Because of such distinct differences in the populations of An. sundaicus, this species has long been suspected to be a species complex (Kalra, 1978; Rao, 1984).

Evidence for recognition of sibling species Species A, B and C (Sukowati et al., 1999) Sukowati and Baimai (1996), for the first time, reported three cytological forms designated as A, B and C differing in their polytene chromosome complement and with heterochromatic variation in their Ychromosomes. The three cytological forms

Anopheline Species Complexes in South and South-East Asia

were identified from the examination of natural populations from different areas in Thailand and in Java and Sumatra, Indonesia. The differences observed between the three forms are summarized in Table 11. A standard photomap of the ovarian polytene chromosomes with a detailed description of each chromosome is given by Sukowati and Baimai (1996). In both Trat and Phangnga, two areas surveyed in southern Thailand, only the A form was found. In Asahan, in northern Sumatra, all three forms were found while in south Tapanali, also in northern Sumatra, only the A and B forms were found in the two surveys conducted. In Lampung, situated in the most southern part of Sumatra, and in south Banten, Panganadaran and Cilacap in Java, only the A form was observed. In a single survey conducted in Purworejo in Java, both A and B were found with a predominance of the A form (90 per cent). In spite of the fact that the cytological forms were found to be sympatric, the authors could not give them the species status because the forms were identified on the basis of Y-chromosome variation, and differences in the polytene chromosomes were not as clear- cut as when there are inversions, for which any heterozygotes are readily available. Sukowati et al. (1999) collected An. sundaicus from bovine and human baits from six localities and the cytological identification revealed A, B and C. Other Indonesian sites with the forms found were as follows: Purowerojo (forms A and B) and Cilacap (form A) in Central Java; Panganduari (form A) in west Java; Lampung (form A) in south Sumatra and Asahan (forms A, B and C), and south Tapanuli (form B) in north Sumatra. An. sundaicus breeds in fresh water in south Tapanuli while it breeds in brackish water in the other five localities. F1 progeny cytologically identified for forms A, B and C were analysed for 11 enzyme systems. Data were analysed by BIOSYS-1 (release 1.7 University of Illinois programme). Genetic

variability in terms of mean heterozygosity, which ranged between 0.101 and 0.179, and polymorphism between 0.267 and 0.533, were compared with those observed for other species complexes. No unique electromorphs were found fixed in any of the three cytological forms studied in these populations, but frequency differences and Wright’s F statistics for the Mpi enzyme distinguished the three cytological forms with a high degree of probability. The data from the Mpi locus strongly indicated a lack of random mating . Therefore, the three cytological forms were considered as three isomorphic species and the Sundaicus as a species complex. A new cytotype (Nanda et al., 2004) Nanda et al. (2004) reported a new cytotype, D, from the Andaman and Nicobar Islands, India. In the polytene chromosomes complement, the X-chromosome resembled that of species A (Xa type), while the chromosome 2 had a prominent loosely diffuse area in Zone 19 proximal to the centromeric regions (2RB type) as in species C (Sukowati and Baimai, 1996). Thus, these specimens did not completely resemble either species A or species C. In the mitotic karyotype, both the X and Y chromosomes were telocentric and the Y-chromosome had two heterochromatic blocks as in species A and C (Table 12). The new cytotype could not be given a new species status, as it was not found sympatric with any of the other forms. Evidence for another sibling species, An. sundaicus s. s. (Dusfour et al., 2004) An. sundaicus specimens collected from two sites, Lundu and Miri in Sarawak, Malaysia, two in Thailand and two in southern Viet Nam were examined for differences, if any, in the sequences of partial regions of cytochrome b and cytochrome oxidase I of the mitochondrial DNA (Dusfour et al., 2004). The specimens from Thailand and Vietnam

Anopheline Species Complexes in South and South-East Asia

77

were found distinctly different from the ones collected from Malaysia, and the genetic divergence and cladistic analysis relationship established that they represent two sibling species: An. sundaicus s. s. from Malaysia and An. sundaicus species A from Viet Nam and Thailand. Formal designation of An. sundaicus from Malaysia and of species A Dusfour et al. (2004) designated specimens from the Sarawak region as An. sundaicus s. s., because Linton et al. (2001) reported An. sundaicus from Lundu province as neotype of this species (as no type specimens of this species exist). Linton et al. (2005) designated species A as An. epiroticus Linton & Harbach based on ITS2, cytochrome b and cytochrome oxidase I sequences. Linton et al. (2005) also reported that no morphological characters were observed which could be used as diagnostic characters at adult, larval and pupal stages between An. sundaicus s. s. and An. epiroticus.

Techniques available for identification of sibling species Mitotic karyotypes The three cytological forms have now been given species status, and cytological techniques can be used to identify these three species. Species B can be differentiated from species A and C by examining the Ychromosome in mitotic karyotypes. As species A and C have similar Y-chromosomes, and if it is clearly known that only A and B or only B and C exist in a given area, observation of the Y-chromosome can give valid identifications. Polytene chromosomes All three species and new cytotype D can be differentiated by examining the X and chromosome 2 in the polytene chromosome complements (Sukowati and Baimai, 1996). The differences observed in the Y-chromosome in the mitotic karyotype and the X-chromosome and 2 (2R) are given in Table 11.

Table 11: Cytological descriptions of forms A, B, C and D in An. sundaicus

Cytological forms/ Species Species A

Species B

Species C? Cytotype D

Polytene chromosomes X* 2R/2** 2Ra# Xa# (standard (standard arrangement) arrangement) 2Ra# Xb#

Xb# Xa#

2Rb# 2Rb#

Mitotic Y-chromosome Y1 Telocentric with 2 heterochromatin blocks Y2 Telocentric but longer than Y1 and has three heterochromatic blocks Y1 Y1

Source: Sukowati and Baimai, 1996; Nanda et al., 2004. * Differences between the Xa and Xb type of chromosomes are slight. The Xb type differs from the standard arrangement at the free end of the X-chromosome. Xa at the tip has four diffuse bands while Xb has two prominent dark bands at the tip. ** The 2 refers to the new arm designation for chromosome 2R. In this paper the authors gave both types of designations to the arms. 2Rb differs from 2Ra in having a loosely diffuse area ni zone 19 (this zone covers the centromeric region). # It should be noted that the designations "a" and "b" used here are not inversion designations, but have been used to indicate different types. Species C exhibits prominent pericentric heterochromatin for all chromosome arms compared with those of forms A and B. An additional block of heterochromatin is observed near the centromeric region of chromosome 2. Sukowati and Baimai(1996) consider that the diffuse heterochromatin observed in zone 19 of polytene chromosome 2R in this form may be because of this extra block of heterochromatin.

78

Anopheline Species Complexes in South and South-East Asia

Electrophoretic variations Variations at the Mpi locus could be used to distinguish the three species (Sukowati et al., 1999) Multiplex PCR assay Dusfour et al. (2004) developed a multiplex PCR assay consisting of four secies specific SCAR (sequence characterized amplified region) primers derived from random amplified polymorphic region. This assay differentiated An. sundaicus s. s. from Malaysia and species A from Thailand and Viet Nam. Species A specimens used in the assay were neither cytogically nor electrophoretically identified, but were from the areas where, in earlier studies, only species A was found. The assay needs to be validated with other sibling species in the complex.

Distribution and biological characters In southern Thailand only species A was found, and in Sumatra, Indonesia, all three species, A, B and C, were found in the northern part, but in the southern part there were only species A and B. Cytotype D was found in laboratory colonies established from larvae collected from brackish and fresh water-breeding sites found in Andaman and Nicobar Islands (Nanda et al., 2004). Salinity in the breeding sites in these islands was as high as 14 gm/l (Das et al., 1998 and Sharma et al., 1999). Recently, Alam et al. (2005), based on D3 and ITS2 sequnce analysis, also concluded that An. sundaicus breeding in fresh water and brackish water sites is identical. The three cytological forms reported by Sukowati and Baimai (1996) were also found in both brackish and fresh waterbreeding sites. Similarly, Dusfour et al. (2004) found An. sundaicus s. s. in fresh water and saline water breeding sites in Malaysia. Thus, breeding in both fresh water and saline water does not appear to be a characterstic of any one sibling species in the complex. In Car Nicobar (Andaman and Nicobar Islands, India) where only cytotype D was found, An.

sundaicus was predominantly zoophagic (Kumari et al., 1993). Cytotype D was found in indoor collections from human dwellings and cattle sheds and also in outdoor collections. It may be noted that An. sundaicus is the sole malaria vector in the Andaman and Nicobar islands. Though only cytotype D was found in this study (Nanda et al., 2004), the authors did not rule out the possibility of another species existing in these islands. Alam et al. (2005), however, found no differences in the D3 and ITS2 sequences of the specimens collected from four isolated islands of the Andaman and Nicobar group of islands. Dusfour, Harbach and Manguin (2004) presented a consolidated account of the bionomics of An. sundaicus s. l. and of the sibling species identified across the range of their distribution in the oriental region. An. epiroticus was collected resting inside human habitations in Viet Nam (Linton et al., 2005). This is an important species complex responsible for the transmission of malaria in South-East Asian countries, hence, the bionomics of all the members of the complex needs to be studied in detail to plan effective control strategies. Now that there are different techniques, mitotic and polytene chromosomal differences, electrophoretic variation, and PCR assay for the identification of sibling species, these techniques should be validated for their specificity and sensitivity throughout the range of An. sundaicus distribution. This is necessary to establish sibling species distribution and their host feeding preferences, vectorial potential and responses to different control measures in different geographical regions.

3.14 The Anopheles stephensi variants Anopheles stephensi belongs to the subgenus Cellia and the Neocellia Series (Harbach, 2004). This species is found in all mainland zones of India but is rarely found at high altitudes in the Himalayas (Rao, 1984). The

Anopheline Species Complexes in South and South-East Asia

79

distribution of An. stephensi extends beyond the west of India to Pakistan, Afghanistan, Iran, Iraq, Bahrain, Oman and Saudi Arabia, and in the east to Bangladesh, south China and Myanmar. It is not reported in the north of India in Nepal nor in the south in Sri Lanka. This species is considered an important vector of malaria in Iran, Pakistan and India. Extensive studies have been carried out on this species, but so far there is no evidence which indicates that this is a species complex. Sweet and Rao (1937) identified two populations, type form and variety mysorensis differing in egg morphometrics, which inhabited urban and rural areas respectively. These populations were designated as races by Sweet, Rao and Subba Rao (1938), and as subspecies by Puri (1949). Stone, Knight and Starke (1959) accepted them as subspecies and included them in the catalogue of mosquitoes. Rutledge, Ward and Bickley (1970) found the two forms sympatric and considered them as variants and not subspecies. Subbarao et al. (1987) reported an additional type, intermediate, and considered all three as “ecological variants”. In addition to egg morphometrics, studies on inversion polymorphism (Mahmood and Sakai, 1984; Subbarao, 1996) have also shown rural and urban populations to be distinct..

Evidence for considering/not considering urban and rural populations as distinct species Egg morphometrics Sweet and Rao (1937) reported two races, An. stephensi type form and An. stephensi

variety mysorensis, on the basis of differences in the egg width and length and number of ridges on the egg float. Subbarao et al. (1987) studied several laboratory colonies established from urban and rural populations of An. stephensi for variation in ridge numbers on egg float. In addition to typical An. stephensi and var. mysorensis strains, some strains were found to have egg float ridge numbers falling between type form and var. mysorensis values. The three categories of egg ridge numbers were : 14 - 22, 12 - 17 and 9 -15. The mode number of ridges among the eggs laid by individual females in these stocks were 16 - 19, 13 - 16 and 10 - 14 respectively. The categories with the highest ridge number corresponded with the type form, and the lowest with var. mysorensis of Rao, Sweet and Subba Rao (1938), and the new category with intermediate numbers was designated as “intermediate”. The authors reported that areas where only mysorensis and “intermediate” were found were the typical rural villages and peri-urban localities. In the heart of Delhi, in two rural areas surrounded by high-income urban residential colonies, all three forms were observed and the type form was in the majority. The authors referred to these areas as semi-urban localities. Subbarao et al. (1987) referred to the three forms as ‘ecological variants’. Spiracular indices Mosquitoes have two pairs of spiracles on the thorax and six pairs on the abdomen. The size of the spiracle is a key factor in the adaptation of mosquitoes to different climatic

Table 12: Egg morphometrics in An. stephensi ecological races Egg Characters

Type form

Var. mysorensis

Length in microns + SD

555+24

476+24

Width in microns + SD

204+26

126+12

Length of float in microns + SD

294+23

218+20

No. ridges on float + SD

18+1.5

13+1.2

Source : Sweet and Rao (1937) , SD- standard deviation

80

Anopheline Species Complexes in South and South-East Asia

stresses. Vinogradskaya (1969) demonstrated that the anterior thoracic spiracular index can be used as a morphological indicator to distinguish xerophilic, mesophilic and hygrophilic mosquitoes. Nagpal et al. (2003) examined the spiracular index of type form and var. mysorensis collected from Jodhpur district, Rajasthan state, India. In type form, the average spiracle length was 0.11 - 0.12 mm and the average spiracle index was 8.09 – 9.23, whereas in mysorensis, the corresponding figures were 0.09 - 0.1 mm and 6.82 - 7.60. These parameters showed consistent differences between the populations of mosquitoes which emerged during the monsoon and those in the summer seasons. The spiracular indices correlated significantly with the number of ridges on the egg float. Thus, using this index the two ecological races can be identified at the adult stage. Crossing experiments Sweet, Rao and Subba Rao (1938) reported failure of egg-laying by a majority of the females in reciprocal crosses between type form and var. mysorensis, and of the eggs laid, a majority failed to hatch. Rutledge, Ward and Bickley (1970) observed varied levels of fertility in reciprocal crosses between three strains established from Iran, Iraq and India. It is not known whether these colonies were established from rural or urban populations. The characters exhibited by these colonies with reference to egg morphology were of mixed type. They resembled mysorensis with respect to egg length but resembled the type form in egg width and egg ridge number. Subbarao et al. (1987) made reciprocal crosses and back crosses between type form and mysorensis colonies established, respectively, from urban and rural areas in India. Only the ridge numbers on the egg float were studied in these crosses. The crosses were fertile and the F1 hybrid progeny were also fully fertile, indicating no post-

copulatory barriers between the populations. A detailed genetic analysis of the ‘ridge number’ character was carried out following the likelihood method of Curtis, Curtis and Barton (1985). The results indicated that the variation in ridge numbers is controlled by more than one genetic factor. The authors explained that intermediate-type strains probably have an intermediate number of polygenes, and the strains maintain intermediate-type ridge numbers in the laboratory strains because of the absence of any selection pressure. Polytene chromosomes and inversion polymorphism A photomap of polytene chromosomes of An. stephensi is presented by Mahmood and Sakai (1985). In this species, six autosomal inversions were reported by Coluzzi, Di Deco and Cancrini (1973a) and 16 polymorphic inversions (12 were new inversions) were reported in Pakistan populations by Mahmood and Sakai (1984). They observed 13 inversions in urban populations and, in contrast, only three in rural populations; the three inversions observed in rural areas were not found in urban localities. Furthermore, the number of females observed with inversions was very low in rural areas (4/225 in Kasur district and 4/168 in Lahore district). In Karachi city, 28 females out of 40 examined were found with inversions. Similarly in India, 10 inversions were observed in urban populations of An. stephensi, while in rural populations only the b inversion on chromosome arm 2 in a low frequency and another inversion h1 were observed in the heterozygous form in one specimen (Subbarao, 1996). The h1 inversion was not observed in urban populations. Break-points of inversions reported by Coluzzi, Di Deco and Cancrini (1973a) and Mahmood and Sakai (1984), and of those observed in Delhi, India, are shown on photomaps of polytene chromosomes by Subbarao (1996). Though rural and urban populations were found to be distinct with reference to

Anopheline Species Complexes in South and South-East Asia

81

inversion polymorphism, no pre-copulatory barriers have been observed so far. One cannot, however, rule out the possibility of the existence of homosequential species within this taxon.

Y-chromosomes and the remainder were of the metacentric type (Y1). However, in Pondicherry, Y1 was found in 31.9 per cent and Y2 in 68.1 per cent of the An. stephensi male population.

Y-chromosome variation Rishikesh (1959) and Aslamkhan (1973) reported the mitotic karyotype of An. stephensi. The two autosomal pairs and the X-chromosome are metacentric, and the Ychromosome is submetacentric. Suguna (1992) reported two types of Y-chromosomes, one submetacentric (as reported by earlier workers) and a new metacentric type from two urban localities in Tamil Nadu, India. In Cuddalore, 91.4 per cent of single female cultures examined had submetacentric (Y2)

Distribution and biological characters In Pakistan, both the forms were reported, and in India, three forms were seen, including the new “intermediate” form (Subbarao et al., 1987). Senior-White (1940) reported both type form and mysorensis from Kolkata, India. Subbarao et al. (1987) did not find type form in rural localities but in semi-urban localities they found all three forms, with a majority of type form. These authors did not survey typical urban populations.

Figure 14: Photomap of An. stephensi polytene chromosomes showing the break-points of the inversions. Inversions included are from Coluzzi et al. (1973a), Mahmood and Sakai (1984) and inversions observed in Delhi populations

(Subbarao, 1996). Re-naming of inversions compared with the names used by Mahmood and Sakai and Coluzzi et al., are as follows: inversions on chromosome 2 (2R)- e as f1, and f as g1; inversions on chromosome arm 5 (2L), d as q1; inversions on chromosome arm 4 (3R)- a as m1, b as n1 and c as o1, and inversions on chromosome arm 3(3L), d as b1, e as c1, f as d1, g as e1, h as f1, i as g1 and j as h1 (Source : Subbarao, 1996). Subbarao (1996) renamed the inversions identified by the earlier workers to have an unified inversion designations for species in the Neocellia Series as proposed by Green (1982) (For details see Chapter 2 in this book). X

2

4

3

5

c a f

1

i

1

p

1

c

p1 c d

g .d a i1 1

f1

o1

q

c

1

f1 e1.g1 d e1.f1

a.b d

c.c1 b d1

c a

c.b1 d1.h1 b g

r1 n1 q

q1 m11.n1.r1 o

q1 1 m

b1

1

h1

h1

b

g1 h1

82

Anopheline Species Complexes in South and South-East Asia

Coluzzi, Di Deco and Cancrini (1973b) reported that 2Rb inversion homozygotes had “mysorensis type” of eggs. Suguna (1981) and Subbarao et al. (1987) observed no such correlation in laboratory and field populations. In rural areas where mysorensis is prevalent, a wide range of breeding sites, such as streams and channels, tanks and ponds, seepages, irrigation wells, etc., were found, and in urban areas the breeding sites were wells, overhead tanks, cisterns, fountains and water collections at construction sites (Rao, 1984). Sweet and Rao (1937) and Rao, Sweet and Subba Rao (1938) reported that An. stephensi type form is an efficient vector, while var. mysorensis is a poor vector. Recently, Nagpal et al. (2003) studied the bioecology of type form and var. mysorensis in an arid zone (Jodhpur, Rajasthan state) in India. The study was carried out during the pre-monsoon, monsoon and post-monsoon seasons. The type form was found breeding indoors in domestic and peri-domestic containers and it was found inside, especially on hanging objects, throughout the year. Var. mysorensis was found breeding in the same sites and resting indoors during the summer, but it moved outside with the onset of rains. The authors do not consider var. mysorensis

to be involved in the transmission of malaria due to its predominantly animal feeding and low parity during the transmission season. However, in Iran, var. mysorensis is considered an important vector. Suguna (1992) reports that in Cuddalore (Tamil Nadu, India) where the submetacentric Ychromosome was found in about 91 per cent of the male population, An. stephensi was implicated in malaria transmission (Suguna, 1981), but not in Pondicherry where the submetacentric type was about 68 per cent. In villages around Delhi, as well as in Delhi city, An. stephensi was incriminated (Sharma et al., 1993). Recently, Rowland et al. (2002) incriminated An. stephensi along with seven other anophelines in rural areas of eastern Afghanistan. Positive mosquitoes were found with P. vivax (CSP210 and 247) and P. falciparum CSP antigens, and the sporozoite rate was 0.35%. Laboratory-feeding experiments revealed that type form takes a shorter period for the development of sporozoites than var. mysorensis (Vasanthi, 1996). There are 16 microsatellite markers developed for this species (Verardi et al., 2002). Now, the taxonomic status of the two ecological races in this species could again be addressed using these markers.

Anopheline Species Complexes in South and South-East Asia

83

4. Prospects for the future

The importance of recognizing species complexes and identifying their members became obvious as early as the 1930s with the discovery of An. maculipennis as a species complex in Europe. During the Second World War, in Italy, G. Davidson (personal communication from C. F. Curtis) had the job of locating the dangerous anthropophagic species An. labranchiae and the less dangerous and generally zoophagic An. maculipennis s.s. so as to site military camps in safe locations. But soon after the war, malaria was eradicated from Europe. Malaria continues to be endemic in more than 100 countries and is a serious health problem in all the South and South- East Asian countries. With the escalating prices of insecticides and drugs, and beset with problems of resistance of vectors to insecticides and of parasites to drugs, almost all countries in this region are experiencing challenges to contain and control malaria. It is now increasingly being accepted by many control programmes that a single strategy for an entire country, and even for a single province/district, is not applicable. Situation-specific, and at times even speciesspecific, strategies need to be employed. Now that almost all important malaria vectors in South and South-East Asia have been identified as species complexes, it is important to map the distribution of the sibling species of the complexes and to establish the role of each sibling species in malaria transmission and monitor their response to control measures. This requires the study of the biological characters of each sibling species.

84

The Culicifacies and Fluviatilis in India and Dirus and Maculatus Complexes in Thailand are the most thoroughly investigated ones in this region. The data generated so far have clearly established the importance of identifying sibling species in malaria control programmes. However, the information generated on these complexes so far is insufficient for planning and implementing effective control strategies suitable for different situations. Since each species has a different gene pool and hence may have different behaviour and genetic potential, data on the following aspects of all members of each complex would be of interest: (i)

Breeding habitats

(ii) Resting behaviour (indoors/outdoors) (iii) Feeding preference with reference to site and host (human/animal; indoors/ outdoors) (iv) Biting rhythms (peak biting time) (v) Susceptibility to plasmodial infections in the laboratory to establish whether the low vectorial capacity is due to genetic/ physiological factors or because of poor longevity, preference to feed on hosts other than humans, etc. (vi) Response to commonly-used insecticides which could indicate the difference in the rate of development of resistance (vii) Molecular mechanisms responsible for resistance (if possible). In addition to these, studies should be encouraged on more subtle, but equally

Anopheline Species Complexes in South and South-East Asia

relevant and not so easily observed, differences such as (a) density regulation of sympatric species in breeding places; (b) factors responsible for distinct distribution pattern; (c) physical and chemical properties of water in different breeding places to determine the factors influencing species preferences; (d) population structure, smallest breeding unit, population bottlenecks, gene flow over the entire range of each species distribution; etc. Serious consideration is now being given to genetic strategies as alternative vector control tools. Efforts are under way to develop genetically manipulated/engineered mosquitoes for use in vector control. Many research groups are working on different aspects to contribute to the development of anopheline strain(s) which will not be susceptible to parasite development, and consequently if these could be made to replace the wild vector population to interrupt malaria transmission. For this final achievement, other necessary aspects essential for the release of such a desirable strain and spread it in nature are being addressed with great vigour and care. With the completion of the sequencing of An. gambiae genome (Holt et al., 2002), it is expected that more novel targets will be identified for anopheline vector control. Volume 219 of Science (2002), in which this sequencing data was published, also contained viewpoints of several distinguished scientists on the implications of genome sequencing in developing vector control strategies and for better understanding of the biology of vectors. For these and for other already existing vector control strategies, it is important to recognize all the species present (whether they are morphologically distinguishable or indistinguishable) and the biological features of each species (as mentioned above). If identification of sibling species is to be done routinely in malaria control activities,

techniques which are simple, accurate and affordable need to be developed. By definition, sibling species are morphologically very similar, but members of a complex should nevertheless be examined for any morphological variation. Members of the Subpictus Complex can easily be distinguished by morphological characters, while for those of the Maculatus Complex, more skill and effort are needed. There are other complexes, for which there are no reports of morphological variations. Examination of polytene chromosomes for species-specific diagnostic inversion genotypes is the cheapest technique now available, but it requires specialized skill and aptitude for microscopic examination. In spite of certain limitations, this technique was efficiently used for studying the Culicifacies and Fluviatilis Complexes in India and Maculatus in Thailand. In the 1980s, there was more interest in the development of allozymes as diagnostic tools than there is now. Advancements and advantages of DNAbased techniques have shifted the interest of researchers towards molecular methods. Initial establishment costs are high and also expertise is required to develop DNA-based methods. However, once these techniques are standardized, the procedures are simple and can routinely be used to identify the species and a large number of samples can be screened on a single day. An informal consultancy meeting on Malaria vector species complexes and intraspecific variations: Relevance for malaria control and orientation for further research was held in Bangkok, Thailand, from 29 October to 3 November 1984. During this meeting several recommendations were made. More than two decades have since passed. The author considers the recommendations of the meeting are still valid and require attention. Therefore, the recommendations are reproduced to emphasize the importance of identifying species complexes and for further research on these complexes.

Anopheline Species Complexes in South and South-East Asia

85

“In order to simplify epidemiological mapping and stratification at the country level, as part of the programme planning process, attempts should be made to link genetically-determined sibling species and associated variants with topographical and vegetational indicators.” “Species distribution does not recognize political boundaries, and it is strongly recommended that collaboration between countries and regions be encouraged in investigations involving species complexes of malaria vectors.” Thus, it becomes imperative that research findings on species complexes should find a place in the planning of national vector control strategies and should not be considered merely as academic achievements or pursuits. In order for this to be accomplished, both researchers and programme planners/managers have to make an effort to: (i) Establish close linkages between organizations/universities where research on species complexes is carried out and authorities/directorates who plan control strategies; (ii) Create a database on species complexes to which national control programme can have an access to (with the advancement in communication techniques, it would not be difficult to create such linkages); (iii) Organize intra-country courses to impart training to personnel in the national programmes to learn methods to identify sibling species and to utilize knowledge on species complexes in malaria entomology. As a follow-up to this meeting, two workshops, one for Indian participants sponsored by the Indian Council of Medical Research in 1994 and the other an intercountry workshop sponsored by the WHO Regional Office for South-East Asia in 1997 were held in New Delhi, India. The Malaria Research Centre (now renamed as National Institute of Malaria Research), Delhi 86

organized both these workshops. In the intercountry workshop, experts from Afghanistan, Bangladesh, India, Indonesia, Myanmar, Sri Lanka and Thailand participated. Both these workshops provided hands-on-experience to the participants on the techniques used in the identification of sibling species. This is an indication of the commitment of the organizing agencies and participating countries to study species complexes. There is need for organizing more such workshops, especially in view of the techniques (molecular methods) that have been developed recently. Another important requirement for any control programme, especially for genetic control strategies, is information on population structure, gene flow and geographical barriers, if any. Workshops covering these aspects and practical training on the tools that can be used for these studies are urgently required in this region. Training on genotyping of populations using microsatellite markers should be organized as markers are available for four important vectors, An. culicifacies, An. dirus, An. maculatus, and An. stephensi. Students from developing countries will greatly benefit studying for their masters and doctoral degrees at universities in developed countries in the specialized aspects of field biology related to malaria vectors. Lastly, it should be noted that sibling species are not a special kind of species and ought to be treated as any other species. This is an appeal to researchers that they should formally designate sibling species with formal binomial nomenclature at the earliest opportunity and drop the provisional designations given to them. It can be assumed that entomologists and programme planners/ managers will not hesitate to accept sibling species with formal designations. For many the concept of biological species/sibling species is still alien and they continue to consider them as races or variant taxa, which have been conclusively proved to be separate biological species that do not generally exchange genes by mating in the field.

Anopheline Species Complexes in South and South-East Asia

5. References and select bibliography (Chapter-wise listing) 1. Introduction Besansky, N. J., Powell, J. R., Caccone, A., Hamm, D. M., Scott, J. A. and Collins, F. H. (1994). Molecular phylogeny of the Anopheles gambiae complex suggests genetic introgression between principal malaria vectors. Proc. Natl. Acad. Sci. USA 9: 6885-8. Besansky, N. J., Lehman, T., Fahey, G. T., Fontenelle, D., Braack, L. E. O.,Hawley, W. A. and Collins, F. H. (1997). Patterns of mitochondrial variation within and between African malaria vectors, Anopheles gambiae and An. arabiensis , suggesst extensive gene flow. Genetics 14: 1817-28. Besansky, N. J., Krzywinski, J., Lehman, T., Simrad, F., Kern, M., Mukabayire, O., Fonteinelle, D. Toure, Y. and Sugnon, N. F. (2003). Semipermeable species boundaries between Anopheles gambiae and An. arabiensis : evidence from mitochondrial sequnce variation. Proc. Natl. Acad. Sci. USA 100: 10818-23. Coluzzi, M. (1988). Anopheline mosquitoes: Genetic methods for species differentiation. In: Malaria principles and practices of malariology (eds W.H. Wernsdorfer and I. McGregor. Churchill Livingstone. Edinburgh/London/ Melbourne/ New York. 1: 411-30. Donelly, M. J., Pinto, J., Girad, R., Besansky, N. J. and Lehman, T. (2004). Revising the role of introgression vs shared ancestral polymorphisms as key processes shaping genetic diversity in the recently separated sibling species of the Anopheles gambiae complex. Heredity 92: 61-8. Garcia, B. A., Caccone, A., Mathiopolis, K. D. and Powell, J. R. (1996). Inversion monophyly in African anopheline malaria vectors. Genetics 143: 1313-20. Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94 : 537-53. Hunt, R. H., Coetzee, M., and Fettene, M. (1998). The Anopheles gambiae complex : a new species from Ethopia. Tras. Roy. Soc. Trop. Med. Hyg. 92: 231-5. Kondrashin, A.V. and Rashid, K.M. (1987). Epidemiological considerations for planning malaria control in South-East Asia Region. World Health Organization, Regional Office for South-East Asia, New Delhi: p. 411. Mayr, E. (1970). Populations, species and evolution. . The Belknap Press of Harvard University Press, Cambridge, Massachusetts: pp. 26-32. Rao, T.R. (1984). The anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518 Sharma, V.P., Chandrahas, R.K., Ansari, M.A., Srivastava, P.K. Razdan, R.K., Batra, C.P., Raghavendra, K., Nagpal, B.N., Bhalla, S.C. and Sharma. G.K. (1986). Impact of DDT

and HCH spraying on malathion transmission in villages with DDT and HCH resistant Anopheles culicifacies. Indian J. Malariol., 23: 27-8. Subbarao, S.K., Adak, T. and Sharma, V.P. (1980). Anopheles culicifacies sibling species distribution and vector incrimination studies. J. Commun. Dis. 12: 102-4. Subbarao, S.K., Vasantha, K. and Sharma, V.P. (1988). Responses of Anopheles culicifacies sibling species A and B to DDT and HCH in India: Implications in malaria control. Med. Vet. Entomol., 2: 219-23. Subbarao, S.K., Adak, T., Vasantha, K., Joshi, H., Raghavendra, K., Cochrane, A.H., Nussenzweig, R.S. and Sharma, V.P. (1988). Susceptibility of Anopheles culicifacies species A and B to Plasmodium vivax and Plasmodium falciparum as determined by immunoradiometric assay. Trans. Roy. Soc. Trop. Med. Hyg., 8: 394-7. Subbarao, S.K., K. Vasantha, H. Joshi, K. Raghavendra, C. Usha Devi, T.S. Satyanarayan, A.H. Cochrane, R.S. Nussenzweig and V.P. Sharma (1992). Role of Anopheles culicifacies sibling species in malaria transmission in Madhya Pradesh, India. Trans. Roy. Soc. Hyg. Trop. Med. Hyg. 86: 613-4. Subbarao, S.K., Nanda, N. and Raghavendra, K. (1999). Malariogenic stratification of India using Anopheles culicifacies sibling species prevalence. ICMR Bull. 29(7): 75-80. Walton, C., Handley, J.M., Collins, F.H., Baimai, V., Harbach, R.E., Deesin, V. and Butlin, R.K. (2001). Genetic population structure and introgression in Anopheles mosquitoes in South-east Asia. Mol. Ecol. 10(3): 569-80.

2. Techniques used in the recognition of Species Complexes Atrie, B., Subbarao, Sarala K., Pillai, M.K.K., Rao, S.R.V. and Sharma, V.P. (1999). Population cytogenetic evidence for sibling species in Anopheles annularis (Diptera: Culicidae). Ann. Entomol. Soc. Amer. 92 (2): 243-9. Audtho, M., Tassanakajon, A., Boonsaeng, V., Tpiankijagum, S. and Panyim. S. (1995). Simple nonradioactive DNA hybridization method for identification of sibling species of Anopheles dirus (Diptera: Culicidae) complex. J. Med. Entomol., 32: 107-11. Ayala, F.J., Powell, J.R., Tracey, M.L., Mourao, C.A. and PerezSalas, S. (1972). Enzyme variability in the Drosophila willistoni group. IV. Genic variation in natural populations of Drosophila willistoni. Genetics, 70: 113-39.

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3.3

The Culicifacies Complex

Abhayawardena, T.A., Dilrukshi, R.K.C. and Wijesuriya, S.R.E. (1996). Cytotaxonomical examination of sibling species in the taxon Anopheles culicifacies Giles in Sri Lanka. Indian J. Malariol. 33: 74-80. Adak, T., Subbarao, S.K., Sharma, V.P. and Rao, S.R.V. (1994). Lactate dehydrogenase allozyme differentiation of species in the Anopheles culicifacies complex. Med. Vet. Entomol. 8: 137-40.

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3.2

The Barbirostris Complex

Baimai, V., Rattanarithikul, R and Kijchalao, U. (1995). Metaphase karyotypes of Anopheles of Thailand and southeast Asia : IV The Barbirostris and umbrosus species groups, subgenus Anopheles (Diptera : Culicidae). J. Am. Mosq. Contr. Assoc. 11 (3): 323–8. Chowdiah, B.N., Avicharan, T.T. and Seetharaman, P.l. (1970). Chromosome studies of Oriental anophelines. Expolientia 26: 315-7. Choochote, W., Sucharit, Supat and Abeyewickreme, A. (1983). Experiments in crossing two strains of Anopheles

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———. (1988b). Studies on the crosses between the sibling species of the Anopheles culicifacies complex. J. Hered. 79: 300-3. ———. (1988c). Response of Anopheles culicifacies sibling species A and B to DDT and HCH in India: implications in malaria control. Med. Vet. Entomol. 2: 219-23. Subbarao, S.K., Vasantha, K., Adak, T. and Sharma, V.P. (1983). Anopheles culicifacies complex: evidence for a new sibling species, species C. Ann. Entomol. Soc. Am. 76: 985-8. Subbarao, S.K., Vasantha, V., Adak, T. and Sharma, V.P. (1987). Seasonal prevalence of sibling species A and B of the taxon Anopheles culicifacies in villages around Delhi. Indian J. Malariol. 24: 9-15. Subbarao, S.K., Nanda, N., Chandrahas, R.K. and Sharma, V.P. (1993). Anopheles culicifacies complex : Cytogenetic characterization of Rameshwaram island populations. J. Am. Mosq. Contr. Assoc. 9: 27-31. Subbarao, S.K., Vasantha, K., Raghavendra, K., Sharma, V.P. and Sharma, G.K. (1988a). Anopheles culicifacies : sibling species composition and its relationship to malaria incidence. J. Am. Mosq. Contr. Assoc. 4: 29-33. Subbarao, S.K., Adak, T., Vasantha, V., Joshi, H., Raghavendra, K., Cochrane, A.H., Nussenzweig, R.S. and Sharma, V.P. (1988b). Susceptibility of Anopheles culicifacies species A and B to Plasmodium vivax and Plasmodium falciparum as determined by immunoradiometric assay. Trans. Roy. Soc. Trop. Med. Hyg. 82: 394-7. Subbarao, S.K., K. Vasantha, H. Joshi, K. Raghavendra, C. Usha Devi, T.S. Satyanarayan, A.H. Cochrane, R.S. Nussenzweig and V.P. Sharma (1992). Role of Anopheles culicifacies sibling species in malaria transmission in Madhya Pradesh, India. Trans. Roy. Soc. Trop. Med. Hyg. 86: 613-4. Suguna, S.G., Tewari, S.C., Mani, T.R., Hiryan, J. and Reuben, R. (1983). Anopheles culicifacies species complex in Thenpennaiyar riverine tract, Tamil Nadu. Indian J. Med. Res. 77: 455-9. Suguna, S.G., Tewari, S.C., Mani, T.R., Hiriyan, J. and Reuben, R. (1989). A cytogenetic description of a new species of the Anopheles culicifacies complex. Genetica 78: 22530. Sunil, Sujatha, Raghavendra, K., Singh, O.P., Malhotra, P., Huang, Y., Zheng, Liangbiao and Subbarao, Sarala K. (2004). Isolation and characterization of microsatellite markers from malaria vector, Anopheles culicifacies. Molecular Ecology notes 4: 440-2. Surendran, S.N., Abhayawardana, T.A., De Silva, B.G., Ramasamy, R. and Ramasamy, M.S. (2000). Anopheles culicifacies Y-chromosome dimorphism indicates sibling species (B and E) with different malaria vector potential in Sri Lanka. Med. Vet. Entomol. 14(4): 437-40. _________ (2002a). Limnological characterization of larval breeding sites of sibling species (B and E) in the Anopheles culicifacies (Diptera: Culicidae) species complex in Sri Lanka. Proceedings of the Institute of Biology. P. 56. _________ (2002b). Differential susceptibility to malathion by two members (B and E) of the Anopheles culicifacies (Diptera: Culicidae) species complex in Sri Lanka. Proceedings of the Sri Lanka Association for the Advancement of Science 58: 153. _________ (2003). Establishment of species E, not B as the major vector of malaria in the Anopheles culicifacies complex in the country. Proceedings of the Sri Lanka association for the Advancement of Science 59: 180.

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3.4

The Dirus Complex

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Mudon, Mon state, Myanmar. J. Vector Ecol. 27(1): 4454. Oo, T.T., Storch, V. and Becker, N. (2003). Anpheles dirus and its role in malaria transmission in Myanmar. J. Vect. Ecol. 28(2): 175-83. Panyim, S., Yasothornsrikul, S. and Baimai, V. (1988). Species specific DNA sequences from the Anopheles dirus complex - a potential for efficient identification of isomorphic species. In Biosystematics of haematophagous insects (ed. M.W. Service) Clarendon Press, Oxford. pp. 193-202. Peyton, E.L. (1989). A new classification for the Leucosphyrus Group of Anopheles (Cellia). Mosq. syst. 21: 197-05. Peyton, E.L. and Harrison, B.A. (1979). Anopheles (Cellia) dirus, a new species of the leucosphyrus Group from Thailand. Mozq. Syst. 11: 40-52. Peyton, E.L. and Harrison, B.A. (1980). Anopheles (Cellia) takasagoensis Morishita 1946, an additional species in the Balabacensis Complex of Southeast Asia (Diptera: Culicidae). Mosq. Syst. 12: 335-47. Peyton, E.L. and Ramalingam, S. (1988). Anopheles (Cellia) nemophilous, a new species of the Leucosphyrus Group from Peninsular Malaysia and Thailand (Diptera: Culicidae). Mosq. syst. 20: 272-99. Prakash, A., Bhattacharyya, DR., Mohapatra, P.K. and Mahanta, J. (2001). Estimation of vectorial capacity of Anopheles dirus (Diptera: Culicidae) in a forest-fringed village of Assam (India). Vector Borne Zoonostic Dis. 1(3): 231-7. Prakash, A., Bhattacharyya, DR., Mohapatra, P.K. and Mahanta, J. (2002). Physico-chemical characteristics of breeding habitats of Anopheles dirus (Diptera: Culicidae) in Assam, India. J. Environ. Biol. 23(1): 95-100. Prakash, A., Bhattacharyya, DR., Mohapatra, P.K. and Mahanta, J. (2005). Malaria transmission risk by the mosquito Anopheles baimaii (formerly known as An. dirus species D) at different hours of the night in North-east India. Med. Vet. Entomol. 19: 423-427. Prakash, A., Walton C., Bhattacharyya, D. R., O’Loughlin, Samantha, Mohapatra, P. K. and Mahanta, J. (2006). Molecular characterization and species identification of the Anopheles dirus and An. minimus complexes in northeast India using r-DNA ITS2. Acta Trop. 100: 15161. Rao, T.R. (1984). The anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518. Rosenberg, R. and Maheswary, N.P. (1982). Forest malaria in Bangladesh II. Transmission by Anopheles dirus. Am. J. Trop. Med. Hyg. 31: 183-91. Rosenberg, R., Andre, R.G. and Somchit, L. (1990). Highly efficient dry season transmission of malaria in Thailand. Trans. R. Soc. Trop. Med. Hyg. 84: 22-28. Sallum, M.A.M., Peyton, E.L. and Wilkerson, R.C. (2005). Six new species of the Anopheles leucosphyrus group, reinterpretation of An. olegans and vector implication. Med. Vet. Entomol. 19: 158-199. Sawadipanich, Y., Baimai, V. and Harrison, B.A. (1990). Anopheles dirus species E: Chromosomal and crossing evidence for another member of the dirus complex. J. Am. Mosq. Contr. Assoc. 6: 477-81. Samboon, P., Prapanthadara, L. and Suwonket, L. (1999). Selection of Anopheles dirus for refractoriness and susceptibility to Plasmodium yoelii nigeriensis. Med. Vet. Entomol. 13(4): 355-61.

Anopheline Species Complexes in South and South-East Asia

Tiwari, S.C., Hiriyan, J. and Reuben, R. (1987). Survey of the anopheline fauna of the western Ghats in Tamil Nadu, India. Indian J. Malariol. 24: 21-8. Trung, H. D., Van Bortel, W., Sochantha, T., Keokanchanh, K. Bret, O. J. T. and Coosemans, M. (2005). Behavioural heterogeneity of Anopheles species in ecologically different localities in Southeast Asia : a challenge for vector control. Trop. Med. Int. Hlth. 10 (3): 251-62. Van Bortel, W., Harbach, R.E., Trung, H.D., Roelants, P., Backeljau, T. and Coosemans, M. (2001). Confirmation of Anopheles varuna in Vietnam, previously misidentified and mistargeted as the malaria vector An. minimus. Am. J. Trop. Med. Hyg., 65: 729-32. Walton, C., Chang, M.S., Handley, J.M., Harbach, R.E., Collins, F.H., Baimai, V. and Butlin, R.K. (2000a). The isolation and characterization of microsatellites from Anopheles dirus mosquitoes. Mol. Ecol. 9(10): 1665-7. Walton, C., Handley, J.M., Collins, F.H., Baimai, V., Harbach, R.E., Deesin, V. and Butlin, R.K. (2001). Genetic population structure and introgression in Anopheles mosquitoes in South-east Asia. Mol. Ecol. 10(3): 569-80. Walton, C., Handley, J.M., Kuvangkadilok, C., Collins, F.H., Harbach, R.E. , Baimai, V. and Butlin, R.K. (1999). Identification of five species of the Anopheles dirus complex in Thailand, using allele-specific polymerase chain reaction. Med. Vet. Entomol. 13(1): 24-32. Walton, C., Handley, J.M., Tun-Lin, W., Collins, F.H., Harbach, R.E., Baimi, V. and Butlin, R.K. (2000b). Population structure and population history of Anopheles dirus mosquitoes in Southeast Asia. Mol. Biol. Evol. 17(6): 96274. Wibow, S., Baimai, V. and Andre, R.G. (1984). Differentiation of four taxa of the Anopheles balabacensis complex using H-banding in sex chromosome (Diptera: Culicidae). Can. J. Genet. Cytol. 26: 425-9. Xu, X., Xu, J. and Qu, F. (1998). A diagnostic polymerase chain reaction assay for species A and D of the Anopheles dirus (Diptera: Culicidae) species complex based on ribosomal DNA second internal transcribed spacer sequence. J. Am. Mosq. Contr. Assoc. 14(4): 385-9. Yasothornsrikul, S., Panyim, S. and Rosenberg, R. (1988). Diagnostic restriction fragment patterns of DNA from the four isomorphic species of Anopheles dirus. Southeast Asian J. Trop. Med. Publ. Hlth. 19: 703-8.

3.5

The Fluviatilis Complex

Adak, T., Singh, O. P., Das, M. K. Wattal, S. and Nanda, N. (2005). Comparative susceptibility of three important malaria vectors, Anopheles stephensi, Anopheles fluviatilis and Anopheles sundaicus to Plasmodium vivax. J. Parasitol. 91: 79-82. Bhombore, S.R., Sitaraman, N.L. and Achuthan, C. (1956). Studies on the bionomics of An. fluviatilis in Mysore state, part II. Indian J. Malariol. 10: 23-32. Brooke Worth, C. and Sitaraman, N.L. (1952). Studies on the bionomics of Anopheles fluviatilis James, 1902 in Mysore state, India. Review of the literature on bionomics of An. fluviatilis. Ind. J. Malariol. 6: 481-91. Chen, B., Butlin, R. K., Pedro, P. M.., Wang, X. Z. and harbach, R. E. (2006). Molecular variation, systematics and distribution of the Anopheles fluviatilis complex in southern Asia. Med. Vet. Entomol. 16: 253-265.

Garros, C., Harbach, R. E. and Manguin, S. (2005). Morphological assessment and molecular phylogenetics of the Funestus and Minimus Groups of Anopheles (Cellia). J. Med. Entomol. 42: 522-36. Gunasekaran, K., Sahu, S.S., Parida, S.K., Sadanandane, C., Jambulingam, P. and Das, P.K. (1989). Anopheline fauna of Koraput district, Orissa state, with particular reference to transmission of malaria. Indian J. Med. Res. 89: 340-3 Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Manonmani, A., Townson, H., Adeniran, T., Jambulinga,, P., Sahu, S. and Vijayakumar, T. (2001). RDNA-ITS-2 polymerase chain reaction assay for the sibling species of An. fluviatilis. Acta Tropica. 1: 7-11. Manonmani, A., Nanda, N., Jambulingam, P., Sahu, S., Vijakumar, T., Ramya vani, J. and Subbarao, S.K. (2003). Comparison of PCR assay and cytotaxonomy for identification of Anopheles fluviatilis sibling species. Bull. Ent. Res. 93: 169-71. MRC (1995, unpublished). Malaria Research Centre Annual Report. Nanda, N., Joshi, H., Subbarao, S.K., Yadav, R.S., Shukla, R.P., Dua, V.K. and Sharma, V.P. (1996). Anopheles fluviatilis complex : Host feeding patterns of species S,T and U. J. Am. Mosq. Contr. Assoc. 12: 147-9. Nanda, Nutan, R.S. Yadav, Sarala K. Subbarao, Hema Joshi and V.P. Sharma (2000). Studies on Anopheles fluviatilis and Anopheles culicifacies in relation with malaria in forest and deforested riverine ecosystems in northern Orissa, India. J. Amer. Mosq. Contr. Assoc. 16(3): 199-205. Rao, T.R. (1984). The anophelines of India. Malaria Research Centre (ICMR), Delhi. Rowland, M., Mohammed, N., Rehman, H., Howitt, S., Mendis, C., Ahmed Kamal, M. and Wirtz, R. (2002). Anopheline vectors and malaria transmission in eastern Afghanistan. Trans. Roy. Soc. Trop. Med. Hyg., 96(6): 620-6. Sahu, S.S., Parida, S.K., Sadanandane, C., Jambulingam, P. and Das, P.K. (1990). of malaria vectors: An. fluviatilis, An. culicifacies, in Koraput district, Orissa. 27: 209-16.

Gunasekaran, K., Breeding habitats annularis and An. Indian J. Malariol.

Senior-White, R. (1946). On the outdoor resting of some species of oriental Anopheles. J. Mal. Inst. India. 6: 425-35. Sharma, S.K., Nanda, N., Dua, V.K., Joshi, H., Subbarao, S.K. and Sharma, V.P. (1995). Studies on the bionomics of Anopheles fluviatilis sensu lato and the sibling species composition in the foothills of Shiwalik range (Uttar Pradesh), India. Southeast Asian J. Trop. Med. Publ. Hlth. 26: 566-72. Sharma, S. K., Tyagi, P.K., Padhan, K., Adak, T., Subbarao, S. K. (2004a). Malarial morbidity in tribal communities living in the forest and plain ecotypes of Orissa, India. Ann. Trop. Med. Parasitol. 98: 459-68. Sharma, S. K., Upadhyay, A. K., Haque, M. A., Singh, O. P., Adak, T., and Subbarao, S. K. (2004b). Insecticide susceptibility status of malaria vectors in some hyperendemic tribal districts of Orissa. Curr. Sci. 87: 1722-6. Sharma, S.K., Tyagi, P.K., Pradhan, K., Upadhyay, A.K., Haque, M.A., Joshi, H., Biswas, S., Nanda, N., Anitha, M., Adak, T., Chitnis, C.E., Chauhan, V.S. and Subbarao, S.K. (2006). Epidemiology of malaria in the forest and plain ecotypes of Sundergarh district, Orissa. Tran. Roy. Soc. Trop. Med. Hyg. 100: 917-25.

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Shukla, R.P., Nanda, N., Pandey, A.C., Kohli, V.K., Joshi, H., Subbarao, S.K. (1998). Studies on bionomics of An. fluviatilis and its sibling species in Nainital district (Uttar Pradesh), India. Indian J. Malariol. 35: 41-7. Singh OP, Chandra D, Nanda N, Raghavendra K, Sunil S, Sharma SK, Dua VK, and Subbarao SK. (2004). Differentiation of members of the Anopheles fluviatilis species complex by an allele-specific polymerase chain reaction based on 28S ribosomal DNA sequences. Am. J. Trop. Med. Hyg., 70 (1): 27-32. Singh, O. P., D. Chandra, N. Nanda , S. K. , Sharma, Pe Than Htun, T. Adak, S. k. Subbarao and A. P. Dash (2006). On the Conspecificity of Anopheles fluviatilis species S with Anopheles minimus species C. J. Biosciences 31 (5): 671677. Subbarao, S.K., Nanda, N., Vasantha, K., Dua, V.K., Malhotra, M.S., Yadav, R.S. and Sharma, V.P. (1994). Cytogenetic evidence for three sibling species in Anopheles fluviatilis (Diptera: Culicidae). Ann. Entomol. Soc. Am. 87: 11621. Vatandoost, H., Shahi, M., Oshaghi, M., A., Naddaf, S., R., Hanafi-Bojd., A., A. and Abaie, M., R.( 2005). Ecology of Anpheles fluviatilis James in a malarious area, Bandar Abbas, Hormozgan Province, Southern Iran (Abstract). Presented at the International conference on 125 Years of Malaria Research : Laveran to Genomic, New Delhi. November 4-6, 2005. Viswanathan, D.K. (1950). Malaria and its control in Bombay state, Chitrashala, Poona, India.

3.6

The Leucosphyrus Complex

Baimai, V. (1988). Population cytogenetics of the malaria vector Anopheles leucosphyrus group. Southeast Asian J. Trop. Med. Publ. Hlth. 19: 667-80. Baimai, V., Harbach, R.E. and Kijchalao, U. (1988). Cytogenetic evidence for a fifth species within the taxon Anopheles dirus in Thailand. J. Am. Mosq. Contr. Assoc. 4: 333-8. Baimai, V., Harbach, R.E. and Sukowati, S. (1988). Cytogenetic evidence for two species within the current concept of the malaria vector Anopheles leucosphyrus in Southeast Asia. J. Am. Mosq. Contr. Assoc. 4: 44-50. Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Maricioto, H., Bangs, M.J. and Baird, J.K. (2002). Epidemic malaria in the Monoreh Hills of Central Java. Am. J. Trop. Med. Hyg. 66(3): 287-92. Chang, M. S., Doraisingam, P., Hardin, S. and Nagum,, N. (19950. Malaria and filariasis transmission in a village/forest setting in Baram district, Sarawak, Malaysia. J. Trop. Med. and Hyg. 98: 192-8. Harbach, R.E., Baimai, V. and Sukowati, S. (1987). Some observations on sympatric populations of the malaria vectors Anopheles leucosphyrus and Anopheles balabacensis in a village-forest setting in south Kalimantan. Southeast Asian J. Trop. Med. Publ. Hlth. 18: 241-7. Hii, J.L.K. (1985). Genetic investigations of laboratory stocks of the complex of Anopheles balabacensis Baisas (Diptera: Culicidae). Bull. Entomol. Res. 75: 185-97. Hii, J.L., Kan, S., Vun, Y.S., Chin, K.F., Tambakau S, Chan, M.K., Lye, M.S., Mak, J.W.. amd Cochrane, A.H. (1988). Transmission dynamics and estimates of malaria vectorial capacity for Anopheles balabacensis and An. flavirostris

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(Diptra: Culicidae) on Banggi island, Sabah, Malaysia. Ann. Trop. Med. Parasitol. 82(1):91-101. Peyton, E.L. (1989). A new classification for the leucosphyrus group of Anopheles (Cellia). Mosq. syst. 21: 197-205. Peyton, E.L. and Harrison, B.A. (1979). Anopheles (Cellia) dirus, a new species of the leucosphyrus group from Thailand (Diptera: Culicidae). Mosq. syst. 11: 40-52. Sallum, M.A.M., Peyton, E.L. and Wilkerson, R.C. (2005). Six new species of the Anopheles leucosphyrus group, reinterpretation of An. olegans and vector implication. Med. Vet. Entomol. 19: 158-99.

3.7

The Maculatus Complex

Baimai, V. (1989). Speciation and species complexes of the anopheles malaria vectors in Thailand. In: Proceedings of the 3rd Conference on Malaria Research, Thailand. 1820th Oct. 1989. pp. 146-62. Baimai, V. and Green, C.A. (1988). Cytogenetics of natural populations of the malaria vectors Anopheles dirus and An. maculatus complexes in Thailand. In: Proceedings of Mahidol University Seminar on Malaria Vaccine Development. 17 - 18 May, 1988 : (eds. A. Sabachavrn, S. Supavej, P. Attanath and M. Ho). pp. 45-51. Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Maricioto, H., Bangs, M.J. and Baird, J.K. (2002). Epidemic malaria in the Monoreh Hills of Central Java. Am. J. Trop. Med. Hyg. 66(3): 287-92. Christophers, S.R. (1931). Studies on the anopheline fauna of India (Parts I-IV). Rec. Malar. Surv. India. 2: 305-32. Christophers, S.R. (1933). The fauna of British India, including Ceylon and Burma. Diptera. Vol. IV. Family Culicidae. Tribe Anophelini. Taylor and Francis, London. 371pp. Crawford, R. (1938). Some anopheline pupae of Malaya with a note on pupal structure. Government Printer, Singapore. 110pp. Ejercito, A. (1934). Anopheles maculatus Theobald, another malaria vector in the Philippines. J. Philipp. Is. Med. Assoc. 14: 342-6. Green, C.A. (1982). Polytene-chromosome relationships of the Anopheles stephensi species group from the afrotropical and oriental regions (Culicidae, Anopheles (Cellia), series Neocellia. pp. 49-61. In: Recent Developments in the Genetics of Insect Disease vectors (eds. W.W.M. Steiner, W.J. Tabachnick, K.S. Rai and S. Narang) Stipes publishing Co. Champaign, Illinois. Green, C.A. and Baimai, V. (1984). Polytene chromosomes and their use in species studies of malaria vectors as exemplified by Anopheles maculatus complex. In: Genetics: New Frontiers. Proceedings XV International Congress of Genetics (eds. V.L. Chopra, B.C. Joshi, R.P. Sharma and H.C. Bansal), Vol. 3, pp. 89-97. Oxford and IBH Publ. Comp., New Delhi. Green, C.A., Rattanarithikul, R. and Charoensub, A. (1992). Population genetic confirmation of species status of the malaria vectors Anopheles willmori and An. pseudowillmori in Thailand and chromosome phylogeny of the maculatus group of mosquitoes. Med. Vet. Entomol. 6: 335-41. Green, C.A., Baimai, V., Harrison, B.A. and Andre, R.G. (1985). Cytogenetic evidence for a complex of species within the taxon Anopheles maculatus (Ditera: Culicidae). Biol. J. Linn. Soc. 4: 321-8.

Anopheline Species Complexes in South and South-East Asia

Green, C.A., Rattanarithikul, R., Pongparit, S., Sawadwongporn, P. and Baimai, V. (1991). A newly-recognized vector of human malarial parasites in the oriental region, Anopheles (Cellia) pseudowillmori (Theobald, 1910). Trans. Roy. Soc. Trop. Med. Hyg. 85: 35-6. Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Kittayapong, P., Clark, J.M., Edman, J.D., Potter, T.L., Lavine, B.K., Marion, J.R. and Brooks, M. (1990). Cuticular lipid differences between the malaria vector and non-vector forms of the Anopheles maculatus complex. Med. Vet. Entomol. 4: 405-13. Kittayapong, P., Clark, J.M., Edman, J.D., Lavine, B.K., Marion, J.R., Brooks, M. (1993). Survey of the Anopheles maculatus complex (Diptera: Culicidae) in Peninsular Malaysia by analysis of cuticular lipids. J. Med. Ent. 30: 969-74. Ma, Y., Li, S. and Xu, J. (2006). Molecular identification and phlogeny of the Maulatus group of Anopheles mosquitoes (Diptera: Culicidae) based on nuclear and mitochondria DNA sequences. Acta Trop. 99: 272-80. Pradhan, J.N., Shrestha, S.L. and Vaidya, R.G. (1970). Malaria transmission in high mountain valleys of west Nepal including first record of Anopheles willmori (James) as a third vector of malaria. J. Nepal Med. Assoc. 8: 89-97. Puri, I.M. (1931). Larvae of anopheline mosquitoes, with full description of those of the Indian species. Indian Med. Res. Mem. 21: 1-227. Rao, T.R. (1984). The Anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518 Rattanarithikul, R. and Green, C.A. (1986). Formal recognition of the species of the Anopheles maculatus group (Diptera: Culicidae) occurring in Thailand, including the descriptions of two new species and a preliminary key to females. Mosq. Syst. 18: 246-78. Rattanarithikul, R. and Harbach, R.E. (1990). Anopheles maculatus (Diptera: Culicidae) from the type locality of Hong Kong and two new species of the maculatus complex from the Philippines. Mosq. syst. 22: 160-83. Reid, J.A. (1968). Anopheline mosquitoes of Malaya and Borneo. Stud. Inst. Med. Res. Malaya 31. Reid, J.A., Wattal, B.L. and Peters, W. (1966). Notes on Anopheles maculatus and related species. Bull. Indian Soc. Malariol. Commun. Dis. 3: 185-97. Rongnoparut, P., Yaicharoen, S., Sirichotpakorn, N., Rattanarithikul, R., Lanzaro, G.C. and Linthicum, K.J. (1996). Microsatellite polymorphism in Anopheles maculatus, a malaria vector in Thailand. Amer. J. Trop. Med. Hyg. 55(6): 589-94. Rongnoparut, P., Sirichotpakorn, N., Rattanarithikul, R., Yaicharoen, S. and Linthicum, K.J. (1999). Estimates of gene flow among Anopheles macularus populations in Thailand using microsatellite analysis. Am. J. Trop. Med. Hyg. 60(3): 508-15. Rowland, M., Mohammed, N., Rehman, H., Howitt, S., Mendis, C., Ahmed Kamal, M. and Wirtz, R. (2002). Anopheline vectors and malaria transmission in eastern Afghanistan. Trans. Roy. Soc. Trop. Med. Hyg., 96(6): 620-6. Torres, E.P., Foley, D.H. and Saul, A. (2000). Ribosomal DNA sequence markers differentiate two species of Anopheles maculates (Diptera: Culicidae) complex in the Philippines. J. Med. Entomol. 37(6): 933-7.

Upatham, E.S., Prasittisuk, C., Ratanatham, S., Green, C.A., Rojanasunan, W., Setakana, P., Theeraslip, N., Tremongkol, A., Viyanant, V., Pantuwatana, S. and Andre, R.G. (1988). Bionomics of Anopheles maculatus complex and their role in malaria transmission in Thailand. Southeast Asian J. Trop. Med. Pub. Hlth. 19: 259-6. Walton, C., Somboon, P., O’ Loughlin, S.M., Zhang, S., Harbach, R.E., Linton, Y.M., Chen, B.K., Duong, S. Nolan, K., Fong, Y.M., Vythilingumi, I., Mohammed, Z.D., Trung, H.D. and Butlin, R.K. (2007). Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA Infec. Genet. Evol. 7(1): 98-102 (Epub 2006 Jun. 19).

3.8

The Minimus Complex

Baimai, V. (1989). Speciation and species complexes of the Anopheles malaria vectors in Thailand. In : Proceedings of the 3rd Conference on Malaria Research, Thailand. 18 20 October 1989. pp. 146-62. Baimai, V. and Green, C.A. (1988). Cytogenetics of natural populations of the malaria vectors Anopheles dirus and An. maculatus complexes in Thailand. In: Proceedings of Mahidol University Seminar on Malaria Vaccine Development 17 - 18 May, 1988 (eds. A. Sabachavrn, S. Supavej, P. Attanath and M. Ho). pp. 45 - 51. Baimai, V., Kijchalao, U. and Rattanarithikul, R. (1996). Metaphase karyotypes of Anopheles of Thailand and Southeast Asia : The Myzomyia series sub-genus Cellia (Diptera: Culicidae). J. Am. Mosq. Contr. Assoc. 12: 97105. Chen, B., Harbach, R.E. and Butlin, R.K. (2002). Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review, distribution and malaria vector status. Med. Vet. Entomol. 16: 253-65. Garros,C., Koekemer, L.L., Coetzee, M. Coosemans, M. and Manguin, S. (2004a). A single multiplex assay to identify major malaria vectors within the African Anopheles funestus and Oriental An. minimus groups. Am. J. Trop. Med. Hyg. 70: 583-90. Garros,C., Koekemer, L.L., Kamau, L. Awola, T. S., Van Bortel, W., Coetzee, M., Coosemans, M. and Manguin, S. (2004b)Restriction fragment lengh polymorphism method for the identification of major African and Asian malaria vectors within the Anopheles funestus and An. minimus groups. Am. J. Trop. Med. Hyg. 70: 260-65 . Garros,C., Ron, M. P., Nguyen, T., Nguyen, H. S., and Manguin, S. (2005). First record of Anopheles minimus C and significant decrease of An. minimus A in central Vietnam. J. Am. Mosq. Contr. Assoc. 2: 139-43 Green, C.A., Gass, R.F., Munstermann, L.E. and Baimai, V. (1990). Population-genetic evidence for two species in Anopheles minimus in Thailand. Med. Vet. Entomol. 4: 25-34. Green, C.A., Rattanarithikul, R., Pongparit, S., Sawadwongporn, P. and Baimai, V. (1991). A newly-recognized vector of human malarial parasites in the oriental region, Anopheles (Cellia) pseudowillmori (Theobald, 1910). Trans. Roy. Soc. Trop. Med. Hyg. 85: 35-6. Harrison, B.A. (1980). Medical entomology studies —XIII. The Myzomyia Series of Anopheles (Cellia) in Thailand, with emphasis on intra-interspecific variations (Diptera: Culicidae). Contrib. Am. Entomol. Inst. (Ann Arbor) 17: 1-195.

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Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Janakara, B., Wajihullah, W.A., Dev, V., Curtis, C.F. and Sharma, V.P. (1995). Deltamethrinimpregnated bednets against Anopheles minimus transmitted malaria in Assam, India. J. Trop. Med. Hyg. 98: 73-83. Jambulingam, P., Sahu, S. S. and Manonmani, A. (2005). Reappearance of Anopheles minimus in Singhbhum hills of East-Central India. Acta Trop. 9: 31-35. Kanda, T., Ogawa, K., Sucharit, S., Pratchayanusorn, N., Lian, C.G. and Harinasuta, C. (1984). Cytogenetic and hybridization studies among 3 strains morphologically varieted and belonging to Anopheles minimus Theobald from Japan and Thailand. Cytologia 49: 865-81. Kengue, P., Trung, H.D., Baimai, V., Coosemans, M. and Manguin, S. (2001). A multiplex PCR-based method derived from random amplified polymorphic DNA (RAPD) markers for the identification of species of the Anopheles minimus group in Southeast Asia. Insect Mol.. Biol. 10: 427-35. Suthas, N., Sawasdiwongphorn, P., Chitprarop, U. and Cullen, J.R. (1986b). The behaviour of Anopheles minimus Theobald (Diptera: Culicidae) subjected to differing levels of DDT selection pressure in northern Thailand. Bull. Entomol. Res. 76: 303-12. Suthas, N., Sawasdiwongphorn, P., Chitprarop, U., Cullen, J.R., Gass, R.F. and Green, C.A. (1986a). A mark release recapture demonstration of host-preference heterogeneity in Anopheles minimus Theobald (Diptera: Culicidae) in a Thai village. Bull. Entomol. Res. 76: 313-20. Phuc, H.K., Ball, A.J., Son, L. Hanh, N.V., Tu, N.D., Lion, N.G., Veradi, A. and Townson, H. (2003). Multiplex PCR assay for malaria vector Anopheles minimus and four related species in the myzomyia series from southeast Asia. Med. Vet. Entomol. 17(4): 423-28. Prakash, A., Walton C., Bhattacharyya, D. R., O’Loughlin, Samantha, Mohapatra, P. K. and Mahanta, J. (2006). Molecular characterization and species identification of the Anopheles dirus and An. minimus complexes in northeast India using r-DNA ITS2. Acta Trop. 100: 15661. Rao, T.R. (1984). The Anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518. Sharpe, R.G., Hims, M.M., Harbach, R.E. and Butlin, R.K. (1999). PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplifcation and single-strand conformation polymorphism. Med. Vet. Entomol. 13: 265-73. Sharpe, R.G., Harbach, R.E. and Butlin, R.K. (2000). Molecular variation and phylogeny of members of the Minimus group of Anopheles subgenus Cellia (Diptera: Culicidae). Systematic Entomology 25: 263-72. Somboon, P., Walton, C., Sharpe, R.G., Higa, Y., Tuno, N., Tsuda, Y. and Takagi, M. (2001). Evidence for a new sibling species of Anopheles minimus from the Ryukyu Archipelago, Japan. J. Am. Mosq. Contr. Assoc., 17: 98-113. Somboon, P., Thongwat, D.,Choochote, W., Walton, C. and Takagi, M. (2005). Crossing experiments of Anopheles minimus species C and putative species E. J. Am. Mosq. Control Assoc., 21(1): 5-9. Sucharit, S. and Komalamisra, N. (1997). Differentiation of Anopheles minimus species complex by RAPD-PCR technique. J. Med. Assoc. Thai. 80(9): 598-602.

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Sucharit, S., Komalamisra, N., Leemingsawat, S., Apiwathnasorn, C. and Thongrungkiat, S. (1988). Population genetic studies on the Anopheles minimus complex in Thailand. Southeast Asian J. Trop. Med. Pub. Hlth. 19: 717-23. Thanaphum, S., Green, C.A., Baimai, V., Gass, R.F. and Gingrich, J.B. (1990). Genetic linkage relationships of eight enzyme/ electromorph loci in Anopheles minimus. Genetica 82: 63 - 72. Trung, H.D., Van Bortel, W., Sochanta, T., Keokenchanh, K,. Briet, O. J. and and Coosemans, M. (2005). Behavioural heterogeneity of Anopheles species in ecologically different localities in Southeast Asia: a challenge for vector control. Trop. Med. Int. Hlth. 10(3): 251-62. Van Bortel and Coosemans (2003). Suggesting new species? Comments on “Evidence for a new species of Anopheles minimus from the Ryukyu Archipelago Japan. J. Am. Mosq. Contr. Assoc. 19 (3): 261-4. Van Bortel, W., Harbach, R.E., Trung , H.D., Roelants, P., Backeljau, T. and Coosemans, M. (2001). Confirmation of Anopheles varuna in Vietnam, previously misidentified and mistargeted as the malaria vector An. minimus. Am. J. Trop. Med. Hyg., 65: 729-32. Van Bortel, W., Trung, H.D., Manh, N.D., Roelants, P., Verle, P. and Coosemans, M. (1999). Identification of two species within the Anopheles minimus complex in northern Vietnam and their behavioural divergences. Trop. Med. Internal. Hlth. 4: 257-65. Van Bortel, W., Trung , H.D., Roelants, P., Harbach, R.E., Backeljau, T. and Coosemans, M. (2000). Moecular identification of An. minimus s.l. beyond distinguishing the members of the species complex. Insect Mol. Biol. 9: 335-40. Van Bortel, W., Trung, H.D., Roelants, P., Backetjall, T. and Coosemans, M. (2003). Population genetic structure of the malaria vector Anopheles minimus A in Vietnam. Heredity 91(5): 487-93. Van Bortel, W., Trung, H.D., Sochanta, T., Keokenchanh, K., Roelants, P., Backetjall, T. and Coosemans, M. (2004). Ecoethological heterogeneity of the members of the Minimus Complex (Diptera:Culicidae) in Southeast Asia and its consequences for vector control. J. Med. Entomol. 41(3): 366-74. Wajihullah, Jana, B. and Sharma, V.P. (1992). Anopheles minimus in Assam. Curr. Sci. 63: 7-9. Yuan, Y. (1987). Studies on the two forms of Anopheles (Cellia) minimus Theobald, 1901 in China (Diptera: Culicidae). Mosq. Syst. 19: 143-5. Zhou, S.S., Tang, L.H., Gu, Z.C. and Wang, Y. (2002). Sequence difference of ribosomal DNA second internal trans spacer in Anopheles minimus in different localities. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. (Article in Chinese). 20(1): 29-31.

3.9

The Philippinensis-Nivipes Complex

Alam, M. T., Das, M. K., Dev, V., Ansari, M. A. and Sharma, Y. D. (2007). PCR-RFLP method for the identification of four members of the Anopheles annularis group of mosquitoes (Diptera: Culicidae). Trans. R. Soc. Trop. Med. Hyg.101(3): 239-44 (Epub 2006 Jun.). Baimai, V., Green, C.A., Andre, R.G., Harrison, B.A. and Peyton, E.L. (1984). Cytogenetic studies of some species complexes of Anopheles in Thailand and Southeast Asia. Southeast Asian J. Trop. Med. Pub. Hlth. 15: 536-46.

Anopheline Species Complexes in South and South-East Asia

Elias, M., Rahman, M., Ali, M., Begam, J. and Chowdary, A.R. (1987). The ecology of malaria carrying mosquitoe Anopheles philippinensis Ludlow and its relation to malaria in Bangladesh. BMRC Bull. 13(1): 192-201 Green, C.A., Harrison, B.A., Klein, T. and Baimai, V. (1985). Cladistic analysis of polytene chromosome rearrangements in anopheline mosquitoes of subgenus Cellia, series Neocellia. Can. J. Genet. Cytol. 27: 123-33. Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae): a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Klein, T.A., Harrison, B.A., Inlao, I. and Boonyakanist, P. (1982). Colonization of Thailand strains of Anopheles nivipes and Anopheles philippinensis. Mosq. News 42: 374-80. Klein, T.A., Harrison, B.A., Baimai, V. and Phunkitchar, V. (1984). Hybridization evidence supporting separate species status for Anopheles nivipes and Anopheles philippinensis. Mosq. News 44: 466-70. Krishnan, K.S. (1961). Vectors of malaria - An. philippinensis Ludlow, 1902. In: Vectors of malaria in India. National Society of India for Malaria and Mosquito Diseases, Delhi: 27-37. Nagpal, B.N. and Sharma, V.P. (1987). Survey of mosquito fauna of northeastern region of India. Indian J. Malariol. 24: 143-9. Prakash, Anil., Bhattacharya, D.R., Mohopatra, P.K. and Mahanta, J. (2000). Mosquito fauna and malaria vectors in Jirampur, district Changlong, Arunachal Pradesh. Indian J. Malariol. 37: 74-81. Praksash, Anil, Bhattacharya, D. R. Mohapatra,, P. K. and Mohanta, J. (2004). Taxonomical observations on Anopheles philippinensis/nivipes group of mosquitoes in north –east India. J. Commun. Dis. 36: 264-270. Praksash, Anil, Bhattacharya, D. R. Mohapatra,, P. K. and Mohanta, J. (2005). Potential of Anopheles philippinensisnivipes complex mosquitoes as malaria vector in north – east India. J. Environ. Biol. 26: 719-724. Prakash, Anil, Walton, C. Bhattacharyya, D. R., Mohapatra, P. K., Mahanta, J. (2006). Characterization of ITS2 rDNA of Anopheles Philippinensis and An. nivipes (Diptera:Culicidae) from north-east India. South-East Asian J. Trop. Med. Hyg. 37: 1134-1138. Rajgopal, R. (1976). Studies on persistant transmission of malaria in Burnihat, Meghalaya. J. Commun. Dis. 8: 235-45. Rao, T.R. (1984). The Anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518. Reid, J.A. (1967). Two forms of Anopheles philippinensis in Malaya. J. Med. Entomol. 4: 175-9.

annularis group in southern Asia. Med. Vet. Entomol. 21: 30-35

3.10

The Punctulatus Complex

Beebe, N.W., Foley, H.D., Saul, A., Cooper, L., Bryan, J.H. and Burkot, T.R. (1994). DNA probes for identifying the members of the Anopheles punctulatus complex in Papua New Guinea. Am. J. Trop. Med. Hyg. 508: 229-34. Beebe, N.W. and Saul, A. (1995). Discrimination of all members of the An. punctulatus complex by Polymerase chain reaction-restriction fragment length polymorphism analysis. Am. J. Trop. Med. Hyg. 53 (5): 478-81. Beebe, N.W., Foley, H.D., Cooper, R.D., Bryan, J.H. and Saul, A. (1996). DNA probes for the An. punctulatus complex. Am. J. Trop. Med. Hyg. 54: 395-8. Beebe, N.W., Ellis, J.T., Cooper, R.D. and Saul, A. (1999). DNA sequence analysis of the ribosomal DNA ITS-2 region for the Anopheles punctulatus group of mosquitoes. Insect. Mol. Biol. 82: 381-90. Booth, D.R., Mahon, R.J. and Sriprakash, K.S. (1991). DNA probes to identify members of the Anopheles farauti complex. Med. Vet. Entomol. 5: 447-54. Bryan, J.H. (1970). A new species of Anopheles punctulatus complex. Trans. Roy. Soc. Trop. Med. Hyg. 64: 28. Bryan, J.H. (1973a). Studies on the Anopheles punctulatus complex. I. Identification by proboscis morphological criteria and by cross-mating experiments. Trans. Roy. Soc. Trop. Med. Hyg. 67: 64-9. Bryan, J.H. (1973b). Studies on the Anopheles punctulatus complex. II. Hybridization of the member species. Trans. Roy. Soc. Trop. Med. Hyg. 67: 70-84. Bryan, J.H. and Coluzzi, M. (1971). Cytogenetic observations on Anopheles farauti Laveran. Bull. Wld Hlth Org. 45: 266-7. Bryan, J.H., Reardon, T. and Spark, R. (1990). How many species are in the Anopheles punctulatus group? Ann. Trop. Med. Parasitol. 84: 295-7. Cooper, L., Cooper, R.D. and Burkot, T.R. (1991). Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems. Rimental Parastiol. 73: 27-35. Cooper, R.D., Waterson, D.G., Bangs, M.J. Beebe, N.W. (2000). Rediscovery of Anopheles (Cellia) clowi (Diptera: Culicidae) is rarely recorded members of the Anopheles punctulatus group. J. Med. Entomol. 37: 840-48.

———. (1968). Anopheline mosquitoes of Malaya and Borneo. Stud. Inst. Med. Res. Malaya 31.

__________, Frances, S.P., Beebe, N.W., Sweeney, A.W. (2002). Speciation and distributions of the members of the Anopheles punctulatus (Diptera: Culicidae) group in Pupae Guinea. J. Med. Entomol. 39: 16-27.

Subbarao, S.K., Vasantha, K. and Sharma, V.P. (1988). Cytotaxonomy of certain malaria vectors of India. In : Biosystematics of haematophagous insects. (ed. M.W. Service). pp. 25-37. Clarendon Press, Oxford.

Foley, D.H. and Bryan, J.H. (1993). Electrophoretic keys to identify members of the Anopheles punctulatus complex of vector mosquitoes in Papua New Guinea. Med. Vet. Entomol. 7: 49-53.

Subbarao, S.K., Kumar, Vasantha, K., Nanda, Nutan, Nagpal, B.N., Dev Vas, and Sharma, V.P. (2000). Cytotaxonomic evidence for the presence of Anopheles nivipes in India. J. Mosq. Am. Contr. Assoc. 16(2): 71-4.

Foley, D.H. and Bryan, J.H. (2000). Showed salinity tolerance invalities a test for the malaria vector Anopheles faruati s.s. on Guadal canal, Sukmore Island. Med. Veter. Entomol. 14: 450-2.

Walton, C., Somboon, P. Harbach, R. E., Zhang, S., Weerasinghe, I., O’ Loughlin, S. M. Phornpida, S. Sochantha, T., TunLin, W., Chen, B. and Butlin, R. k. (2007). Molecular identification of mosquito species in the Anopheles

Foley, D.H., Cooper, R.D. and Bryan, J.H. (1995). Allozyme analysis reveals a new species within the Anopheles punctulatus complex in Western Province, Papua New Guinea. J. Am. Mosq. Contrl. Assoc. 11: 122-7.

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Foley, D.H., Meek, S.R. and Bryan, J.H. (1994). The Anopheles punctulatus group of mosquitoes in the Solomon islands and Vanuatu surveyed by allozyme electrophoresis. Med. Vet. Entomol. 8: 340-50. Foley, D.H., Paru, R., Dagoro, H. and Bryan, J.H. (1993). Allozyme analysis reveals six species within the Anopheles punctulatus complex of mosquitoes in Papua New Guinea. Med. Vet. Entomol. 7: 37-48. Hartas, J., Whelan, P., Sriprakash, K.S. and Booth, D. (1992). Oligonucleotide probes to identify three sibling species of the Anopheles farauti Laveran complex (Diptera: Culicidae). Trans. Roy. Soc. Trop. Med. Hyg. 86: 210-2. Kondrashin, A.V. and Rashid, K.M. (1987). Epidemiological considerations for planning malaria control in South-East Asia Region. World Health Organization, Regional Office for South-East Asia, New Delhi, p. 411. Mahon, R.J. and Miethke, P.M. (1982). Anopheles farauti No. 3, a hitherto unrecognized biological species of mosquito within the taxon An. farauti Laveran (Diptera: Culicidae). Trans. Roy. Soc. Trop. Med. Hyg. 76: 8-12. Rozeboom and Knight (1946). The punctulatus complex of Anopheles (Diptera: Culicidae). J. Parasitol. 32: 95-131. Schmidt, E.E., Foley, D.H., Hartel, G.F., Williams, G.M. and Bryan, J.H. (2001). Descriptions of the Anopheles (Cellia) farauti complex of sibling species (Diptera : Culicidae) in Australia. Bull. Entomol. Res. 91: 389-410. Sweeney, A.W. (1987). Larval salinity tolerances of the sibling species of Anopheles farauti. J. Am. Mosq. Contr. Assoc. 3: 589-92.

3.11

The Sinensis Complex

Kanda, Tozo, Takai, K., Oguma, Y., Chiang, G.L., Cheong, W.H., Sucharit, Joesoef, A.M. and Imajo, S. (1981). Evolutionary genetics of the Anopheles hyrencus group, the Leucosphyrus group and the Pyretophorus group in East Asia and the Pacific area. In : Cytogenetics and genetics of vectors (eds. R. Pal, J.B. Kitzmiller and T. Kanda). Koansta Ltd., Tokyo. Li, B.W., H.L. Lu, K.T. Yao, and D. Liu. 1991. Restriction fragment length differences of genomic repetitive DNA from five sibling species of the Anopheles hyrcanus group. Chinese J.Parasitol. Para. Dis. 9: 8-11. Lee, H.I., Lee, J.S., Shin, E.H., Lee, W.J., Kim, Y.Y. and Lee, K.R. (2001). Malaria transmission potential by Anopheles sinensis in the Reublic of Korea. Korean J. Paraistol. 39 (2): 185-92 (from abstract). Ma, S.F. 1981. Studies on the Anopheles (A.) sinensis group of mosquitoes in China, including four new sibling species, [in Chinese] Sinozoologia 1: 59-74. Ma, Y. J., Qu, F. Y., and Xu, J. J. (1998). Sequence differences of rDNA-ITS-2 and species diagnostic PCR assay of Anopheles sinensis and Anopheles anthropophagus from China. J. Med. College PLA 13: 123-8. Ma, Y. J., Qu, F. Y., Xu, J. J. and Song, G. H. (2000). Differences in sequences of ribosomal DNA second internal transcribed spacer among three members of Anopheles hyrcanus complex from the Republic of Korea. Entomol. Sinica. 7: 36-40. Ma, Y. Xu, J. (2005). The Hyrcanus Group of Anopheles (Anopheles) in China (Dipter: Culidae): Species Discrimination and Phylogenetic Relationships Inferred by Ribosomal Internal Transcribed Spacer 2 Sequences. J. Med. Entomol. 42 (4): 610-9.

Baimai, V., Rattanarithikul, R. and Kijchalao, U. (1993). Metaphase karyotypes of Anopheles of Thailand and southeast Asia : 1. The Hyreanus group. J. Am. Mosq. Contr. Assoc. 9(1): 59-67.

Min, G.S., Choochote, W., Jitpakdi, A., Kim, S.J., Kim, W., Jung, J. and Junkum, A. (2002). Intraspecific hybridization of Anopheles sinensis (Diptera: Culicidae) strains from Thailand and Korea. Mol. Cells. 14(2): 198-204.

Chow, Y. (1970). Biomics of malaria vectors in the western pacific region. Southeast Asian. J. Trop. Med. Publ. Helt., 1: 4057.

O’Connor, C.T. (1980). The Anopheles lyrcanus group in Indonesia. Mosq. Syst. 12: 293-305.

Choochote, W., Jitpakdi, A., Rongsriyam, Y., Komalamisra, N., Pitasawat, B. and Palakul, K. (1998). Isoenzyme study and Hybridization of two strains of Anopheles sinensis (Diptera: Culicidae) in northern Thailand. Southeast Asian J. Trop. Med. Publ. Hlth. 29(4): 841-8. Coleman, R.E., Kiattibut, C., Sattabongkot, J., Ryan, J., Burbett, D.A., Kim. H.C., Lee, W.J. and Klein, T.A. (2002). Evaluation of anopheline mosquitoes (Diptera: Culicidae) from the Republic of Korea for Plasmodium vivax Circumsporozoite protein. J. Med. Entomol., 39(1): 2447. Gao, Q., Beebe, N.W., and Cooper, R.D. (2004). Molecular identification of the malaria vectors Anophelesl anthropophagus and Anopheles sinensis (Diptera: Culicidae) in central China using polymerase chain reaction and appraisal of their position within the Hyranus group. J. Med. Entomol. 41(1): 5-10 Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Kanda, T. and Oguma, Y. (1977). Hybridization between Anopheles sinensis and Anopheles sineroides. Mosq. News. 37(1): 115-7. Kanda, T. and Oguma, Y. (1978). Anopheles engarensis, a new species related to sinensis from Hokkido Island, Japan. Mosq. Syst. 10: 45-52.

100

Oguma, Y. (1978). Crossing studies among six strains of Anopheles sinensis. Mosq. News. 38(3): 357-66. Oguma, Y. and kanda, T. (1970). The salivary gland chromosomes of Anopheles koreicus. Jap. J. Sanit. Zool. 21: 114. Oguma, Y. and kanda, T. (1976). Laboratory colonization of Anopheles sinensis Weidman 1828. Jap. J. Sanit. Zool. 27: 319-24. Ree, H.I., Hwang, U.W., Lee, I.Y. and Kim, T.E. (2001). Daily survival and human blood index of Anopheles sinenesis the vector species of malaria in Korea. J. Am Mosq. Contr. Assoc. 1791): 67-72. Rongsriyam, Y., Jitpakdi, A., Choochote, W., Samboon, R., Tookyang, B. and Suwonkerd, W. (1998). Comparative susceptibility of two forms of Anopheles sinensis Wiedmann 1828 (Diptera: Culicidae) to infection with Plasmodium falciparum, P. vivax, P. yoelii and determination of misleading factor for sporozoite identification. Southeast Asian J. Trop. Med. Publ. Hlth. 29: 159-67. Sleigh, A. C., X.L. Liu, S. Jackson, P. Li, and L.Y. Shang. 1998. Resurgence of vivax malaria in Henan Province, China. Bull. W.H.O. 76: 265-70. Stricman, D., Miller, M.E., Kim, H.C. and Lee, K.W. (2000). Mosquito surveillance in the demilitarized zone, Republic of Korea during an outbreak of P. vivax of malaria. J. Am. Mosq. Contr. Assoc. 16: 100-13.

Anopheline Species Complexes in South and South-East Asia

Wilkerson, R., C., Li, C., Rueda, L. M., kim, H., Klien, T. A., Song, G. and Stricman, D. (2003). Molecular confirmation of Anopheles (Anopheles) lesteri from the Republic of South Korea and its genetic identity with An. (Ano.) anthropophagus from China (Diptera: Cculicidae). Zootaxa 378: 1-14.

3.12

The Subpictus Complex

Abhayawardana, T.A., Wijesuriya, S.R.E. and Dilrukshi, R.K.C. (1996). Anopheles subpictus complex: Distribution of sibling species in Sri Lanka. Indian J. Malariol. 33: 53-60. Amerasinghe, F.P., Amerasinghe, P.H., Peiris, J.S.M. and Wirtz, R.A. (1991). Anopheline ecology and malaria infection during the irrigation development of an area of the Mahaweli project, Sri Lanka. Am. J. Trop. Med. Hyg. 45: 226-35. Amerasinghe, P.H., Amerasinghe, F.P., Wirtz, R.A., Indrajith, N.G., Somapala, W., Pereira, L.R. and Rathnayake, A.M.S. (1992). Malaria transmission by Anopheles subpictus (Diptera: Culicidae) in a new irrigation project in Sri Lanka. J. Med. Entomol. 29: 577-81. Atrie, B. (1994). Cytogenetic studies of different field populations of Anopheles annularis Van Der Wulp and Anopheles subpictus Grassi. Ph.D. thesis, Dept. of Zoology Dept. University of Delhi, Delhi, India. Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Kelley-Hope, L. A., Yapabandara, A. M. G. M., Wickramasinghe, M. B., Perera, Karunaratne, S. H. P. P., Fernando, W. P., Abeyasinghe, R. R., Herath, P. R. j., Galappaththy, G. N. L. and Hemingway, J. (2005). Spatiotemporal distribution of insecticide resistance in Anopheles culicifacies and Anopheles subpictus in Sri Lanka. Trans. Roy. Soc. Trop. Med. Hyg. 99: 751-61. Kulkarni, S.M. (1983). Detection of sporozoites in Anopheles subpictus in Bastar District, Madhya Pradesh. Indian J. Malariol. 20: 159-60. Nanda, N., Das, C.N.S., Subbarao, S.K., Adak, T. and Sharma, V.P. (1987). Studies on the development of Plasmodium vivax in Anopheles subpictus. Indian J. Malariol, 24: 13542. Panicker, K.N., Geetha Bai, M., Bheema Rao, U.S., Viswam K. and Suryanarayanamurthy, U. (1981). Anopheles subpictus vector of malaria in coastal villages of South-East India. Curr. Sci. 50: 694-5. Rao, T.R. (1984). The Anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518. Reid, J.A. (1966). A note on Anopheles subpictus Grassi and An. indefinitus Ludlow (Diptera: Culicidae). J. Med. Entomol. 3: 327-31. Reuben, R. and Suguna, S.G. (1983). Morphological differences between sibling species of the taxon Anopheles subpictus Grassi in India, with notes on relationships with known forms. Mosq. syst. 15: 117-26. Singh, S.P., Raghavendra, K., Nanda, N. and Subbarao, S.K. (2004). Morphotaxonomic studies to identify the members of the Anopheles subpictus Grassi (Diptera: Culicidae) species complex in riverine villages of district Sonepat, Haryana state, India. J. Commun. Dis. 36: 35-40. Subbarao, S.K., Vasantha, K. and Sharma, V.P. (1988). Cytotaxonomy of certain malaria vectors of India, pp. 2537. In : Biosystematics of haematophagous insects M.W. Service (ed.)., Clarendon Press, Oxford.

Suguna, S.G. (1982). Cytological and morphological evidence for sibling species in Anopheles subpictus Grassi. J. Commun. Dis. 14: 1-8. Suguna, S.G., Gopala Rathinam, K., Rajavel, A.R. and Dhanda, V. (1994). Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex. Med. Vet. Entomol. 8: 88-94. Sundararaman, S., Soeroto, R.M. and Siran, M. (1957). Vectors of malaria in mid-Java. Indian J. Malariol. 11: 321-38. Van Hell, J.C. (1952). The Anopheline fauna and malaria vectors in South Celebes. Documenta Med. Geogr. Trop. 4: 45-6.

3.13

The Sundaicus Complex

Alam MT, Das MK, Ansari MA, Sharma YD (2005). Molecular identification of new sibling species of human malaria vector Anopheles sundaicus from Andaman and Nicobar islands of India. Acta Trop 1738: 1–9. Barcus, M.J., Laihad, F., Sururi, M., Sismadi, P., Marwoto, H., Bangs, M.J. and Baird, J.K. (2002). Epidemic malaria in the Menoreh hills of Central Java. Am. J. Trop. Med. Hyg., 66(3): 287-92. Collins, R. T., Jung, R. K., Anoez, H., Sutrisno, R. H. and Putut, D. (1979). A study of the coastal malaria vectors, Anopheles sundaicus (Rodenwaldt) and Anopheles subpictus Grassi in south Sulawesi, Indonesia. Geneva: World Health Organization. WHO/Mal. 79. Das, M.K., Nagpal, B.N. and Sharma, V.P. (1998). Mosquito fauna and breeding habitats of anophelines in Car Nicobar islands. Ind. J. Malariol. 35: 197-205. Dash, A. P., Hazra, R. K., Mahapatra, N., Tripathy, H. K. (2000). Disappearance of malaria vector Anopheles sundaicus from Chilka lake area of Orissa state in India. Med. Vet. Entomol. 14: 445-9. Dusfour, I., Harbach, R. E. and Manguin, S. (2004). Bionomics and systematics of the Oriental Anopheles sundaicus complex in relation to malaria transmission and vector control. Am. J. Trop. Med. Hyg. 71(4): 518-24. Dusfour, I., Linton, Y. M., Cohuet, A., Harbach, R. E., Baimai, V. Trung, H. D., Seng, C. M., Matusop, A. and Manguin, S. (2004). Molecular Evidence of Speciation Between Island and Continental Populations of Anopheles (Cellia) sundaicus (Dipter: Culicidae), a Principal Malaria Vector Taxon in Southeast Asia. J. Med. Entomol. 41(3): 28795.Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 53753. Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Kalra, N. L. (1978). National Malaria Eradication Programme, India – its problems, management and research needs. J. commun. Dis. 10: 1-20. Kirnowardoyo, S. (1988). Anopheles malaria vector and control measures applied in Indonesia. Southeast Asian J. Trop. Med. Pub. Hlth. 19: 713-6. Kirnowardoyo, S. and Gambiro, P.Y. (1987). Entomological investigations of an outbreak of malaria in Cilacap on south coast of Central Java, Indonesia during 1985. J. Commun. Dis. 19: 121-7. Kumari, R. and Sharma, V.P. (1994). Resting and biting habits of Anopheles sundaicus in Car Nicobar island. Indian J. Malariol. 31: 103-14.

Anopheline Species Complexes in South and South-East Asia

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Kumari, R., Joshi, Hema, Giri, A. and Sharma, V.P. (1993). Feeding preferences of Anopheles sundaicus in Car Nicobar Island. Indian J. Malariol. 30: 201-6. Linton, Y. M., Harbach, R.E., Chang, M. S., Anthony, T. G. and Matusop, A. (2001). Morphological and molecular identity of Anopheles (Cellia) sundaicus (Diptera: Culicidae), the nominotypical member of a malaria vector species complex in Southeast Asia. Syst. Entomol. 26: 357-66. Linton,Y. M., Dusfour, I., Howard, T. M., Ruiz, F., Minh, N. D., Trung, H. D., Sochanta, T., Coosemans, M. and Harbach, R. E. (2005). Anopheles (Cellia) epiroticus (Diptera: Culicidae), a new malaria vector species in the Sundaicus Complex. Bull. Entomol. Res. 95: 329-39 Nanda, N., Das, M.K., Wattal, S., Adak, T. and Subbarao, S.K. (2004). Cytogenetic characterization of Anopheles sundaicus (Diptera: Culicidae) population from Car Nicobar island, India. Ann. Entomol. Soc. Am. 97(1): 171-6. Rao, T.R. (1984). The Anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518. Reid, J.A. (1968). Anopheline mosquitoes of Malaya and Borneo. Stud. Inst. Med. Res. Malaya. 31 : 1-502. Sharma, S.K., Adak, T., Haq, S. and Kar, I. 1999. Observations on relationshops of salinity with the breeding habitats of Anopheles sundaicus (Diptera: Culicidae) at Car Nicobar (Andaman & Nicobar islands), India. Mosq. Borne Dis. Bull. 16: 33-6. Singh, N., Nagpal, B.N. and Sharma, V.P. (1985). Mosquitoes of Kutch, Gujarat. Indian J. Malariol. 22: 17-20. Sukowati, S. and Baimai, V. (1996). A standard cytogenetic map for Anopheles sundaicus (Diptera: Culicidae) and evidence for chromosomal differentiation in populations from Thailand and Indonesia. Genome 39: 165-73. Sukowati, S. and Bairmai, V., Haran, S., Dusuki, Andri, H. and Efriwati (1999). Isoenzyme evidence for three sibling species in the Anopheles sundaicus complex from Indonesia. Med. Vert. Entomol. 13: 408-14. Sundararaman, S., Soeroto, R.M. and Siran, M. (1957). Vectors of malaria in Mid Java. Indian J. Malariol. 11: 321-38.

3.14

The Anopheles stephensi variants

Aslamkhan, M. (1973). Sex chromosomes and sex determination in the malaria mosquito, Anopheles stephensi. Pak. J. Zool. 5: 127-30. Coluzzi, M., Di Deco, M. and Cancrini, G. (1973a). Chromosomal inversions in Anopheles stephensi. Parassitologia. 15: 129-36. Coluzzi, M., Di Deco, M. and Cancrini, G. (1973b). Ulteriori osservazioni sulla lunghezza dell’uovo in Anopheles stephensi in relazione al polimorfismo cromosomico. Parasitologia, 15: 213-5. Curtis, J., Curtis, C.F. and Barton, N.H. (1985). Methodology for testing the hypothesis of single locus control of host resistance. In : Genetic Control of Host Resistance to Infection and Malignancy (ed. by E. Skamene), Alan R. Liss, New York pp. 65-70. Harbach, R. E., (2004). The classification of the genus Anopheles (Diptera : Culicidae) : a working hypothesis of phylogenetic relationships. Bull. Entomol. Res., 94: 537-53. Mahmood, F. and Sakai, R.K. (1984). Inversion polymorphisms in natural populations of Anopheles stephensi. Can. J. Genet. Cytol. 26: 538-46.

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———. (1985). An ovarian chromosome map of Anopheles stephensi. Cytobios 43: 79-86. Nagpal, B.N., Srivatsav, A., Kalra, N.L. and Subbarao, S.K. (2003). Spiracular indices in Anophelesl stephensi : a taxonomical tool to identify ecological variants. J. Med. Entomol. 40(6): 747-9. Puri, I.M. (1949). Anophelines of the oriental region. In : Malariology (ed. M.F. Boyd), W.B. Saunders, Philadelphia. pp. 483-505 Rao, T.R. (1984). The Anophelines of India. Malaria Research Centre (ICMR), Delhi. p 518. Rao, B.A., Sweet, W.C. and Subba Rao, A.M. (1938). Ova measurements of An. stephensi type and An. stephensi var. mysorensis. J. Malar. Inst. India. 1: 261-6. Rishikesh, N. (1959). Chromosome behaviour during spermatogenesis in Anopheles stephensi. Cytologia 24: 447-58. Rowland, M., Mohammed, N., Rehman, H., Howitt, S., Mendis, C., Ahmed Kamal, M. and Wirtz, R. (2002). Anopheline vectors and malaria transmission in eastern Afghanistan. Trans. Roy. Soc. Trop. Med. Hyg., 96(6): 620-6. Rutledge, L.C., Ward, R.A. and Bickley, W.E. (1970). Experimental hybridization of geographic strains of Anopheles stephensi (Diptera: Culicidae). Ann. Entomol. Soc. Am. 63: 1024-30. Senior-White, R. (1940). Anopheles stephensi in Calcutta. J. Mal. Inst. India. 3: 349-61. Sharma, S.N., Subbarao, S.K., Choudhury, D.S. and Pandey, K.C. (1993). Role of An. culicifacies and An. stephensi in malaria transmission in urban Delhi. Indian J. Malariol. 30: 155-68. Stone, A.K., Knight, L. and Starcke, H. (1959). A synoptic Catalog of the Mosquitoes of the World (Diptera, Culicidae). Thomas Say Foundation, Entomological Society of America, Washington. Subbarao, S.K. (1996). Genetics of malaria vectors. Proc. Natl. Acad. Sci. India. 66 (B), SPL issue: 51-76. Subbarao, S.K., Vasantha, K., Adak, T., Sharma, V.P. and Curtis, C.F. (1987). Egg-float ridge number in Anopheles stephensi: ecological variation and genetic analysis. Med. Vet. Entomol. 1: 265-71. Suguna, S.G. (1981). Inversion (2)R1 in Anopheles stephensi, its distribution and relation to egg size. Indian J. Med. Res. 73 (Supplement): 124-28. ———. (1992). Y-chromosome dimorphism in the malaria vector Anopheles stephensi from south India. Med. Vet. Entomol. 6: 84-6. Sweet, W.C. and Rao, B.A. (1937). Races of An. stephensi Liston, 1901. Indian Med. Gaz. 72: 665-74. Sweet, W.C., Rao, B.A. and Subba Rao, A.M. (1938). Crossbreeding of An. stephensi type An. stephensi J. Mal. Inst. India. 1: 149-54. Vinogradaskaya, O.N. (1969). Geographic distribution of mosquitoes – vectors of infections (on the basis of their xerophilly and hygrophilly). Izadatelstva Medishina, Moscow var. mysorensis. Vasanthi, V. (1996). Field and laboratory studies on selected ecological and behavioural aspects of variants of Anopheles stephensi Liston from south India. Ph.D thesis, University of Madras, India. Veradi, A., Donnelly, M.J., Rowland, M. and Townson, H. (2002). Isolation and characterization of microsatellite loci in the mosquito Anopheles stephensi Liston (Dipera: Culicidae). Molecular Ecology Notes 2(4): 488.

Anopheline Species Complexes in South and South-East Asia

Vector-borne diseases are a major health problem in the South-East Asia Region and in other parts of the world. There are about 4 500 mosquito species in existence; species belonging to the Anopheles genus transmit malaria. Combating malaria is part of the Millenium Development Goals, and vector control is a key strategy both regionally and globally. Therefore, the review and dissemination of information on vector species is critically important. Most of the anophelines that are involved in the transmission of malaria in South and South-East Asia have been identified as species complexes. Members of a species complex are reproductively isolated evolutionary units with distinct gene pools and, hence, they differ in their biological characteristics. In 1998, WHO published Anopheline species complexes in South-East Asia. New identification tools have been developed since then, and therefore this updated edition is being published. It summarizes work that has been done on anopheline cryptic species and will be highly valuable to researchers, field entomologists and malariacontrol programme managers.

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