Anopheline Species Complexes in South and South-East Asia WHO Library Cataloguing-In-Publication Data World Health Organization, Regional Office for South-East Asia

SEARO Technical Publication No. 57

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 malaria- control programme managers. AAnnoopphheelliinnee SSppeecciieess CCoommpplleexxeess iinn SSoouutthh aanndd SSoouutthh--EEaasstt AAssiiaa

ISBN 978-92-9022-294-1

9 7 8 9 2 9 0 2 2 2 9 4 1 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

ii 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 iii

Foreword

ector-borne diseases continue to be a Differences in the biological characteristics of major health problem in the world. The members of the complexes have an important Vworsening malaria situation during the bearing on malaria transmission dynamics. It 1980s led the World Health Organization is, therefore, imperative to determine sibling (WHO) to declare the control of malaria as a species composition and their bionomics as global priority. The World Declaration on well as their roles in the transmission of Malaria, adopted in Amsterdam in October malaria. 1992, committed WHO Member States to the In 1998, WHO published as a technical worldwide intensification of control efforts publication* Anopheline species complexes in against this disease. Accordingly, a global South-East Asia authored by Dr Sarala K. Malaria Control Strategy was developed which Subbarao. This book has received much laid emphasis on the following key elements: appreciation both from researchers and case management; capacity building for programme managers. Since its publication, control; containment of epidemics; and basic several papers on species complexes and applied research. Halting the incidence identification tools, especially molecular-based of malaria is also highlighted as one of the tools, formal designation of members of targets to be achieved under the United complexes, and the phylogenetic relationship Nations Millennium Development Goals between members of a complex and also (MDGs). between the complexes have been published. It is very important that vector control, as a In view of the importance of species complexes part of the global as well as the regional malaria in malaria control operations, an updated control strategy, should succeed. Its success edition has been prepared to provide the latest would depend on a systematic review of the information on this important subject. This is available information on vector species and part of our commitment to highlight and their biology, and of the vector control options disseminate the knowledge on species and their selective use. Most of the complexes which is so vital to malaria control anophelines that are involved in the strategy, especially when target-specific transmission of malaria in the South and South- selective and sustainable vector control is East Asian countries have been identified as urgently needed. In addition to the South-East species complexes. Species complexes are of Asia Region, the present edition covers the common occurrence among anopheline taxa. work done on the species complexes prevalent More than 30 Anopheles taxa have been in the South Asian countries as well. It presents identified so far as species complexes and they a clear summary of the work done on are important vectors of malaria in different anopheline cryptic species, and I am sure it parts of the world. Members of a species will be very useful for field malaria complex, commonly known as sibling species, entomologists, malaria control programme are reproductively-isolated evolutionary units managers and basic researchers working on with distinct gene pools and, hence, differ in species complexes. their biological characteristics.

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.

vi Anopheline Species Complexes in South and South-East Asia 1. Introduction

osquitoes are ubiquitous and have East Asian countries have been identified as a tremendous reproductive species complexes, which include Annularis, Mpotential and great adaptability to Barbirostris, Culicifacies, Dirus, Fluviatilis, different ecological conditions. Adding to Leucosphyrus, Macaulatus, Minimus, their innate ability to adapt, humans are Philippinenisis-niyipes, Punctualatus, Sinensis, providing them with conditions, which are Subpictus and Sundaicus. highly congenial for their multiplication. Maculipennis was the first complex There are about 4500 mosquito species in described in the genus Anopheles. The different parts of the world, belonging to 34 discovery of this complex resolved the genera in the family Culicidae, order Diptera, epidemiological paradox that prevailed in the class Insecta and phylum Arthropoda. 1930s when there was malaria transmission Some of the mosquito species transmit in Europe and North America. In some areas diseases and consequently form an important in southern Europe, there was no malaria in target for control in public health spite of the presence of An. maculipennis, programmes. Species belonging to the genus which led to the expression “anophelism Anopheles transmit malaria. Approximately without malaria”. Detailed studies on the 424 formally designated anophelines have biological and cytogenetic characters of these been identified morphologically, out of which populations have now identified eight sibling only about 70 species are considered to be species in this taxon in Europe. Recognizing the main vectors of malaria in the world. The the significance of species complexes in total number of species has now reached malaria epidemiology, Mayr (1970) goes on more than 500 because of the identification to state that, “Perhaps the most celebrated of biological species within morphologically case of sibling species is that of the malaria indistinguishable taxa. mosquito complex in Europe,” referring to the Maculipennis Complex. So far, about 30 Among many Diptera genera, such as complexes have been described in different Drosophila, Simulium, Anopheles, Aedes, regions of the world. The number of sibling Sciara and Chironomus, the populations within species varies in each complex and a total of a morphologically defined species do not about 145 species have been identified interbreed. These morphologically-similar, among these complexes (Table 1). reproductively-isolated species within a taxon are known as cryptic, sibling or isomorphic Among the members of the Maculipennis species, and the taxon as a whole as a species Complex in Europe, An. atroparvus, An. complex. Sibling species are found in other labranchiae, An. messeae, An. sacharovi and animal groups also. Mayr (1970) gives a An. subalpinus were vectors because they detailed account of the groups where sibling would at least sometimes bite humans, while species have been found. Most of the An. maculipennis sensu stricto, An. melanoon anophelines that are implicated in the and An. beklemishevi were non-vectors transmission of malaria in the South and South- because they were generally entirely

Anopheline Species Complexes in South and South-East Asia 1 zoophilic. Some of these species breed in (Besansky et al., 1994, 1997; Garcia et al., fresh water and others in brackish water. In 1996) have suggested the possibility of gene the Gambiae Complex in Africa, while five flow occurring between members of the sibling species were recognized as vectors Gambiae Complex. Clear evidence for with varying levels of efficiency in transmitting unidirectional introgression leading to gene malaria, An. quadriannulatus was found to be flow from An. arabiensis to An. gambiae a non-vector (Coluzzi, 1988). Later, within came from a multilocus molecular marker An. quadriannulatus, two zoophagic sibling study (Besansky et al., 2003) and a novel species, A and B, were more recently population genetic analysis (Donelly et al. recognized (Hunt, Coetzee and Fetenne, 2004). It is intriguing that gene flow is not 1998). In India, of the five An. culicifacies uniform throughout the genome, i.e. sibling species, species A, C, D and E are genomes are mosaic with respect to gene flow vectors while species B is a non- vector (Garcia et al., 1996; Besansky et al., 2003). (Subbarao, Adak and Sharma, 1980; Similarly, introgression was observed between Subbarao et al., 1988, 1992; and Subbarao, members of the Dirus Complex found in Nanda and Raghavendra, 1999). In areas South-East Asia (Walton et al., 2001). where An. culicifacies A and B are sympatric, The fact that introgressive hybridization DDT spraying in many areas has caused an occurs between sibling species, this may again epidemiological impact on the transmission raise the issue of whether the sibling species (Sharma et al., 1986) due to reduction in are full biological species or not and whether species A, which is a vector (Subbarao et al., such introgression would change the 1988) and is more susceptible to DDT than biological characterstics of these species and species B (Subbarao, Vasantha and Sharma, affect vector control strategies. There is, 1988). These are a few examples that however, so far no evidence that An. gambiae demonstrate the differences between sibling and An. arabiensis have mixed and the species within a species complex and characterstic differences of these two species highlight the importance of identifying sibling have disappeared, and also very few hybrids species in malaria control programmes. were found in nature in spite of their The genetic distinctness of each sibling sympatric association over large areas of their species comes from the definition of the distribution (Besansky et al., 2003). Similarly, biological species concept whereby each only a single hybrid of An. culicifacies species species is an actually interbreeding natural A and B was found among several thousands population that is reproductively isolated from of specimens screened over large other such populations. Reproductive geographical areas where these two species isolation between sibling species is are sympatric (Subbarao, unpublished). These maintained either by pre- or post-mating observations indicate that introgressive barriers or both. The post-mating barrier is hybridization, even if it occurs between sibling expressed in the form of non-viability of species, is a rare event and pre-mating hybrid progeny at immature stages or hybrid isolation barriers are strong. sterility or both. The pre-mating barrier(s) is Thus, the sibling species should continue due to failure in copulation because of to be considered as full biological species and physical incompatibilities or behavioural there does not seem to be any likelihood of differences in mating procedures. Thus, each any of the sibling species changing their sibling species has a specific mate recognition biological characters in the near future that system that is distinctly different from that of should lead to changes in vector control the other sibling species in the complex. strategies that are being contemplated. Coluzzi Population genetic studies involving (1988) highlights the importance of chromosomal inversions and DNA markers identification of sibling species by saying that

2 Anopheline Species Complexes in South and South-East Asia failure to recognize sibling species of Malaria control strategies are not uniform and anopheline taxa may result in failure to at different times and in different areas, distinguish between a vector and a non-vector; programme organizers demand specific hence, the assessment of the impact of control strategies to cope with local situations. To meet measures may be seriously misleading if they these challenges, there is a need to generate are carried out on a morphologically defined field data to establish the prevalence of species taxon which could be a mixture of two or more at the lowest administrative units possible for sibling species. The discovery of sibling species the implementation of effective control adds a new dimension to vector control. strategies. These aspects are covered in Chapter 4. The references cited are listed by With this background, an effort was chapter and by complex in Chapter 5. made to compile the information available on anopheline species complexes prevalent Table 1 : Anopheline species complexes in South and South-East Asia in a single identified so far document. The main objective of this effort Complexes No. of Distribution in species zoogeographical was to bring to the notice of researchers, field identified regions workers and programme organizers, up-to- Coustani 2 Afrotropical date information available on species Gambiae 7 Afrotropical complexes prevalent in South and South-East Funestus 9* Afrotropical Asian countries. Marshallii 4 Afrotropical Nili 4 Ethiopian The first edition of the document was Lungae 3 Australasian published in 1998. The present document Punctulatus 11 Australasian contains the distribution of malaria vectors in Annulipes 7 Palearctic Claviger 2 Palearctic South Asia, South-East Asia and neighbouring Maculipennis 8 Palearctic countries (Figure 1). Most of these anophelines 5 Nearctic implicated in malaria transmission are species Quadrimaculatus 5 Nearctic complexes. The species complexes and sibling Albitarsis 4 Neotropical and Nearctic species discovered in each of the complexes, Crucians 6 Neotropical the formal designations given to sibling species Freeborni 2 Neotropical Nuneztovari 2 Neotropical and their prevalence in the South and South- Pseudopunctipennis 2 Neotropical East Asian countries are shown in Tables 2a Punctimacularus 2 Neotropical and 2b. The techniques currently being used Oswaldi 2 Neotropical for the identification of species complexes are Annularis 2 Oriental described in Chapter 2 and the details of the Barbirostris 3 Oriental Culicifacies 5 Oriental complexes are given in Chapter 3. The Dirus 7 Oriental complexes covered in this chapter are: Fluviatilis 4 Oriental Annularis, Barbirostris, Culicifacies, Dirus, Gigas 3 Oriental Fluviatilis, Leucosphyrus, Maculatus, Minimus, Leucosphyrus 4 Oriental Philippinensis-nivipes, Sinensis, Subpictus and Lindesayi 4 Oriental Maculatus 9 Oriental Sundaicus. An. stephensi, though not yet Minimus 5 Oriental identified as a species complex, is included Philippinensis-nivipes 3 Oriental because it is an important vector and is a Sinensis 4 Oriental complex of different variants/ecological races. Subpictus 4 Oriental For each of the complexes, information on the Sundaicus 4+1** Oriental types of evidence used for the identification Source: Information in this table has been compiled from Harbach (2004) and other published and unpublished of sibling species, the number of sibling species documents. identified, the techniques that have been * The Funestus Group consists of nine species that are developed for the identification of sibling morphologically similar at adult stage and, of these, four belonging to the Funestus Subgroup are morphologically species and the distribution and biological indistinguishable at all stages. characteristics of the members are presented. ** New cytotype

Anopheline Species Complexes in South and South-East Asia 3 Table 2a : Species complexes recognized and number of sibling species identified in South Asian countries

Anopheles Sri Lanka Iran Afghanistan Pakistan India Nepal Bhutan Bangladesh Complexes Annularis (2) + + + 2 + + + A, B Barbirostris (4) + + + + + + Culicifacies (5) 2 11251 B, E A A A, B A, B, C, D, E B + + Dirus (7)2 2++1 D, E D Fluviatilis (4) 1 + + 4 + + + T S, T, U, V Leucosphyrus (4)2 Maculatus(9)2 1 3243+1 B B, H, I B, H B, C, H, I B, H, I B Minimus (5) + 1 + + A Philippinensis/ 2 + + nivipes (3) n(A), p Punctulatus (11) Sinensis (4) Subpictus (4) 2 + + 4 + + + A, B A, B, C, D Sundaicus(4+1)2 1+ cytotype D

4 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 Myanmar Thailand Laos Viet Nam Cambodia Malaysia Indonesia Timor- Philippines Complexes Leste Annularis (2) + + + + + + + + + Barbirostris (4) + + + + + 3 + A, B, C Culicifacies (5) + 1 1 1 BBB Dirus (7)2 15 1121 D A, B, C. A A B, F B D, F Fluviatilis (4) + Leucosphyrus (4)2 123 A A, b A, B, b Maculatus (9)2 37+3111+2 A, B, C A, B, C, A, B, I B B B D, J G, H, I, K Minimus (5) C 4 2 2 1 + + A, B, A, C A, C A C, D Philippinensis/ + 3 n, p n, p n, p + + + nivipes (3) n (A, B), p Punctulatus (11) 2 f, c Sinensis (4) + 2 + + + 1 s, sin 1 Subpictus (4) + + + + + + + + Sundaicus(4+1)2 + 1 1113+ A A A s. s 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 Leucosphyrus Maculatus Sundaicus Complex Complex Complex Complex dirus (A) leutens (A) sawadwangporni (A) epiroticus (A) cracens (B) leucosphyrus s.s. (B) maculatus s.s.(B) sundaicus sensu stricto (s. s.) scanloni (C) dravidicus (C) baimaii (D) greeni (D) elegans (E) notonandai (G) nemophilous (F) willmori (H) pseudowillmori (I) dispar (J)

Anopheline Species Complexes in South and South-East Asia 5 N VIETNAM An. aconitus 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* An. dirus An. minimus An. sundaicus An. aconitus* An. barbirostris* An. jeyporensis* An. maculatus* An. nivipes* An. philippinensis* An. subpictus* An. vagus* An. annularis An. barbirostris An. maculatus An. minimus An. subpictus An. sundaicus TIMOR-LESTE CAMBODIA Sulawesi LAOS An. dirus An. minimus An. sundaicus An. minimus An. aconitus* An. maculatus* An. nivipes* An. philippinensis* An. vagus* PHILIPPINES Borneo BRUNEI Java East THAILAND project no. IC4-CT-2002-100041-Project Coordinator Professor project no. IC4-CT-2002-100041-Project Malayasia An. dirus An. maculatus An. minimus An. aconitus* An. annularis* An. leucosphyrus* An. philippinensis* An. sundaicus* SINGAPORE An. balabacensis An. donaldi An. flavirostris An. leucosphyrus An. sundaicus MALAYSIA Sumatra Malvecasia An. dirus An. minimus MYANMAR Malaysia Peninsular An. campestris An. letifer An. maculatus An. sundaicus An. nigerrimus* K BHUTAN SEA-VBC-82; For all other countries – Kondrashin and Rashid (1987), Rao (1984), from all other countries – Kondrashin For SEA-VBC-82;

An. dirus An. fluviatilis An. minimus An. dirus An. minimus An. aconitus* An. annularis* An. philippinensis* An. sundaicus* BANGLADESH An. culicifacies An. annularis* SRI LAN A An. subpictus* 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 NEPAL An. fluviatilis An. annularis* PAKISTAN An. culicifacies An. stephensi An. fluviatilis MALDIVES Malaria vectors prevalent in South Asia, South-East Asia and neighbouring countries An. annularis An. culicifacies An. maculatus An. stephensi Figure 1: Figure AFGHANISTAN INDIA An. culicifacies An. dirus An. fluviatilis An. minimus An. stephensi An. sundaicus An. annularis* An. jeyporensis* An. philippinensis* An. varuna* Primary vectors * Secondary vectors/local importance Map not to scale IRAN An. culicifacies An. d'thali An. fluviatilis An. pulcherimus An. sachorovi An. stephensi An. superpictus An. maculipennis s.s. Malaysia — Indra Vythalingam; Cambodia, Laos and Viet Nam – Data from the INCO-DEV Cambodia, Laos and Viet Malaysia — Indra Vythalingam; Source: L. Kalra; Timor-Leste— Marc Coosemans—Sylvia Manguin; Nepal and Bhutan— N. published and unpublished documents.

6 Anopheline Species Complexes in South and South-East Asia 2. Techniques used in the recognition of Species Complexes

Group(s) of individuals within a species Table 3: Techniques used in the identification sometimes exhibit distinct differences with of species complexes reference to resting habitats, preference to z Morphological variations feed on a host, the rate of development of z Crossing experiments resistance to insecticides, susceptibility to z Mitotic and meiotic karyotypes acquiring infection, and so on. All these – Structural variations differences may indicate the presence of – Heterochromatin variations isomorphic species within a taxonomic z Polytene chromosomes species (defined morphologically), but these z Electrophoretic variations differences cannot confer species status on z Cuticular hydrocarbon profiles populations. Hence, genetic techniques that z Molecular approaches can demonstrate reproductive isolation within – DNA or RNA probes a morphologically similar taxonomic species z Allele specific polymerase chain reaction (ASPCR) are needed. Table 3 gives the methods which – Restriction fragment length polymorphism are available to researchers. Crossing (RFLP) – Random amplified polymorphic DNA (RAPD) experiments, chromosomal variations and – Sigle strand conformational polymorphism electrophoretic variations at enzyme loci have (SSCP) been extensively used in studies to recognize – Heteroduplex analysis (HDA) species complexes. The chromosomal variation and electrophoretic variation at described in several papers. Reviews by enzyme loci provide evidence for the White, Coluzzi and Zahar (1975), Miles (1981), recognition of species complexes; later the Green (1985), Coluzzi (1988), Green et al. variations are also used in the development (1985), Service (1988), Subbarao (1996) and of diagnostic techniques/assays to identify Black and Munstermann (1996, 2004) are a sibling species. Cuticular hydrocarbon few that describe and discuss these techniques. analysis and molecular approaches are Readers are also referred to a WHO document generally used to develop diagnostic assays prepared by Zahar (1996). This is a review of for the identification of sibling species which literature published between 1974 and 1994 have already been recognized by other on vector bionomics and the epidemiology techniques. Sibling species by definition are and control of malaria. The document also species without easily observable includes a compilation of species complexes morphological differences. A careful of the South-East Asia and West Asia regions. examination may sometimes reveal Another article recommended is by Harbach morphological differences that are minute (2004). This article is an update of the internal and may be restricted to a particular stage in classification of the genus Anopheles, which the life-cycle. was earlier reported by the same author in The techniques (Table 3) and the 1994 (Harbach, 1994). The article lists species, principles behind these techniques have been species complexes, subgroups, groups and

Anopheline Species Complexes in South and South-East Asia 7 series recognized (formally and informally) so Crossing experiments far in the genus. Reviews by Green (1985) and The assortative mating observed between Coluzzi (1988) are strongly recommended for sibling species in nature due to pre-mating all those who work in this area. Reviews by barrier (s) generally breaks down in the Besansky, Finnerty and Collins (1992), Hill and laboratory and different sibling species mate Crampton (1994), Collins et al. (2000), at random and produce hybrid progeny. Kryzywinski and Besansky (2003) and Black Genetic differences between sibling species and Munstermann (2004) are also are expressed in the form of non-viability of recommended for those who intend to use or hybrid progeny at immature stages, hybrid develop molecular techniques for the sterility or both. Hybrid males in one or both identification of sibling species, and by Philipps crosses are sterile and hybrid females are et al. (1988) for cuticular hydrocarbon analysis. generally fertile. Therefore, hybrid sterility is White (1977) and Service (1988) discussed and used as the criterion in designating populations described the role of morphological characters as separate species. Hybrid males exhibit in the investigation of species complexes. For partial development of reproductive organs the benefit of readers a few salient points are (the extent of development ranges from mentioned below. atrophied testes and vas deferens to fully Morphological variations developed testes but without sperm; accessory glands and ejaculatory duct are generally Morphological characters that are often used normal) and do not produce progeny when to identify adults of anopheline species are crossed. For species which do not mate in largely confined to scale pattern and colour laboratory cages, artificial mating methods can and their distribution. Characters that are be adopted. Thus, laboratory crossing used in the description of immature stages experiments demonstrate post-mating barriers are sculpture of eggs, setation and and establish the species status of the pigmentation of larvae, and the forms of isomorphic populations. An. gambiae was first paddles and trumpets as well as chaetotaxy recognized as a species complex from the of pupae. Spermatheca and spiracular results observed between two strains which morphology are also used in the identification were crossed to study the genetics of resistance of species. In addition to light microscope to an insecticide (Davidson and Jackson, examination for the specific characters, 1962). It may be noted that though these post- scanning and transmission electron mating barriers are studied between members microscopes are also used to study of the complexes, they are not necessarily morphological variations. Morphometrics has required to give populations species status. proved useful in studying some species Furthermore, species which exhibit a pre- complexes when used in conjunction with mating isolating mechanism need not statistical analyses. For details see White necessarily have any post-mating barrier, as (1977) and Service (1988). has been observed between species B and C White (1977) states that morphological of the Culicifacies Complex (Subbarao, studies should not come too early in the Vasantha and Sharma, 1988). F1 hybrid males process of detecting anopheline sibling of reciprocal crosses between species B and species since it might be misleading to C are fully fertile. Dobzhansky (1970) reports characterize taxa which have not been that viable and fertile hybrids may be obtained identified by trustworthy techniques such as in experiments between undoubtedly distinct cross-breeding experiments or cytological or species that maintain complete reproductive biochemical characterizations. isolation in nature. Therefore, the reproductive status of hybrid males is not

8 Anopheline Species Complexes in South and South-East Asia always diagnostic in the recognition of species Chromatids after division remain attached, complexes. While studying post-mating causing thickening of chromosomes which barriers, a point to be remembered is that for results in the appearance of long ribbon-like colonies established from species-specific structures with dark and light horizontal diagnostic characters such as fixed inversions, portions representing band and interband enzyme electromorphs of progeny from single regions respectively. The dark and light female cultures have to be used. A laboratory regions represent differential condensation of colony established from natural populations chromosomes. The banding pattern of each may be a mixture of two or three sympatric chromosome is specific in a given species; sibling species. thus, each species differs from others in characteristic banding pattern. Any changes Cytogenetic techniques in the pattern can be easily detected. In the polytene chromosome complement, only Polytene chromosomes euchromatic regions are seen and the Anopheline females in the semi-gravid stage heterochromatic portions of the have the best polytene chromosomes in chromosomes which are under-replicated are ovarian nurse cells (Coluzzi, 1968). Larvae not seen. Therefore, in anophelines, the short at the IV instar stage have polytene arm of the X-chromosome and Y- chromosomes in salivary glands. For those chromosome are not seen in the polytene anopheline species which do not have good complement. In some species a definite ovarian polytenes, larval salivary chromocentre is seen. In such cases, all the chromosomes can be used ( but salivary gland chromosome arms are seen attached to the polytene chromosomes are not very good in chromocentre by their centromeric ends. most anophelines). The advantage with adult Generally, this is not the case with females is that ovaries can be removed and anophelines and chromosome arms are seen fixed in modified Carnoy’s fluid (1:3 glacial separately; occasionally, the two arms of a acetic acid:methanol) and can be used at any chromosome are seen attached at time. Another advantage is that the same the centromeric ends. Homologous female can be studied for other parameters chromosomes exhibit high affinity for pairing such as host preference, presence of and, therefore, are seen as a single sporozoites/sporozoite antigen, susceptibility chromosome. to insecticides, etc. In anopheline cytogenetics literature, two The recommended references for the types of designations are seen for the polytene preparation of polytene chromosomes are: chromosome arms: (i) the two arms of a for ovarian polytene chromosomes from adult chromosome are designated as right (R) and females, Green and Hunt (1980), and for left (L) arms; this system is followed by salivary gland polytene chromosomes from Drosophila cytogeneticists and is adapted by IV instar larvae, Kanda (1979). Hunt and many anopheline cytogeneticists; and (ii) the Coetzee (1986) describe storing of field- new nomenclature for arm designation is that collected mosquitoes in liquid nitrogen for suggested by Green and Hunt (1980). In this, correlated cytogenetic, electrophoretic and each arm is given a separate number—2,3,4 morphological studies. The preparation of and 5—for autosomal arms and the polytene chromosomes from adult females euchromatic arm of the X-chromosome seen is not difficult. in the polytene complement is designated as Polytene chromosomes are the result of X. Taking An. gambiae belonging to the repeated replication of chromosomes at Pyretophorous series as an arbitrary standard, interphase without nuclear division, the the two arms of chromosome 2, 2R and 2L process being known as endomitosis. 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 recognition systems (Peterson, 1980). In 5 respectively in this nomenclature. populations where heterozygotes for an inversion are absent, the inversion is said to During evolution in anophelines, whole- be fixed and the two banding patterns on arm translocations have occurred. This came polytene chromosomes, inversion and its to notice when banding patterns of different standard alternate arrangement become species were compared and studied. In An. diagnostic tools for the identification of culicifacies and An. fluviatilis belonging to the species. The occrrence of a small proportion Myzomyia series and An. stephensi, An. of heterozygotes can be due to: (i) breakdown annularis and An. maculatus belonging to the of pre-mating barrier(s) between two species Neocellia series, the arm association is 2-5 (which is rare); and (ii) an inversion which is and 3-4. In the Myzomyia series, for the An. fixed in one species is polymorphic (floating) funestus group of species, the arm association in another species and the two species are is 2-4 and 3-5, indicating another sympatric (e.g. An. culicifacies species A and translocation event (Green and Hunt, 1980). D) (Vasantha, Subbarao and Sharma, 1991). With this arm- designation system such events can be incorporated and it is best suited for In both cases, the number of studies on the cladistic analysis of species heterozygotes is far less than the number belonging to a subgroup, group or series. expected where an inversion is polymorphic Researchers who use polytene chromosomes with random mating. These situations can in their work are recommended to read be analysed statistically by calculating the Green and Hunt (1980) for the arguments expected number based on Hardy-Weinberg and justification for the new arm designations. equilibrium and applying a Chi-square test. Thus, the population cytogenetic analysis Most of the species complexes identified demonstrates and distinguishes intraspecific so far have been by the examination of and interspecific occurrence of inversions. polytene chromosomes of wild populations. Paracentric inversions are very common in It may be noted that sibling-species the natural populations of anophelines. The having homosequential polytene advantage of paracentric inversions in species chromosome banding pattern exists among identification studies is that they act like single anophelinespecies complexes, e.g. An. gene loci and the alternate arrangements as labranchiae and An. atroparvus, two members codominant alleles. There are inversions that of the Maculipennis Complex (Coluzzi, 1970) are polymorphic within a species and species B and E of the Culicifacies (intraspecific) and the three forms of these Complex (Kar et al., 1999). A point to be inversions, the two homozygotes, standard kept in mind by all those who work with and inverted, and the heterozygotes, are polytene chromosomes is that inversion easily recognized on polytene chromosomes, fixation is an accidental association with while there are inversions that are fixed and speciation. As emphasized by Green and different between species (interspecific). Baimai (1984), where variation occurs in polytene chromosomes due to inversions, it The total absence or significantly may provide useful markers for speciation and deficient proportion of heterozygotes for an subspeciation events, but in its absence one inversion in a population indicates can say nothing about such events among reproductive isolation within a taxon; hence, individuals bearing the same chromosomal the taxon may be considered as a species rearrangements. complex. Thus, the examination of polytene chromosomes of field-collected adult females In spite of the limitations that provides unequivocal evidence for the homosequential species exist in anophelines existence of different species- specific mate and that polytene chromosome complements

10 Anopheline Species Complexes in South and South-East Asia can only be examined in semi-gravid adult chromosomes have been found as females or in salivary glands of IV instar larvae, telocentric, acrocentric or subtelocentric, or this is the easiest and cheapest tool now submetacentric (depending on the position available for the recognition of species of the centromere in the chromosome) in complexes and for routine use in the different species of anophelines. The paper identification of members of a complex in of Levan (1964) is recommended for entomological studies. This tool is, however, chromosome nomenclature. The best mitotic more laborious than the DNA- based tools in chromosomes are found in the neurogonial large-scale entomological studies. cells of the brain in early IV instar larvae and meiotic chromosomes in the reproductive Asynapsis in polytene complements in organs of newly-emerged adults. The hybrids is used as one of the criteria in recommended references for the preparation determining species status. The degree of of mitotic and meiotic chromosomes are: asynapsis may vary, and thus has to be used Breeland (1961), French, Baker and Kitzmiller with caution. In hybrids between members (1962) and Baimai (1977). of the Culicifacies Complex, the chromosomes remain synapsed, except in the Structural variations due to the position inversion heterozygote region where a loop of centromere and quantitative variations in or asynapsis is observed (Subbarao et al., heterochromatin blocks are commonly 1983). observed. The variation in autosomes and X-chromosomes, which are found in the Inversions are designated with lower-case homozygous state, demonstrate reproductive letters of the English alphabet and are specific isolation between the populations if for each chromosome arm, that is, an heterozygotes for the variation are not found inversion a on chromosome arm 2 bears no or are found in deficient numbers. This is relationship to a similarly denoted inversion similar to the situations described for on another arm, that is, inversion a on arm paracentric inversions under polytene 3. The standard arrangement is designated chromosomes (see above). Unlike the + before the letter indicating the inversion banding pattern due to paracentric inversions, concerned and these may or may not be variations at a given position in the written as a superscript, e. g. 2+a or 2+a. chromosome can exist as more than two Green (1982a) for the Myzomyia Series and alternatives. Data have to be generated from Green (1982b) for the Neocellia Series have single female progeny of wild-caught females suggested a unified method for designating or larvae. Structural variations in the Y- inversions for species belonging to these chromosome, though very common in Series, as it provides an efficient means of anophelines, do not by themselves reveal the storing data and its subsequent retrieval. This genetic structure of the population, as the has been followed for several members variations in a population give no indication belonging to these series (An. fluviatilis, An. whether they are intra- or inter-specific culicifacies, An. annularis, An. maculatus, An. because the Y-chromosome is inherited from philippinensis, etc.). father to son and is found in hemizygous Mitotic and meiotic karyotypes condition. However, one can test the All anophelines studied so far have three pairs association of the Y-chromosome with other of chromosomes—two pairs of autosomes genetic variations, where linkage which are either metacentric or sub- disequilibrium between Ys and other metacentric and a pair of sex chromosomes— characters would indicate the presence of which are homomorphic (XX) in females and different sibling species. Species E in the heteromorphic (XY) in males. X- and Y- Culicifacies Complex was identified by

Anopheline Species Complexes in South and South-East Asia 11 associating sporozoite positivity in females single locus can exist and in each species with Y-chromosome variants in their sons (Kar more than one allele may be fixed. In An. et al., 1999). melanoon and An. sacharovi (two members of the Maculipennis Complex), Hk-190 and Hk- Heterochromatic variants, revealed by 1100 alleles at the hexokinase locus are fixed special staining techniques using Giemsa, in the former sibling species and Hk-195 and Hoechst, etc., on autosomes and X- Hk-197 alleles in the latter. Therefore, chromosomes are distinct and are also electromorphs found to be diagnostic for each diagnostic in the identification of sibling taxon can be used only to identify individuals species. Both structural and heterochromatic from populations already sampled (Miles, variants in mitotic karyotypes are not 1981) as geographical variation within a convenient as routine entomological tools for species can exist. the identification of sibling species in field studies as progeny of wild-caught females If a single fully diagnostic enzyme system have to be examined for the variants. cannot be identified (i.e. without any However, these techniques can be used to polymorphism), several enzyme systems study the larval ecology of sibling species. which differ in their frequencies of alleles between populations can be identified and Enzyme electrophoretic variations used in association, which will reduce the Enzyme electrophoresis is extensively used error in identification. For species complexes in the study of species complexes. The with several members, enzyme systems that technique involves the detection of the are diagnostic for different members of the protein bands of an enzyme system with same species complex can be identified and different mobilities as a function of electric used in the form of a biochemical key, as has charge and molecular structure. On a gel been developed for the Maculipennis and zymogram of an enzyme system, Gambiae Complexes (for details, see Coluzzi, electrophoretic variations in the form of bands 1988). with different mobilities represent proteins Electrophoretic variations at enzyme loci coded by different alleles (allozymes). These are not only useful for the identification of alleles, being codominant, behave like isomorphic species but can also be used for paracentric inversions and the two the correct identification of morphologically homozygotes and heterozygotes can be identifiable species. An. minimus, An. differentiated. Variations at a locus thus aconitus and An. varuna are found sympatric enable the detection of the reproductive and are morphologically very similar. Hence, isolation between populations resulting from errors are made in the identifications. For positive assortative matings within a Thailand populations, alleles at the Malate population. Because of the simplicity of the dehydrogenase-1 (Mdh-1) locus were found procedures for the processing and to be diagnostic (Green et al., 1990). Mdh- interpretation of data, this technique permits 1100 fixed in An. minimus differentiates it from large-scale sampling of natural populations An. aconitus and An. varuna which have the and is very useful as a diagnostic tool in the Mdh-1157 allele. Less than 0.1 per cent An. routine identification of species. For details minimus had the Mdh-1157 allele and a few on techniques, Ayala et al., (1972), Black and specimens had the Mdh-1115 allele. Mdh-1115 Munstermann (1996, 2004) and Steiner and is diagnostic for An. pampana, but this species Joslyn (1979) are recommended. However, is very rare in Thailand (Green et al., 1990). one has to remember that unlike inversions, where only one inverted arrangement with A point to be kept in mind when working reference to a standard arrangement exists, with electrophoretic variations is that in order more than two electrophoretic forms at a to determine the mobilities of the bands of

12 Anopheline Species Complexes in South and South-East Asia samples to be identified, samples of known identification of sibling species. Several reference standards should be run on the same reviews are now available in the literature gel. Alternatively, non-enzymatic protein detailing various techniques and their standards have to be run along with the advantages in the identification of isomorphic samples. For the identification of the Minimus species (Besansky, Finnerty and Collins, 1992; Complex sibling species, human haemoglobin Hill and Crampton, 1994; Black and (homozygous for normal haemoglobin) and Munsterman, 2004; Collins et al., 2000; and known laboratory colony material were run Krzywinski and Besansky, 2003). on the same gel as reference standards (Green The first of the DNA methods used to et al., 1990). Mosquitoes collected from the identify species was the use of DNA probes. field if not used immediately should be stored Clones containing specific DNA segments of frozen (£40°) to retain their enzyme activity the undefined highly repeated component of prior to electrophoresis. the genome are identified by differential Cuticular hydrocarbon profiles screening of genomic libraries with homologous and heterologous genomic Cuticular hydrocarbon analysis for sibling- DNAs. DNA segments from these clones are species identification involves determining labelled and used as probes. The paper of species-specific differences in the Post and Crampton (1988) on DNA probes hydrocarbons contained in the wax layer of for the Simulium damnosum Complex and insect cuticle. The wax layer lies beneath Black and Munstamann (2004) give the outermost cuticular layer. Carlson and procedures (with illustrations) used in the Service (1979) were the first to use this isolation of species-specific DNA probes for technique to identify An. gambiae s.s. and An. the identification of sibling species. Initially, arabiensis (two members of the Gambiae radioactive probes were used. Simple non- Complex). The review by Philipps et al. radioactive probe assays for squash-blot (1988) is recommended for details on the hybridizations have been developed for the procedure. This technique has now been identification of members of the Gambiae used to identify members of several species (Hill et al., 1991), Punctulatus (Cooper, complexes in mosquitoes and other Cooper and Burkot, 1991) and Dirus (Audtho haematophagous insects. et al., 1995) Complexes. Johnson, Cockburn It may be noted that this technique can and Seawright (1992) have improved the be employed to identify members of an procedure to clean up the background in already recognized species complex but is not squash-blot hybridizations. Non-radioactive recommended to be used to recognize new probe methods remove the hazards of complexes. The most important criterion for radioisotopes and make the assays simple and designating different species is reproductive usable under field conditions. The advantage isolation which is not possible with this with DNA probes, as with isozymes, is that technique because it is not easy to species can be identified at all stages of the differentiate between intra- and inter-specifc mosquito life-cycle. And if kits are variations in the hydrocarbon profiles of developed, as they have been for the individual specimens. Furthermore, this tool Gambiae Complex (Hill, Urwin and uses gas liquid chromatography with Crampton, 1992), probes can be used with expensive equipment. much more ease in field laboratories. Hill, Urwin and Crampton (1991) have shown that DNA methods by producing synthetic probes, the cost of Advancements in DNA recombinant each assay can be brought down to between technology have facilitated the development US$ 0.04 and US$ 0.33 depending on the of simple and rapid molecular tools for the labelling method used.

Anopheline Species Complexes in South and South-East Asia 13 With the improved DNA technologies members of a complex are identified. The and reduction in the cost of reagents and taxon An. (Nysorhynchus) albitarsis was, equipment, polymerase chain reaction (PCR) however, identified as a complex of four assays have become popular for species sibling species by the examination of natural identification. The PCR assay was developed populations from Paraguay, Argentina and in 1985 by Saiki et al. (1985). PCR-based Brazil, using RAPD-PCR (Wilkerson et al., methods essentially require variation in 1995). Though RAPD-PCR is simple, this neucleotide sequence across species. The technique has inconsistent reproducibility major advantage of this technique is that it and therefore has been of limited use for the requires only miniscule amounts of DNA. identification of sibling species. During the reaction period of 2-3 hours, a Allele-specific PCR (ASPCR) assays mostly particular region of the genome is amplified exploit variation in the rDNA cistron. In 1-100 million times, and this can then be anophelines this is X-linked (Rai and Black, simply visualized on agarose gel after 1999). It consists of tandem repeated arrays electrophoresis and staining. The most of conserved genes (18S, 5.8S and 28S) commonly used PCR-based methods are: punctuated by rapidly evolving non-coding restriction fragment length polymorphism of internal transcribed spacers, ITS1 between PCR-amplified product (PCR-RFLP), single- 18S and 5.8S and ITS2 between 5.8S and strand conformational polymorphism (SSCP), 28S. Each gene cluster is separated by Heteroduplex analysis (HDA), and allele- intergenic spacers (IGS). Within interbreeding specific PCR (ASPCR). The ASPCR is populations the arrays undergo rapid essentially cheaper and quicker than the other homogenization through concerted methods and does not involve special evolution, which drives new sequence treatment following PCR. However, the variations to fixation, leading to species- position of neucleotide variations is critical specific differences. For members of many in designing primers for ASPCR so that all the species complexes, differences in ITS2 and allele-specific amplicons can be distinguished variable regions within 28S rDNA gene have separately on a gel. Black and Munstermann been used to develop ASPCR assays for (2004) show in detail the steps involved in members of the Culicifacies Complex (Curtis PCR assay with illustrations. and Townson, 1998; Singh et al., 2004a), for Basically, there are two types of PCR the Fluviatilis Complex (Manonmani et al., strategies: one surveying the genome 2001; Singh et al., 2004b), for the Dirus randomly and the second targeting specific Complex (Walton et al., 1999) and for several regions of the genome, such as ribosomal other species complexes prevalent in the DNA (rDNA) or mitochondrial DNA Afrotropical, and Neotropical regions. (mtDNA). The randomly amplified Portions of the genes from mitochondrial polymorphic DNA PCR (RAPD-PCR) belongs genome, COI and COII, are also used to to the first category. This technique does not develop diagnostic PCR assays as has been require prior knowledge of the genome, and done for members of the Culicifacies an added advantage is that commercial kits Complex (Goswamy et al., 2006). For An. are available with large number of random minimus species A and C, Kengue et al. (2001) decamer primers. Arbitrary regions of the used the RAPD marker assays of Sucharit and genome are amplified using a single decamer Komalamisra (1997) to develop a robust primer. Amplified products by each primer multiplex ASPCR. This assay distinguished are analysed for differences between the other anophelines, An. aconitus, An. varuna species concerned. This technique was and An. pampanai, which are morphologically developed by Williams et al. (1990). DNA very close to An. minimus and are found tools are generally developed once the sympatric with An. minimus.

14 Anopheline Species Complexes in South and South-East Asia In PCR-RFLP, the amplified product is cut of a probe and a test DNA molecule. The by certain restriction enzymes which produce number and type of mismatched nucleotides a different pattern of digested products when within a given HDP determine the mobility run on agarose gel. A single nucleotide change of the DNA duplex on an electrophoresis gel may alter the restriction enzyme site and (Tang and Unnash, 1997). The target DNA thereby a different pattern of bands may be and probe DNA are PCR products of chosen revealed. A PCR-RFLP targeted at ITS2 rDNA loci, which are hybridized and run on a gel. was developed by Van Bortel et al. (2000) The probe DNA is chosen from a closely which distinguishes An. minimus A and C and related species. The advantage of this four related species, An. aconitus, An. technique is that without going through the pampanai, An. varuna and An. jeyporiensis. sequencing of loci, intra- and inter-specific Digestion of the PCR-amplified D3 region of variation can be detected. 28S rDNA with HPA II endonuclease DNA sequences are also being used to distinguished An. funestus from An. veneedeni establish the phylogenetic relationship of the (Koekemoer, Coetzee and Hunt, 1998), and species between the subgroups and of those digestion of COII-amplified product with Dde within a subgroup/complex. Portions of genes I distinguished species E from species B and from mitochondrial genome, COI, COII, C of the Culicifacies Complex (Goswamy et Cytochrome b, etc., and from nuclear rDNA al., 2005). regions, D2, D3, ITS1, ITS2, etc., are used Hiss et al. (1994) developed a technique (Chen, Butlin and Harbach, 2003; Dusfour based on single-strand conformation et al., 2004 and Garros, Harbach and polymorphism (SSCP) of Orita et al. (1989) Manguin, 2005a and b). Sallum et al. (2002) as a diagnostic tool. SSCP is a highly sensitive examined the phylogenetic relationship of 32 technique and detects point mutations with species of the subfamily Anophelinae, which an efficiency of 99% in products with 100- included species from the genera Anopheles, 300bp length. As the product length Bironella and Chagasia. increases, the efficiency is reduced. This Microsatellite markers are di-, tri- and technique does not require prior knowledge tetra-nucleotide sequence arrays of variable of DNA sequence data. In this technique length found frequently in the genomes. the amplified product is denatured to single These sequences are identified from genomic strands at 95 oC for five minutes and then libraries using labelled repeat probes. Once placed immediately into an ice bath (0-4 oC) the sequences from the positive clones are so that single-strand duplexes are formed selected from unique neucleotide sequences from intrastrand base pairing. Variation in flanking the repeated sequences, specific the confirmation of intrastrand duplexes is primers for each marker are developed for visualized by a gel retardation assay. Sharpe PCR assays. For An. gambiae s. s., markers et al., (1999) developed an SSCP assay for were also developed from genome sequence the identification of An. minimus species A analysis (for this species the entire genome and C, An. aconitus and An. varuna, and sequence data is now available at http:// Koekmoer et al.(1999) used D3 region of www.ensemble.org/Anopheles_gambiae) rDNA to distinguish four members of the An. instead of going through the labour-intensive funestus group, An. funestus, An. vandeeni, screening process mentioned above. An. rivulorum and An. leessoni. Microsatellite loci generally have higher Heteroduplex analysis (HDA) is another mutation rates than other regions of the technique used in the identification of closely- genome and, therefore, are highly related species. HDA detects the polymorphic. Beacause these are neutral electrophoretic retardation of heteroduplex markers, they are preferred for use in products (HDPs) formed between the strands population genetic studies. There are a few

Anopheline Species Complexes in South and South-East Asia 15 anophelines for which these markers have (Wondji, Simard and Fontenelle, 2002 and been developed— An. gambiae s.s. (Zheng Stump et al., 2005). In years to come, some et al., 1993, 1997), An. maculatus of these forms may be given a species status. (Rongnoporut et al., 1996), An. funestus ( Green constructed phylogeny using cladistic Sinkins et al., 2000), An. dirus (Walton et al., analysis based on paracentric inversions seen 2000a), An. stephensi (Veradi et al., 2002) in ovarian polytene chromosomes for and An. culicifacies ( Sunil et al., 2004). members of the Series Myzomyia (Green, Microsatellite markers developed for one 1982, 1995) and for the Series Neocellia member can be used for other members of (Green et al., 1985). the complex. They can also be used for closely Programs are now available for related species as has been done for members calculating gene frequencies, average of the Leucosphyrus Complex using those heterozyosity, per cent polymorphic loci developed for An. dirus s.s (Walton et al., observed and expected heterozygote values 2000a). based on the Hardy-Weinberg equilibrium, These markers were initially used for the Wright’s fixation indices and genetic distance genetic mapping of refractory gene(s) and and identity, and for establishing phylogenetic morphological markers (Zheng et al., 1993, relationships. Computer programmes can 1996) and for developing a fine-scale genetic also be used for analysing populations that map (Zheng et al., 1997) of An. gambiae. are polymorphic for many inversions, as has Now they are being used in population been done for An. gambiae s.s. and An. genetic analysis and ecological studies in An. arabiensis (Garcia et al., 1996) and An. gambiae s.s. and also for other members of annularis (Atrie et al., 1999). the Gambiae Complex (Besansky et al., 1997; Currently, more than 240 programmes Kamau et al., 1999 and Donelly and have been listed at http://www.nslij- Townson, 2000). Similarly, microsatellites are genetics.org/soft. The BIOSYS-1 program in being used for the population genetic analysis FORTRAN of Swofford and Selander (1981) of An. maculatus ( Rongnoparut et al., 1999), and its later vesions were used for the analyses An. dirus ( Walton et al., 2000b) and An. of many of the anopheline species. The most culicifacies (Subbarao, unpublished). commonly used programmes are—TFPGA, It is important to highlight the power of Arlequin, POPGENE, GDA, GENEPOP, simple polytene chromosome analysis using GeneStrut, GeneAlEx, etc. MEGA (Molecular paracentric inversions as a tool in population Evolutionary Genetic Analysis) is a software genetic studies and in establishing phylogentic for computational molecular evolutionary relationships. This simple tool has brought out genetics based on nucleotide/protein the complexities of population structure sequences. Those interested on population within the An. gambiae s.s. by identifying five genetic analysis may visit http:// chromosomal forms — Bamako, Mopti, dorakmt.tripod.com/genetics/popgen.html to Savanah, Forest and Bissau in West Africa learn about the basics of population genetics, (Coluzzi, Petrarca and DiDeco, 1985). Now, links to freeware, related literatures and other DNA markers together with cytogenetic tools useful links. are being used to clarify this complexity

16 Anopheline Species Complexes in South and South-East Asia 3. Species Complexes

3.1 The Annularis Complex further reports on details of these species are available. Anopheles annularis Van Der Wulp 1984 belongs to the subgenus Cellia, Annularis Species A and B (Atrie et al., 1999) Group in the Neocellia Series. The other Nine inversions – wI, i1, j1 and k1 on members in this Group which are prevalent chromosome arm 2; j1 and z on arm 3; h1 in South-East Asia are An. nivipes, An. and s1 on arm 4; and k on arm 5 – were philippinensis, An. pallidus and An. schueffneri found polymorphic in six districts in five states (Harrison 1988, Harbach, 2004). in India. The total absence of heterozygotes for inversion j1 and its standard arrangement An. annularis has a wide distribution in on chromosome arm 2 in villages in the Oriental region. It is found in Afghanistan, Ghaziabad and Shahjahanpur districts in the Pakistan, Nepal, India, Bangladesh, Myanmar, state of Uttar Pradesh in India led to the Philippines, China and Sri Lanka (Rao 1984; recognition of two sibling species A and B. Kondrashin and Rashid, 1987). This species The deficiency of heterozygotes for three is considered to be an important vector in other inversions – 2i1, 2k and 4h – was also Nepal and in certain parts in India (Dash et observed. However, the partitioning of al., 1982; Rao, 1984; Gunasekaran et al., polymorphic forms of these inversions 1989). It is a secondary vector in certain between the two sibling species characterized localities though it has a wide distribution by the +j1 and j1 arrangements indicated and is sometimes found abundantly (Rao, balanced polymorphisms of the inversions in 1984). An. annularis has been incriminated each of the presumed sibling species. This recently in the Thai-Combodia border area further supported the conclusion that An. (Baker et al., 1987) and is also a vector in annularis is a species complex and Myanmar (Bang, 1985). In Sri Lanka, this heterozygote deficiencies are due to the species has been found playing a role in the difference in the frequencies of these transmission of malaria in a new irrigation inversions in the two sibling species. development area (Ramasamy et al., 1992). Techniques available for Evidence for identification of sibling identification of sibling species species Polytene chromosomes Two sibling species from Nepal (WHO, 1983) This technique is based on the difference in the banding pattern of polytene The total absence of heterozygotes for a chromosomes due to a paracentric inversion. paracentric inversion and its standard Species A is characterized by the +j1 arrangement on the X-chromosome was arrangement and species B by the j1 taken as evidence for the presence of two arrangement on chromosome arm 2. A sibling species in An. annularis in Nepal. No

Anopheline Species Complexes in South and South-East Asia 17 photomap of polytene chromosomes with the these two sibling species (Atrie et al., 1999). break points for the diagnostic inversion is The assays were developed on the specimens given by Atrie et al. (1999). The X- collected from different areas based on the chromosome and the autosomal arms 3, 4 distribution pattern reported for sibling and 5 are homosequential in both species. species A and B by Atrie et al. (1999).

Breakpoints of nine polymorphic Distribution and biological characters inversions observed in these species are also In the two districts of Shahjahanpur and marked on the photomap. The photomap is Ghaziabad in Uttar Pradesh, India, species A presented in Figure 2. Polytene chromosome and B were found sympatric. In all the other maps for An. annularis are also given by Green states surveyed, namely, Rajasthan, Haryana, et al. (1985). The 2+j1 arrangement in Assam and Orissa, only species A has been species A is homosequential with the one found. An. annularis samples from presented by Green et al. (1985). Of the nine Shahjahanpur district, where both species A inversions observed in India, four inversions and B are prevalent, were found totally - 2w, 3z, 4h1 and 5k - were found in zoophagic when blood meals of these species populations from Taiwan, Philippines, were examined by counter-current Thailand and Bangladesh (Green et al., electrophoresis (Atrie, 1994). Both the species 1985). were found in the riverine, non-riverine and The mitotic karyotype of both the species canal-irrigated ecotypes found in the villages was found to be the same. Both the in Shahjahanpur. However, in other districts autosomes and the sex chromosomes were where only species A was found, the same submetacentric (Atrie, 1994). ecotypes were observed, and collections were also made from hilly-forested areas PCR-RFLP (Atrie et al., 1999). Two PCR-RFLP assays, one based on endonuclease restriction sites in the ITS2 In India, An. annularis is considered a sequence and the other based on those in vector only in certain states. Only species A the D3 sequence of rDNA, have been was found in Sundergarh, Orissa state, and developed by Alam et al. (2006). In the ITS2 Kamrup in Assam state where An. annularis sequence, species A showed three restriction is considered a vector. However, species A sites each for MspI, MyaI and Eco24I enzymes was also found in Haryana, Rajasthan and and species B showed restriction sites for Uttar Pradesh where An. annularis is not MspI, HintI and NruI enzymes. In the D3 considered a vector. Furthermore, species A sequence, species A had a unique restriction was totally zoophagic. Thus, the identification site for Alw26I while species B had the site of An. annularis as a species complex did not for KpnI. With the D3 sequence two enzymes explain why it is a vector only in certain areas are needed for the accurate identification of in India. two sibling species. MspI restriction sites were An. annularis is a vector of local found in both the species in the ITS2 importance in Nepal and Bangladesh, and a sequence and fragments differing in lengths secondary vector in India and Sri Lanka. The were produced in the two species following status of An. annularis as a vector is not known the digestion. Therefore, ITS2-MspI could be in Bhutan (Figure1). Recently, in Afghanistan, used as a diagnostic system to identify sibling it was incriminated with a Plasmodium vivax species A and B of the Annularis Complex. sporozoite rate of 0.58% (Rowland et al., However, the two assays have not been 2002) in villages with river-irrigated rice fields. correlated with the cytological identification The distribution of these two sibling species, which was the basis for the identification of A and B, has not been studied so far in any of

18 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)

X 2 345

1 29 2 7 30 38 8 39 20 21 2 30 3 31 39 40 3 8 21 4 9 22 40 41 4 22 31 5 23 32 9 41 10 42 5 6 23 32 10 42 24 33 11 43

11 43 12 33 44 24 34

25 45

46 45 44 12 13 34 35

25 13 26 14

26 35 27 36 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 1.64 + 0.49, male —1.19 + 0.23) and laid examine the biological characters of these two 143 + 47.54 eggs/female, while the CHB species and investigate whether there are strain weighed, female — 0.97 + 0.23, male more species in this complex. — 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. 3.2. The Barbirostris In the CHB female x CHP male cross, Complex eggs were laid but none hatched, while in the reciprocal CHP female x CHB male cross An. barbirostris belongs to the subgenus there was 60.8 per cent hatching. The two Anopheles, Barbirostris Subgroup, Barbirostris parental strains had more than 80 per cent Group in the Myzorhynchus Series (Harbach, hatch. In CHP x CHB cross, there was 47.1 2004). There are two Subgroups, Barbirostris per cent of pupation and only 16.7 per cent and Vanus, in this Group. The Barbirostris emerged. When F1 females from this cross Subgroup includes barbirostris, campestris, were crossed to CHP males, no eggs were donaldi, franiscoi, hodgkini and pollicaris; and laid in spite of 70.4 per cent insemination. the Vanus Subgroup includes ahomi, F1 males had abnormal genitalia with very barbumbrosus, reidi, manalangi and vanus short claspers, and atrophied testes and (Reid, 1968). accessory glands. Polytene chromosomes

Anopheles barbirostris is reported from from the salivary glands of the F1 were India, Pakistan, Bangladesh, Nepal, Myanmar, homosequential with the maps described by Thailand, Malaysia, Laos, Cambodia, Viet Chowdayya et al. (1970), but exhibited Nam, Indonesia, Sri Lanka and South China inconsistent asynapsis along the autosomes (Rao, 1984). This species has been suspected while the X-chromosomes showed complete as a malaria/filaria vector in Indonesia and synapsis. Based on these results, the authors Thailand. considered An. barbirostris to be a species complex. Evidence for identification of sibling species Mitotic karyotypes from Thailand and Indonesia (Baimai, Rattanarithikul and Crossing experiments (Choochote, Kijchalao, 1995) Sucharit and Abeyewickreme, 1983) Four forms of metaphase karyotypes were The evidence showing An. barbirostris to be observed in mosquitoes collected from wild a species complex came from the progeny of populations. The karyotype consisted of two reciprocal crosses carried out between two pairs of autosomes, metacentric chromosome laboratory strains (Choochote, Sucharit and 2 and sub-metacentric chromosome 3 and Abeyewickreme, 1983). Two strains, the the X- and Y- chromosomes which differed

Chumphon strain (CHP) established from in size and shape. Three forms, A (X2, X3, mosquitoes collected from Bang Luke Y1), B (X1, X2, X3, Y2) and C (X2, X3, Y3,), were

Canton, Chumphon Province (southern observed in Thailand and one form, D (X2,

Thailand) and the Chon Buri strain (CHB) Y4), in Indonesia. The collections made in from Chan Buri province (central Thailand) Thailand were extensive and covered the were used in this study. The two strains were entire country. The D karyotype was not reared in the laboratory by using the forced found in any of these collections and was mating technique. restricted to Indonesia. The authors concluded that the X,Y variations found in Both the strains differed in average body Thailand could be inter- or intra-specific weight and number of eggs deposited. The variations. CHP strain always weighed more (female —

20 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 communication). The CHP and CHB strains (Sukowati, Andris and Sondakh, 2003) reported by Chuchote, Sucharit and Mitotic karyotypes of the progeny of wild- Abeyewickreme (1983), cytotypes decsribed caught An. barbirostris females from their four by Baimai, Rattanarithikul and Kijchalo (1995) geographically isolated populations were and three possible species reported by examined. The mitotic karyotypes differed in Sukowati, Andres and Sondakh (2003) have X and Y- chromosomes, the variation being to be correlated with each other. in the amount and distribution of constitutive heterochromatin in giemsa-stained preparations. The authors reported that Form 3.3 The Culicifacies

A (X1, X2, X3, Y1) is widely distributed in Complex Indonesia, and found sympatric with form B Anopheles culicifacies Giles belongs to the (X1, X2, X3, Y2) and form D (X2, X3, Y4) in Tara- tara 2, North Sulawesi; Konga, Flores and subgenus Cellia, Culicifacies Subgroup, Tanjung Bunga, Flores, and form A was found Funestus Group in the Myzomyia Series (Harbach, 2004). An. culicifacies sensu lato sympatric with form B and form C (X2, X3, Y3) only in Boru-Boru, Flores. It may be noted (s. l.) has a wide distribution in India and extends to Ethiopia, Yemen, Iran, Afghanistan that in Indonesia, Form A included X1 variation too. Neither form C nor D were and Pakistan in the west, and Bangladesh, Myanmar, Thailand, Cambodia and Viet Nam found with the X1 chromosome inspite of in the east. It is also found in Nepal and being sympatric with forms A and B. Y3 and southern China to the north and extends to Y4 chromosomes were associated with the C and D forms respectively. Mitotic Sri Lanka in the south (Rao, 1984). Recently, chromosomes of the four forms found in this species was reported from Cambodia Indonesia are shown in Figure 3. (Van Bortel et al., 2002). An. culicifacies is an important vector of malaria in India and Thus, there appear to be at least three Sri Lanka and in the countries west of India. distinct cytotypes probably representing inter- The history of malaria control in these specific variation. All three forms were countries concerns mainly the control of An. homosequential in their polytene culicifacies. chromosome banding pattern. S. Sukowati (personal communication) considers these to Significant differences were observed in be three distinct sibling species in this the bionomics of An. culicifacies in various complex. Species A was found highly regions, including differences in seasonal zoophagic and species B and C were abundance, diurnal activity, man-biting anthropophagic. Species C was found biting behaviour and vectorial potential (Rao, during daytime. In the areas where these 1984). As early as 1947, due to such distinct are found, filariasis due to Wucheraria differences in biological characters, it was bancrofti and Brugia malayi and malaria are suggested that the species culicifacies may prevalent. An. barbirostris is considered to cover a range of ’biological races’ (Senior be a vector of malaria and filaria. Studies on White, 1947). An. culicifacies has now been polytene chromosome difference, allozyme recognized as a complex of five sibling variation and DNA methods need to be species, provisionally designated as species initiated to give specific taxonomic status to A, B, C, D and E. these populations and establish relationships with the prevalent diseases. Studies have Evidence for identification of sibling been initiated to develop a PCR assay for the species differentiation of these three species Four sibling species, A, B, C and D in this (Supratman Sukowati, personal complex, were identified following the

22 Anopheline Species Complexes in South and South-East Asia observation of a total absence or significant addition to a and b on the X-chromosome; deficiency of heterozygotes in natural thus, species B had Xab; 2g1 arrangement populations for the alternate arrangements (Subbarao et al., 1983). In Gujarat and observed in polytene chromosomes due to Madhya Pradesh, two chromosome 2 paracentric inversions. The fifth species, arrangements, g1+h1 and +g1h1, within Xab species E, was identified by correlating Y- populations were seen. In a large sample chromosome polymorphism of sons and the examined, only four double-inversion sporozoite positivity of mothers. Laboratory heterozygotes (2g1+h1 /+g1h1) were colonies established from the progeny of observed; therefore, Xab; 2g1+h1 and Xab; single females with species-specific inversions 2+g1h1 were considered as two were used subsequently to study post-zygotic reproductively isolated populations. The new isolation mechanisms between the sibling population with Xab; 2+ g1h1 was designated species. as species C (Subbarao et al., 1983).

Species A and B (Green and Miles, 1980) Species D (Subbarao, Vasantha and Green and Miles (1980) found two distinct Sharma, 1988a; Suguna et al., 1989; polytene X-chromosomes in the An. Vasantha, Subbarao and Sharma, 1991) culicifacies s. l. population in the village Okhla In a few populations in northern India, the i1 near Delhi, India. One X-chromosome was inversion on chromosome arm 2 was found with two paracentric inversions a and b and polymorphic in species A, and it was found the other with a banding pattern that fixed in the southern Indian populations of resembled the polytene chromosome An. culicifacies species A (Subbarao, 1984). photomap described by Saifuddin, Baker and The latter population with the X+a+b; 2i1 Sakai (1978). No heterozygotes for these arrangement was found sympatric with inversions were found. The absence of species B. The evidence for reproductive heterozygotes was taken as an evidence of isolation between species A (X+a+b; reproductive isolation between the two 2+g1+h1) and the population with the populations which were considered as two X+a+b; 2i1 h1 arrangement came from two distinct species. The population with the locations (inversion i1 includes the g1 inversion standard arrangement, X+a+b, was region and the distal breakpoint is the same designated as species A and that with Xab for both the inversions). In northern and arrangement as species B. Green and Miles central India, a deficiency of heterozygotes in the same paper (1980), by examining was found for i1 inversion in the X+a+b laboratory colonies of An. culicifacies s. l., populations (Subbarao, Vasantha and reported species A and B from Pakistan and Sharma, 1988a; Vasantha, Subbarao and species B from Sri Lanka. Following this Sharma, 1991) while in southern India a total report, Subbarao, Adak and Sharma (1980) absence of heterozygotes was found between by examining field populations, confirmed species A (X+a+b; 2+g1+h1) and the the presence of species A and B within An. population with the X+a+b; 2i1+h1 culicifacies in the villages of Haryana and arrangement (Suguna et al., 1989). The Uttar Pradesh, both states bordering Delhi. X+a+b population with the new chromosome 2 arrangement 2i1+h1 was Species C (Subbarao et al., 1983) designated as species D. The explanation for The examination of polytene chromosomes the heterozygotes (+i1/ i1) of i1 inversion of An. culicifacies from villages around Delhi observed in northern India is that this and in the states of Gujarat and Madhya inversion is floating in species A with varied Pradesh, India, revealed the presence of two frequencies in different populations and is fixed inversions on chromosome arm 2. fixed in species D (Vasantha, Subbarao and Species B was fixed for the g1 inversion in Sharma, 1991).

Anopheline Species Complexes in South and South-East Asia 23 Species E (Kar et al., 1999) mechanisms were also observed. From one In Rameshwaram island, Tamil Nadu (a state of the reciprocal crosses between species A in southern India), An. culicifacies females and B, Miles (1981) observed hybrid male had the Xab; 2 g1+h1 inversion arrangement sterility. In this study, crosses were carried on polytene chromosomes, which is out by the forced copulation technique diagnostic for species B. However, the between the progeny of single females. biological characters of this population were Hybrid males from the species B female x A different than those observed for species B male cross were fertile. Mahmood, Sakai and on the mainland. This prompted a detailed Akhtar (1984) reported fertility of hybrid examination. Because homosequential males varying from 10 per cent to 96 per cent species exist in nature, Subbarao et al. (1993) depending on the species B strain used in investigated the mitotic karyotype variations. the cross between B female and A male. The progeny of the field-collected females Subbarao, Vasantha and Sharma (1988b) revealed the Y-chromosome to be observed almost total sterility with an polymorphic with acrocentric and occasional hatch of 1 to 5 per cent in the B submetacentric types. Because the Y- female x A male cross, in contrast to 90 per chromosome has patroclinal inheritance, the cent hatch in the A female x B male cross. variation observed could not be used to Crosses by these authors were carried out in indicate reproductive isolation. Correlation cloth cages by normal matings. However, in with sporozoite positivity i.e. total absence crosses where a low hatch was observed in of sporozoite positives among females whose the B female x A male cross, hybrid males sons had acrocentric Y-chromosome and were sterile, having fully developed presence of sporozoite positives among reproductive organs but without any sperm. mothers whose sons had submetacentric Y- In the reciprocal cross (A female x B male), chromosome, was taken as evidence for the reproductive organs were partially assortative mating between acrocentric and developed, testes and vas deferens were submetacentric populations. The two either totally absent, atrophied or reduced, populations were considered as two while the ejaculatory duct and accessory sympatric species. The vector population glands were normal (Miles, 1981; Subbarao, with the submetacentric Y-chromosome was Vasantha and Sharma, 1988b). The results designated as species E and the non-vector from the crosses between species A and C population with acrocentric type Y- were similar to those between species A and chromosome retained the original B, except that in the C female x A male cross, designation of species B (Kar et al., 1999). no egg hatch had been observed so far (Subbarao, Vasantha and Sharma, 1988b). There seem to be at least five specific Reciprocal crosses between species B and C mate recognition systems operating in An. produced fully fertile F1 hybrid males and culicifacies, leading to reproductive isolation females, suggesting that there is no post- and lack of gene flow between the zygotic barrier between B and C. Species E populations; hence, there are at least five resembled species B and C in the crosses to sibling species in this complex. species A, and there was no post-mating barrier in the crosses with species B and C Post mating isolation mechanisms (Kar et al., 1999). (Mahmood, Sakai and Akhtar, 1984; Subbarao, Vasantha and Sharma, 1988b; Techniques available for Kar et al., 1999) identification of sibling species In addition to the pre-mating isolation The techniques available for the identification mechanisms, post-mating isolation of sibling species are summarized in Table 4.

24 Anopheline Species Complexes in South and South-East Asia Table 4: Techniques for the identification of An. culicifacies sibling species*

Polytene Mitotic LDH Cuticular Species- PCR-RFLP ASPCR Species chromosome karyotype- enzyme hydro- specific inversion Y-chromosome alleles carbon DNA genotypes profile probes

A X+a+b; 2+g1+h1; Submetacentric Fast Identified Yes Yes Yes +i1/i1 B Xab; 2g1+h1 Acrocentric Slow Identified Yes Yes Yes Submetacentric C Xab; 2+g1h1 Acrocentric Slow Identified as B as B Yes Submetacentric 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

Polytene chromosomes In a situation such as (c), a population The use of diagnostic fixed inversions genetic analysis will indicate the presence of readable on the polytene chromosomes species D but individual specimens cannot (Subbarao, Vasantha and Sharma, 1988a) has be identified as species D (Vasantha, been the only technique available until Subbarao and Sharma 1991). recently for the identification of sibling species The photomaps of the polytene in this complex. This technique has been chromosome complement of An. culicifacies extensively used to study the biological have been reported by Saifududdin, Baker characters and establish the role of the sibling and Sakai (1978). Chromosome maps with species in malaria transmission. The problems breakpoints of inversions a and b on the X- in using this technique are: chromosome are given in Green and Miles (i) Species B and species E are (1980), and for those on the X-chromosome 1 1 1 homosequential, hence cannot be and of g , h and i on chromosome arm 2, differentiated. in Subbarao et al. (1983) and Subbarao, Vasantha and Sharma (1988a). The (ii) With reference to species A and D and photomaps from the latter paper are i1 inversion, one encounters any of the reproduced here as Figure 4. following situations in a population: In addition to fixed inversions in this 1 1 (a) +i and i homozygotes without any complex, six inversions, five on chromosome heterozygotes, suggesting that species arm 2 (j1, k1, l1, i1 and o1) and r on arm 3 A and D are present; were found polymorphic in species A, and in 1 (b) + i1, i1 and + i1/ i1 (heterozygotes) in species B, l on arm 2 and r on arm 3 were expected proportions, suggesting that seen in natural populations. The breakpoints only species A is present and i1 is of all these inversions are mapped on polymorphic; photomaps by Vasantha, Subbarao and Sharma (1991). (c) similar to (b) but with a significant deficiency of heterozygotes, Mitotic karyotypes suggesting that species A and D are Initial studies carried out on limited samples present, and in species A i1 is for mitotic karyotypes in sibling species polymorphic. revealed Y-chromosome characters to be

Anopheline Species Complexes in South and South-East Asia 25 diagnostic for the identification. analysed by gas liquid chromatography. The Submetacentric Y-chromosomes in species A three species were found to be significantly and C and acrocentric in species B were different in their cuticular hydrocarbon observed by Vasantha et al. (1982; 1983). composition by multi-variate analysis of Suguna et al. (1983) also observed variance. The best separation between the corresponding differences in species A and B species was obtained using 27 peaks in a populations from southern India. Adak et al. discriminant analysis. Using the (1997) reported both acrocentric and chromatographic characteristics of these submetacentric Y-chromosome poly- peaks, each specimen analysed was assigned morphism in species B and C populations in to the group to which its probability of several areas surveyed. Species E has a membership was the greatest. With this, the submetacentric Y-chromosome (Kar et al., average correct identification was 78 per cent. 1999). Thus, the Y-chromosome cannot be Analysis of a small number of field samples used as a diagnostic tool for the identification also exhibited intraspecific variability similar of members of the complex except for to that observed in laboratory cage samples. species E. Mitotic karyotypes of specie B and E are given in Figure 5. DNA probes Three highly repetitive DNA sequences, Rp Electrophoretic variations 36, Rp 217 and Rp 234, were selected from Out of the nine enzyme systems studied, a genomic library of species B (Gunasekera electrophoretic variation in lactate et al., 1995). Radio-labelled fragments of Rp dehydrogenase (LDH) was found to be 36, Rp 217 and Rp 234 gave positive signals diagnostic (Adak et al., 1994). Two in dot-blot hybridization assays with sibling electromorphs, fast (F) and slow (S), were species A, B and C. The hybridization signal represented at the Ldh locus. The frequency given by species A was much less than that of LdhF in species A and D varied between given by species B and C. Species A can be 0.94 and 1.00, as against 0.0 and 0.19 in distinguished from species B and C when species B and C. Because of the low-level single-mosquito extracts are diluted 200-fold polymorphism observed within each species, and assayed by dot-blot hybridization with the power of this technique in the any of the three probes, which then give a identification of sibling species was evaluated negative signal for species A. These probes by the authors using three indicators: have not been evaluated in different sensitivity, specificity and predictive value. geographical regions. Overall, the probability of correct separation of species pairs by LDH enzyme was PCR-RFLP determined as 94.6 per cent. In species E, The use of restriction endonclease Rsa I for LdhS was found (Kar et al., 1999), thus, this the ITS2 amplicon and Alu I for mitochondrial species falls into the same category as species cytochrome oxidase (CO) subunit II grouped B and C. As Ldh is autosomal and is expressed species A and D in one category and B, C at all stages of the life-cycle, Ldh allozyme and E into another (Goswamy et al., 2005). method is useful in the identification of sibling The COII amplicon digested with Dde 1 species in certain sympatric associations. distinguishes species E from species B and C.

Cuticular hydrocarbon profiles ASPCR Cuticular hydrocarbon profiles were Three PCR assays from the rDNA cistron were examined in species A, B and C (Milligan et developed for the identification of sibling al., 1986). Cuticular wax extracted from species of the Culicifacies Complex. One was single specimens from pure stocks was developed from the variable D2 domain

26 Anopheline Species Complexes in South and South-East Asia (Cornel et al.,unpublished) and the second Distribution and biological from the D3 variable domain (Singh et al., characteristics 2004) of the 28S rDNA cistron. Both these In India all five species of the Culicifacies assays distinguish species A and D from Complex have been identified. Species A species B, C and E but fail to distinguish identified in Yemen (Akoh, Beidas and White, species within each of these groups. Primers 1984) and Iran (Zaim et al., 1993) has been for both these have been evaluated against found sympatric with species B in Pakistan field-collected and cytotaxonomically- (Mahmood, Sakai and Akhtar, 1984). identified specimens. A third assay reported Recently, An. culicifacies has been by Curtis and Townson (1998) was developed incriminated as one of the eight species from the ITS2 region of the 28S rDNA cistron. responsible for malaria transmission in The assay distinguishes species A from species Afghanistan (Rowland et al., 2002). A few B. Other species have not been tested with specimens from Afghanistan were these primers and this method has not been cytotaxonomically identified several years ago evaluated on field samples. Recently, as species A. Larger samples from different sequence analysis of ITS2 region revealed that regions of the country need to be examined species D is similar to species A and species for sibling species composition. Extensive C and E to species B (Goswami et al., 2005). surveys carried out in Sri Lanka using polytene Two allele-specific PCR assays, AD-PCR chromosomes (Abhayawardena et al., 1996) and BCE-PCR, were developed from the COII and DNA probe (de Silva et al., 1998) region of mitochondrial DNA to distinguish the identified only species B. Surendran et al. species. The strategy in this assay is that once (2000) in Sri Lanka found both acrocentric the two groups, i.e. species A and D and and sub-metacentric Y-chromosome types in species B, C and E are identified either by An. culicifacies population and by analogy allele-specific PCR of the D3 or D2 regions or with the situation in Rameshwaram island as by the PCR-RFLP assays of the ITS2 and COII proposed by Kar et al. (1999) they assumed regions (Goswamy et al., 2005), one can use that the acrocentric population was species specific assays developed from the COII region B and sub-metacentric type was species E. i.e. the AD-PCR assay to distinguish species A Recently, cytologically-identified An. and D and the BCE-PCR assay to distinguish culicifacies sub-metacentric and acrocentric species B, C and E (Goswamy et al., 2006). and unidentified sensu lato specimens from This assay was evaluated on An. culicifacies Sri Lanka were assayed by the PCR method collected from different areas of India, and of Goswami et al. (2006). Specimens simultaneously, identifications were correlated belonging to the sub-metacentric and with cytological identification based on acrocentric categories were identified as species-specific diagnostic inversions. species E and B respectively. Among the cytologically-unidentified specimens, both Microsatellite markers species B and E were also found (Surendran, About 31 microsatellite markers were Sri Lanka, and K. Raghavendra, MRC, India, developed from the An. culicifacies species personal communication). This establishes A. Some of the markers tested were found that in Sri Lanka, species B and E are polymorphic in species A, B and C. The allele sympatric as in Rameshwaram island in India. number varied from 2-12 (Sunil et al., 2004) Baimai, Kijchalao and Rattanarithikul (1996)

Anopheline Species Complexes in South and South-East Asia 27 reported species A and B from the Chiang Ramanathapuram district, species B and E Mai province of Thailand. These were found sympatric. The distribution of identifications were based on mitotic species E in other areas is yet to be mapped. karyotypes described by Vasantha et al. A map showing the distribution of sibling (1982; 1983). Species A was identified as species in India is reproduced from Subbarao the karyotype which had a submetacentric (1991) as Figure 6. In this map, the recently Y-chromosome and species B as that which found species E is also shown. had an acrocentric Y-chromosome. Later, it The sibling species were not only found was pointed out that Y-chromosome to have definite distribution patterns but, in polymorphism had been observed in species a given area, the prevalence of a species B and also in species C (Adak et al., 1997). varied according to the seasonal Taking into consideration that species B, environmental changes. Species A was found which is found in the eastern districts of the predominant throughout the year. An state of Uttar Pradesh and in the state of increase was observed in the proportion of Assam in India, the population in Thailand species B in the post-monsoon months in may be species B with Y-chromosome villages around Delhi, where A and B were polymorphism. In Cambodia, Van Bortel et sympatric (Subbarao et al., 1987). Similar al. (2002) reported the presence of species B observations were made in the district of based on the ITS2 sequence analysis. Surat, in Gujarat state, where species B and Following this, the authors also suggest that C were found sympatric. Species C of Gujarat An. culicifacies from the Sichuan province in behaved like species A did in and around China is also species B. This information on Delhi (Subbarao, unpublished), and also in the distribuition of sibling species clearly the district of Sundergarh, Orissa state ( indicates that the distribution of species A Nanda et al., 2000). extends to countries to the west of India while that of species B to the east of India. Biological variations such as host- specificity, susceptibility to malarial parasites In India, where all the five sibling species and response to insecticides have also been are prevalent, species B was found almost noticed between the species. An. culicifacies everywhere throughout the country wherever s.l. is predominantly zoophagic and feeds on An. culicifacies was encountered. Species B man only when the cattle population is low was found exclusively in some areas, whereas (Rao, 1984). The four species, A, B, C and D, in other areas it was found sympatric with A were found to be predominantly zoophagic. or C or D or E alone or in combination However, species A was found with a (Subbarao, Vasantha, Sharma, 1988a; relatively higher degree of anthropophagy Subbarao, 1991, Kar et al., 1999). Species A (about 3.5 per cent), compared to species B and B are sympatric in northern and southern in several areas (Joshi et al., 1988). Species India, with the predominance of species A in C and D were also found to have low the north and species B in the south. anthropophagy (<1 per cent). In However, in the eastern states of north India, Rameshwaram island, where species B and species B predominates or is the only species E are sympatric, An. culicifacies s.l. was found present. Species B and C were predominant to be much more anthropophagic than in in the western and eastern regions, while other areas (Jambulingam et al., 1984, Hema species D was found in sympatric association Joshi, personal communication). By analogy, with A and B in the north-western region, as species B has low anthropophagy, species and with A, B and C in central India and in a E can be considered to be contributing to high few places in the state of Tamil Nadu in the anthropophagy in Rameshwaram island. south, where, in Rameshwaram island and also in a few blocks on the mainland in

28 Anopheline Species Complexes in South and South-East Asia The biting rhythms were distinct among colony was identified as species B several species A, B, C and D (Satyanarayan, 1996). generations after its establishment. In The biting activity of species A, B and C was laboratory feeding experiments on P. vivax- found all through the night, while no biting infected blood (Adak, Kaur and Singh, 1999) activity of species D was observed after and P. vincei petteri and P. yoelii yoelii-infected midnight. The peak biting activity of species mice (Kaur, Singh and Adak, 2000), species A and B was in the second quarter of the A had significantly higher oocyst rate night, between 2200 hours and 2300 hours; compared to species B and C and species B while the peak biting activity of species C was was found the least susceptible. Recently, a found to be in the first quarter of the night, strain totally refractory to P. vivax and partially between 1800 and 2100 hrs., in the month to P. falciparum was isolated from species B of April. However, the peak biting activity of (Adak et al., 2006). In this strain malaria species C shifted to the second quarter of the parasites are encapsulated very early in the night in December in one of the study areas. oocyst development leading to the death of Whether this is true in all areas where species the parasites. The low vectorial potential of C is prevalent needs to be examined. No species B in malaria transmission was also seasonal change was observed for species A confirmed from our observation where there and B. Though the peak activity of species D is no malaria, or its incidence is low, as in the was between 2100 hours and 2200 hours, eastern districts of Uttar Pradesh and in 30 per cent to 40 per cent of this species bite northern Bihar in India, where species B is in the first quarter (this is significantly higher predominant (Subbarao et al., 1988a). The than that observed for species A and B). identification of species E, a vector species, now explains and resolves the In northern India, where species A and epidemiological paradox that sporozoite- B are sympatric, in the cytologically-identified positive specimens were found in An. specimens of species A, sporozoites were culicifacies s. l. in Rameshwaram island found (Subbarao, Adak and Sharma, 1980). (Sebesan et al., 1984), and specimens from Using the two-site immunoradiometric assay the same area were identified as species B technique of Zavala et al. (1982), species A, by polytene chromosome examination. C and D were found to be vectors of P. vivax Similarly, in Sri Lanka, only species B (Green and P. falciparum malaria and species B a poor and Miles, 1980; Subbarao 1988; vector if at all (Subbarao et al., 1988b; 1992). Abhayawardena, Dilrukshi and Wijesuriya, The cumulative sporozoite rates for species 1996) and several sporozoite-positive A, C and D are 0.51 per cent, 0.3 per cent specimens were found (Amerasinghe et al., and 0.4 per cent respectively in India 1991 & 1999). The recent molecular (Subbarao and Sharma, 1997). In Pakistan, identification of An. culicifacies from Sri Lanka where species A and B are sympatric, species as species B and E (mentioned earlier in this A was incriminated and is considered the section) also resolved the epidemiological major vector (Mahmood, Sakai and Akhtar, paradox confronted with the cytotaxonomic 1984). In the same paper, the authors identifications of this species. In Iran, where reported that though species B could not be only species A is prevalent, it was incriminated incriminated under field conditions, in for P. vivax sporozoites antigen in May and laboratory feeding experiments both species An. pulcherrimus for P. vivax antigen in the A and B supported sporogony of P. vivax and September and October collections by P. falciparum. Species B used in this study was immunoradiometric assay in the same study supplied by the Malaria Research Centre, (Zaim et al., 1993). In Afghanistan, in river- Delhi, India. This colony was established from irrigated rice-growing villages, of the eight An. culicifacies s.l. collected from an area species incriminated, An. culicifacies had the where both A and B are sympatric. The

Anopheline Species Complexes in South and South-East Asia 29 lowest sporozoite rate of 0.20 per cent (only breeding in rock pools and sand pools along An. splendidus had lower rate than An. river margins. These studies need to be culicifacies) (Rowland et al., 2002). An. extended to more areas, and sporozoite culicifacies s.l. is resistant to DDT and HCH positivity has to be examined in both species in most parts of India, and in a few areas to B and E in field collections too. Species E malathion as well (Subbarao, 1988). Recently, was observed to be more resistant to this species was reported to be showing malathion than species B (Surendran et al., resistance to pyrethroids in Gujarat (Singh et 2003). Both species B and E were found al., 2002) and in Tamil Nadu (Mittal et al., equally susceptible to deltamethrin and 2002). lambdacyhalothrin. The Sri Lankan research groups are now planning to carry out these Species A remains more susceptible to studies in detail with field-collected DDT than species B in areas where both A specimens and identifications by molecular and B are sympatric and DDT has been methods of Goswami et al. (2005 & 2006) withdrawn for long periods (Subbarao, (N. Surendran, personal communication). Vasantha and Sharma, 1988c). In areas with species B and C sympatric association, species Taking into consideration the biological C developed resistance to malathion at a differences observed among sibling species, faster rate than did species B (Raghavendra an insecticide spray strategy for the control et al., 1991), and species A at a slower rate of An. culicifacies in India has been proposed than species B (Raghavendra et al., 1992). (Subbarao and Sharma, 1997). Later, In Gujarat, where An. culicifacies recently Subbarao, Nanda and Raghavendra (1999) developed resistance to pyrethroids, stratified the country into seven major sympatric species B and C exhibited a similar divisions based on the prevalence of An. pattern in the resistance levels (Singh et al., culicifacies sibling species. An. fluviatilis sibling 2002). species’ prevalence was also taken into consideration while proposing specific control Surendran et al. (2002a & b; 2003) strategies in each of these divisions. It is studied the biological variations between Y- considered that the implementation of this chromosome polymorphic types. Though the strategy will lead to more efficient use of authors refer in their reports to these insecticides and this will reduce the polymorphic types as species B and E, these expenditure on insecticides and their impact studies were carried out before the two forms on the environment. In Karnataka state, were correlated either with sporozoite southern India (Ghosh et al., 2005), positivity, as has been done by Kar et al. selectively-introduced larvivorous fish into (1999), or with molecular identifications of tanks and wells brought down the densities Goswami et al. (2005 & 2006). As mentioned of An. culicifacies, the major vector, and earlier in this section, the two Y-chromosome consequently malaria incidence in several polymorphic forms in Sri Lanka have recently villages. The concentration on tanks and wells been identified as species B and E by and omission of streams was based on the molecular assays. Surendran et al. (2002a) finding that species A, the vector species, reported that species E larvae were found in breeds predominantly in wells and tanks and a wide variety of breeding sites and this species B, the non-vector species, in streams. species developed oocysts when fed on P. This study emphasizes the advantage of vivax and P. falciparum-infected patients sibling species identification and taking (Surendran et al., 2002b). In species B, account of their biological characters while oocysts were not developed and it was found implementing vector control strategies.

30 Anopheline Species Complexes in South and South-East Asia Figure 4: Schematic representation of polytene chromosomes of Anopheles culicifacies sibling species

X chromosome

a ab + +b

b b a b b a a a

Chromosome arm 2

1 1 1 1 1 g h 1 h 1 h g 1 + + i + g + + h i1 + i1

h1 1 1 h 1 h h h1 h1 h1 h1 1 i 1 g g1 i1 g1

g1 g1 g1 i1 i1

Species A Species D Species B Species C

Anopheline Species Complexes in South and South-East Asia 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 AB AB AB AB AB A B AB A BCD A BD B B ABC A B B B ABC A B ACD BCB BC A B BC A ABCD A BCD BC BC ABCD A BCD A BC BC A BC A BC BC BC BC A BC D ABC BC A BC D A BC BC BC BC ABCD BC BC AB BC BC BC ABC ABC B C BC B AB AB Species A A B AB A B B ABCD Species B B BD AB Species C BE Species D Species E

32 Anopheline Species Complexes in South and South-East Asia 3.4 The Dirus Complex (iii) An. balabacensis Perlis form is now species B and An. balabacensis Fraser’s The Dirus Complex belongs to the subgenus Hill form is now species F of the Dirus Cellia, Leucosphyrus Group in the Complex. Neomyzomyia Series (Harbach, 2004), and (iv) An. balabacensis Taiwan form has been occurs in the Oriental region. There has been raised to the species status by Peyton and a considerable interest in the Leucosphyrus Harrison (1980) and has been designated Group as three members of this group, as An. takasagoensis Morishita. This is now Anopheles dirus, An. balabacensis and An. included as a member in the Dirus Complex. leucosphyrus, are important malaria vectors in South and South-East Asia. However, there (v) The mosquitoes now given the name An. has been much confusion, in the literature balabacensis and included as one of the regarding the taxonomy of this group. Peyton members of the Leucosphyrus Complex (1989) resolved the confusion and included (Table 5) are those belonging to the taxon 20 species and two forms belonging to the described from Sabah, east Malaysia. Neomyzomyia series in the Leucosphyrus (vi) An. elegans earlier described under the Group based on the following morphological Elegans Subgroup has been redescribed characteristics. In the adult stage they possess and is now placed in the Dirus Complex a very broad conspicuous white-scaled band (Sallum, Peyton and Wilkerson, 2005) covering the apex of the hind tibia and the (Table 5). base of hind tarsomere 1 and speckled legs. The wings have many discrete pale- and dark- Table 5: Species now (2005) * included in the scaled spots on all veins and with four or more Leucosphyrus Group dark spots present on vein Cu-A and terminal Leucosphyrus subgroup abdominal segments always with some scales. 1. An. baisasi 2. Con Son island, Viet Nam Form Following a detailed morphological study, Leucosphyrus Complex Sallum, Peyton and Wilkerson (2005) 3. An. balabacensis Baisas reclassified the species belonging to the 4. An. introlatus Colless Leucosphyrus Group under three Subgroups: 5. An. leutens (leucosphyrus A) 6. An. leucosphyrus s.s. (leucosphyrus B) Leucosphyrus, Hackeri (earlier referred to as Dirus Complex Elegans) and Riparis (new). Leucosphyrus and 7. An. dirus (species A) Dirus are the two Complexes within the 8. An. cracens (species B) Leucosphyrus Subgroup (Table 5). 9. An. scanloni (species C) 10. An. baimaii (species D) The name, An. balabacensis, has in the 11. An. elegans (species E) past been frequently used in a misleading way 12. An. nemophilus (species F) until Peyton and Ramalingam (1988) and 13. An. takasagoensis Peyton (1989) amended the Leucosphyrus Hackeri (elegans) subgroup Subgroup as follows: 1. An. hackeri Edwards (i) It now includes what was described as 2. An. pujutensis Colless 3. An. sulawasi Waktoedi An. balabacensis in the South-East Asian 4. An. leucosphyrus Sumatra form mainland but is now referred to as An. dirus (Peyton and Harrison 1979). Riparis subgroup, new subgroup 1. An. cristatus King & Baisas (ii) The Balabacensis Complex referred to by 2. An. macarthuri Colless Baimai, Harrison and Somchit (1981), 3. An. riparis King & Baisas Baimai et al. (1984) and Hii (1985) no 4. Negros form longer exists. 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 form as species B. A cross between B female found in India, Nepal, Bangladesh, Myanmar, and A male produced sterile hybrid males. Thailand, Indonesia, Malaysia, Viet Nam, Mitotic karyotypes of these two strains were Cambodia, south China, and Taiwan. This described by Baimai, Harrison and Somchit complex includes seven members recognized (1981). and two more suspected (details in the following sections). Peyton and Ramalingam Species C and D (Baimai et al., 1987) (1988) provided the morphological and A strain from Kanchanaburi, Thailand, geographical descriptions of the Dirus derived from a single female culture and Complex for the first time. They described mitotically different from species A and B the morphological characters of this complex (Wibow, Baimai and Andre, 1984) was which distinguish it from other members of designated as species C as males in one the Leucosphyrus Group. direction in crosses with species A, and in crosses with species B it produced no The most significant adult character is: progeny. Species A female and C male cross accessory sector pale (ASP) spot on the costa produced sterile F males. The strain from and usually also on the subcosta (an 1 Ranong and Phangna, Thailand, with a mitotic occasional male in some species belonging karyotype different from those of species A, to the Leucosphyrus Complex exhibits an ASP B and C was designated as species D as it spot on the costa, but always in less than 6 produced no progeny in reciprocal crosses per cent of specimens in any population). with species A, B and C. Additional adult characters that serve to characterize this complex are: presector dark Natural populations comprising mixtures (PSD) spot on wing vein R with one or more of the chromosomally identified species A, pale spots on at least one wing (except B, C and D were examined for occasional specimens of An. nemophilous) electrophoretic variations of six enzyme and hind tarsomere 4 with a distinct basal systems to associate chromosomal forms with pale band or dorsal patch. allozyme genotypes (Green et al., 1992). Data were analysed by GENESTAT of Black

Evidence for identification of sibling and Krafsur (1985). The mean value of FIS for species An. dirus sensu lato (sympatric species) was +0.28 (SD 0.02). The partitioning of The cytological (mitotic karyotype) analyses electromorph data for the chromosomal of natural and laboratory colonized forms reduced the mean F to +0.03 (SD populations, crossing experiments between IS 0.01), suggesting that positive assortative populations and morphological variations mating is a characteristic of each form (values observed in natural populations have led to of F range from +1.0 for total absence of the recognition of members of this complex. IS heterozygotes in mixtures of two or more Species A and B (Hii, 1985) homozygotes to -1.0 for total absence of homozygotes). Furthermore, significant The unidirectional F hybrid male sterility 1 deviations observed by chi-square analysis observed between the Bangkok strain were removed by partitioning. This indicates identified as An. dirus by Peyton and Harrison that different electromorphs at each locus are (1979) and the Perlis form strain was the first associated with one or the other of the evidence that An. dirus is a complex of two chromosomal forms, which supports the sibling species (Hii, 1985). The Bangkok strain finding that chromosomal forms are was designated as species A and the Perlis reproductively isolated populations.

34 Anopheline Species Complexes in South and South-East Asia Species E (Sawadipanich, Baimai and and An. introlatus (a member of the An. Harrison, 1990) leucosphyrus complex). A colony established from mosquitoes An. takasagoensis Morishita collected from south-western India, and An. balabacensis Taiwan form was elevated which produced sterile F1 hybrid males in all reciprocal crosses with species A, B, C and to species status by Peyton and Harrison D, was designated as species E. The mitotic (1980) and designated as An. takasagoensis. karyotype was described as different from The mitotic karyotype of this species was those of species A and B, and resembling that described by Baimai, Harrison and Somchit of species D. (1981). At that time it was described as a member of the An. balabacensis complex. Sallum, Peyton and Wilkerson (2005) This species is not sympatric with other examined morphologically more than 8000 members of the An. dirus complex and is specimens from seven countries for found only in Taiwan (Peyton and taxonomic revision of the Leucosphyrus Ramalingam, 1988). This species is included Group. After describing larval, pupal and in the An. dirus complex because of its adult morphological characters of the possession of diagnostic morphological provisionally designated members of the characters of An. dirus. Dirus Complex, they have given formal designations to sibling species A, B, C, D and In addition to taking structural and E (see Table 5 for the formal designations). heterochromatin variations in mitotic karyotypes and hybrid sterility in interspecific Species F (Baimai, Harbach and crosses as evidence for designating species, Kijchalao, 1988) the asynapsis observed in polytene From the mosquitoes collected from the Thai- chromosomes of F1 progeny of interspecific Malaysia border, an isofemale line selected crosses was also taken into consideration (Hii, due to its new cytotype was designated as 1985; Baimai et al., 1987; Baimai and Green, species F as it produced hybrid males with 1988; Sawadipanich, Baimai and Harrison, atrophied testis lobes in crosses with species 1990). A, B, C and D and also with An. balabacensis Two suspected new species from Sabah. Morphologically, it resembled the Fraser’s Hill form of An. balabacensis The ITS2 sequence analysis revealed two recognized by Colless (1956; 1957). The types of sequences of species C, from mitotic karyotype resembled that of species northern Thailand (site 12) and another of B in having acrocentric sex chromosomes species C from southern Thailand (site 14) (Table 6 and Figure 7) but the (Walton et al, 1999). The population genetic heterochromatic short arm is smaller than that analysis using 11 microsatellite markers of of species B. Autosome III is metacentric northern and southern populations of species because of a large block of heterochromatin C in Thailand, showed significant differences while this chromosome is submetacentric in in the genetic structure (Walton et al., 1999 species B and also in other species. & 2001). Because of the geographical separation of these populations, it is not Peyton and Ramalingam (1988) have possible to distinguish the hypothesis of the formally described species F (Fraser’s hill existence of two species from that of form) as An. nemophilus after comparing the geographical variation within species. voucher specimens with the cytotype of species F. This paper has a detailed From the sequence analysis of morphological description of An. nemophilus amplifications from PCR of the ITS2 region, which is compared with An. dirus (Species A) 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 The tips of chromosomes X, 2R and 2L species D from China as reported by Xu, Xu can be used as diagnostic characters for the and Qu (1998). The authors suggest that identification of species. Species B has a fan- species D from China may represent yet shaped tip on the X-chromosome, which is another species in this complex. quite distinct (Hii, 1985; Baimai, Poopittayasataporn and Kijchalao, 1988). Techniques available for Inversion a on the X-chromosome in species identification of sibling species A is polymorphic with the inversion in a The techniques available and their diagnostic minority compared with the standard features for the identification of sibling species arrangement. Species D is highly polymorphic are given in Table 6. Additional information, with at least a single paracentric inversion in for which there was no space in the table, each autosome (break points are given in and the relevant references are given below. Baimai, Poopittayasataporn and Kijchalao, 1988). In species D and E hybrids, the X- Mitotic karyotypes chromosome was found totally synapsed; Baimai (1989) gives diagrams of the mitotic therefore, it is inferred that it is also fixed for karyotypes of all sibling species and their the a inversion of species D (Sawadipanich, distribution in South-East Asia (this figure is Baimai and Harrison, 1990). The same paper reproduced as Figure 7). All species have two also reports that species E possesses floating pairs of autosomes, metacentric autosome II inversions in at least two autosomes, but no and submetacentric autosome III, and X and details are given of these inversions. Y sex chromosomes. Variations in the Though initially mitotic karyotypes were structure and blocks of heterochromatin in used in the identification of sibling species, chromosomes are used in the identification. Baimai, Poopittayasataporn and Kijchalao In addition to the variations shown in Table (1988) report that polytene chromosomes are 6, the following features can be used in the now being extensively used, and find this diagnosis: method more convenient. (i) Species B has centromeric Electrophoretic variations heterochromatin in the autosomes (Baimai and Traipakvasin, 1987). Green et al. (1992) reported that Observation of five types of X- electrophoretic phenotypes were suitable for chromosome and four types of Y- the identification of sibling species. In this chromosome suggests extensive study, two sets of samples were used: (i) polymorphism in this species. chromosomally-identified samples which included species A, B, C and D; and (ii) (ii) Species F has a block of centromeric specimens identified by DNA probes which heterochromatin in autosome III which included sympatric populations of species A makes the chromosome resemble the and D. Species A and D could be identified metacentric type (Baimai, Harbach and with an accuracy of 99 per cent using GCD Kijchalao, 1988). The short arms of the and AAT enzyme systems. Species B could sex chromosomes are smaller than those be identified by slow electromorph(s) of ACP, of species B. while in species A, C and D, a fast electromorph was observed. As a marker, Polytene chromosomes human whole blood with normal A standard map of polytene chromosomes haemoglobin was used (photographs of gels of speices A has been prepared and has been with a scale were presented to show the used in comparative studies of the actual distances moved by alleles of different chromosomes of other species (Baimai, enzyme systems and haemoglobin). Poopittayasataporn and Kijchalao, 1988).

36 Anopheline Species Complexes in South and South-East Asia Egg morphology under light and scanning polymorphism (Yasothormsrikul, Panyim and electron microscopes (Damron-gphol and Rosenberg, 1988) in species A, B, C and D. Baimai, 1989) Seven enzymes (Ava II, Alu I, Bgl II, Hae III, The egg size of species A (length 0.524mm x Hinf 1, Mbob and Sau 3A I) produced unique width 0.124 mm) and C (0.548mm x patterns for each species while other enzymes 0.143mm) was intermediate between that of produced unique patterns for one or two species B (0.570mm x 0.146mm) and species species. D (0.515mm x 0.144mm). The number of Allele-specific PCR (ASPCR) assay rows of cells formed by tubercles in the outer chorionic membrane between the frill and Xu, Xu and Qu (1998) reported a PCR assay the float was found to be species-specific using three primers, one derived from highly (Table 6). Size variation and cells formed by conservative 5.8S coding sequences and two tubercles could be observed under the light from the internal transcribed spacer (ITS2) microscope. The tubercles that formed region of ribosomal DNA. This assay aggregates on deck surface were species- distinguished species A from species D with specific (Table 6) and were examined under 374 base pairs and 663 base pair length the scanning electron microscope. amplicons respectively. Walton et al. (1999) reported a multiplex Pupal setae under scanning electron PCR assay with primers designed from microscope ribosomal DNA internal transcribed spacer- An. dirus Bangkok strain (now identified as 2 (ITS2) sequences of different species. This species A) and An. balabacensis Perlis form assay identifies species A (An. dirus s.s.), B, (now identified as species B) were C, D or F (An. nimophilous) found in Thailand distinguished with 100 per cent accuracy by in a single reaction. Under the assay examining pupal setae under the scanning conditions developed, no products were electronmicroscope (Choochote et al., 1987). generated with DNA from individuals of An. An. dirus species A has a stout and simple leucosphyrus, An. macarthuri or An. pujuteasis pupal seta 9-IV on both right and left sides, found in Sarawak. An. hackeri belonging to while in species B it is long and slender and the Elegans (now Hackeri) Subgroup gave two usually has side branches. There is no report faint bands of intermediate length between on the evaluation of this observation in the those produced by An. dirus B and C, and field population. thus would not result in a false positive identification. Using this assay, 179 field- DNA probes collected An. dirus s.l. from 15 sites distributed Panyim, Yasothornsrikul and Baimai (1988) throughout Thailand and Malaysia were report four species-specific DNA sequences, identified as one of the five species expected. and Audtho et al. (1995) have developed a The results were in agreement with the simple system for the use of DNA probes, chromosomal identification of specimens or horse-radish peroxidase - labelled DNA with known distributions of the species. The probes - and a chemiluminscent detection fragment size of species D from Thailand in system. The sensitivity of the system was such this assay was 306 bases while the sizes for that it could detect 1-5 ng of target DNA, species A, B, C and An. nemophilus were 562, 32 which was comparable to P labelled probes. 514, 349 (or 353) and 223 bp respectively. This technique successfully identified species The authors further state that species D from A, B, C and D from field collections. Thailand differs from that described as species D by Xu, Xu and Qu (1998) from RFLP profiles China. Twenty restriction enzymes were used to study restriction fragment length

Anopheline Species Complexes in South and South-East Asia 37 Manguin et al. (2001) and Huong et al. (1989) is reproduced as Figure 7. For the (2001) also developed PCR assays which distinct pattern observed in the distribution distinguished species A, B, C and D of the of sibling species in Thailand, Baimai et al. Dirus Complex. The method of Huong et al. (1988) do not suggest any geographical/ (2001) used a cocktail of four primer sets for topographical reasons. identificaton, and the authors report that it is Species A is widespread throughout a sensitive method requiring a small amount Thailand except in the south. It occurs of DNA. They also report a short method, exclusively in the central and north-eastern i.e. homogenizing the mosquito in 1x PCR regions. Species B and C have a restricted buffer and use of the supernatant directly for distribution. Species B is found only in the PCR identification. It is a simple and rapid southern peninsular region of Thailand and method for the identification of field- extends into peninsular Malaysia. Species B collected specimens. is also reported from Sumatra island, Microsatellite markers for population Indonesia. Species C is reported only from analysis Thailand (Kanchanaburi, Nakhon Si Thammarat and Phat-thalung). Species D is Walton et al. (2000a) isolated 11 commonly found on the north-western side microsatellite markers from An. dirus species of Thailand. Along the Thai-Myanmar border A and tested for polymrophism in species C it is found in sympatric association with and D. Walton et al. (2000b) used these species A. It is exclusively found in Myanmar markers to study populations of species A, C and Bangladesh (Baimai et al., 1988) and and D to address the issues of gene flow north-eastern states of India (Baimai, 1989). within each species. All the three species were The distribution of species D in India was found well differentiated from each other at predicted based on its distribution in Thailand the microsatellite loci used. The population and Myanmar. Recently, in four north-eastern genetic analysis using microsatellite markers states of India (Assam, Arunachal Pradesh, also showed mtDNA introgression between Meghalaya and Nagaland) using Walton et species A and D (Walton et al., 2001). al. (1999) PCR assay, Prakash et al. (2006) Distribution and biological confirmed the presence of species D. Species characteristics D is reported from Yunan province, southern China, but as mentioned earlier, this may be The association of An. dirus with malaria different from the one reported from Thailand transmission in forests and forest-fringe areas and the other neighbouring countries. Species is well established in Bangladesh (Rosenberg E is exclusively found in south-western India and Maheshwari, 1982), India (Rao, 1984), in the Kyasanur area of Shimoga hills in and Thailand (Rosenberg, Andre and Karnataka state (Tiwari, Hiriyan and Reuben, Somachit, 1990). 1987; Bhat, 1988). Species F (An. nimophilus, Baimai et al. (1988) report that the Peyton and Ramalingam, 1988) is found on distinct differences exhibited by these species the Thai-Malaysia border and is also reported suggest some implications for Plasmodium from the monsoon forests of the mountain transmission, and therefore recommend the areas of south-eastern, southern and western identification of these sibling species during Thailand and peninsular Malaysia malariometric studies. (Rosenberg, Andre and Somachit, 1990). An. takasagoensis is exclusively found in Taiwan. Approximate information on the distribution of sibling species in Thailand is Distinct seasonal variations in relative given below (for details see Baimai 1988, and frequency were observed in sympatric species Baimai et al., 1988). The map from Baimai (Baimai et al., 1988). In areas where species

38 Anopheline Species Complexes in South and South-East Asia B and C are sympatric, species B was found Thailand. In Binh Thuan province, south- in abundance at the beginning of the rainy central Viet Nam, several An. dirus specimens season (May to October) while species C was were found positive for P. falciparum abundant at the end of the season. In species sporozoite antigen by ELISA (Van Bortel et A and D sympatric areas, species A is more al., 2001). The authors considered An. dirus abundant at the end of the wet season, to be the main vector in the area and compared to species D which is abundant in suggested that despite the fact that other the middle of the wet season. All-night secondary vectors were found in the area, human-bait catches indicated that all the four vector control should target against An. dirus. species bite throughout the night with the They further proposed that one round of bed- peak biting times being strikingly different in net treatment preceding the transmission the four species studied (for details on study season of An. dirus would be sufficient to sites and other information, see Baimai et al., control malaria in the area. 1988). The peak biting activities were: In India, in the north-eastern states, An. Species A — 2100-2300 hours; Species B — dirus D is abundant and implicated 1900-2100 hours; Species C — 1800-2000 epidemiologically in malaria, but there is no hours; and Species D — 0100-0300 hours. information or report to say that species E is A study was carried out at Mae-tao-kee involved in malaria transmission. In the state near Maesod in north-western Thailand of Assam in the north-eastern part of India, (Green et al., 1991) to examine the vectorial An. dirus (species D) was found to be highly potential of the members of the complex. In anthropophagic, biting both indoors and this area, An. dirus species A and D were outdoors equally (Dutta et al., 1996). In the sympatric and were found in association with forest areas of Assam, the vectorial capacity four members of the Maculatus Complex and of An. dirus was the highest, 0.779 and 0.649 An. minimus species A. The man-biting rates for P. vivax and P. falciparum respectively, of species A and D were 1.26 and 0.61 (these during the hot monsoon season (June- were lower than those observed for An. September) and about 10-fold lower values pseudowillmori, a member of the Maculatus were seen during the cool-dry season (March- Complex, and An. minimus species A), but May) (Prakash et al., 2001). Prakash et al. had the highest sporozoite rates of 6.4 per (2005) calculated the effective entomological cent and 2 per cent respectively. The only inoculation rate (EEIR) for An. baimaii other species which was found positive for (formerly An. dirus species D) in a forest-fringe sporozoites was An. pseudowillmori (0.5 per village in Dibrugarh district, Assam state, in cent) in this study. different seasons. With an overall sporozoite rate of 1.9 per cent, in the monsoon season Baimai (1989), in a review on the (June-September), 0.39 infective bites/ members of the Dirus Complex, reported that person/night and in the post-monsoon season a study carried out at Mae Sot, Tak province, (October-November), 1.47 infective bites/ and Tung Song, Thailand, revealed that person/night were observed for this species. species A, C and D were efficient vectors of Though biting was observed all night, the P. vivax and P. falciparum malaria. (It is not maximum infective bites were in the second mentioned in the review whether this quarter of the night. As 21 per cent of the conclusion was drawn from the total infective bites were recorded before epidemiological investigation in the area or 2100 hours, the authors suggested that whether they were incriminated.) In the same appropriate protective measures are needed review it is mentioned that there was no to supplement the impact of insecticide- evidence to show that species B was a vector treated nets (ITN) against An. baimaii in north- under field condition and species F was not east India. The national malaria control known as a vector of human malaria in

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 Inversion Enzyme DNA Egg morphology Mitotic ‘a’ on systems probes Tubercles RFLP PCR polytene appearance of X- chromo- Rows of cells Aggregates X- and Y- somes between frill on deck chromosomes and float surfaces

An. dirus s.s Telocentric X+a/a Gcd 100 pMU- 2.5-3.5 rows Moderately ?? (Species A) Aat 100 A40, broad large and 1#5 widely spaced

An. cracens Acrocentric X+a Acp (slow) pMU-B5 3.5-4.5 rows Same as A ?? (species B) long and closely and narrow spaced

An. scanloni Telocentric X+a -pMU-2-3 rows long Large and ?? (species C) C19.2 and narrow closely spaced

An. baimaii Telocentric Xa Gcd 128 pMU-D9 2.5-4 rows Small and ?? (species D) Aat 123 or broad and closely 100 irregular spaced shaped

An. elegans Telocentric Xa ------(species E) (Y-small rod or dot like)

An. nemophilus Acrocentric ------? (Species F)

An. Telocentric ------takasagoensis

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 Somboon et al. (1999) isolated lines fully ITN programme in the north-eastern states refractory and fully susceptible to Plasmodium of India. yoelii nigerriensis (an African rodent malaria parasite) after 17 generations of mass In a study carried out to examine the selection. Most of the mosquitoes of the behavioural heterogeneity of Anopheles refractory line inhibited the parasite species in six different localities in South-East development by encapsulating oocysts, which Asia, Trung et al. (2005) found An. dirus s. s. appeared as melasized spots in the gut. But in the central province of China and in this line showed normal susceptibility to Cambodia. This species was found to be human malaria parasites, P. falciparum and P. highly anthropophagic, exophilic, exophagic vivax. 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 3.5 The Fluviatilis Complex sprays. A detailed review of literature on the Anopheles fluviatilis James belongs to the distribution and bionomics of all members subgenus Cellia, the Minimus Subgroup and of this complex is given by Sallum, Peyton the Funestus Group in the series Myzomyia and Wilkerson (2005). (Harbach, 2004). The Fluviatilis and Minimus Prakash et al. (2002) also studied the larval Complexes, An. flavirostris and An. lessoni, ecology of An. dirus in the rain forest area of are members in the Minimus Subgroup. An. Assam. An. dirus-positive shady ground pools fluviatilis has a wide distribution in the showed higher mean values of total alkalinity, Oriental region and parts of the West Asia hardness and chloride content, while stream- subregion (Rao, 1984). In the Oriental region, side pools showed lower pH and dissolved it is found in Pakistan, Afghanistan, India, oxygen and higher total alkalinity and Nepal, Bangladesh, Myanmar, Thailand and hardness, compared to negative breeding sites south China, and in the West Asia region it is of similar type. In Mudon, a coastal area in found in Iran, Iraq, eastern and southern Mon state in south Myanmar, An. dirus was Arabia, Oman and Bahrain. It is considered found breeding in wells, a situation not an important vector only in India, Pakistan encountered in other parts of Myanmar (Oo, and Nepal (Rao, 1984). Distinct differences Storch and Becker, 2002) and from any other observed in densities, preference to feed on country in South-East Asia so far. Shade a host and infection rates between the vegetation and debris on the surface of well- populations led Rao (1984) and several earlier water were important factors influencing the workers, Senior White (1946), Viswanathan abundance of larval and pupal density. In (1950), Brooke Worth and Sitaraman (1952), Mudon area, the malaria incidence is high, and Bhombore, Sitaraman and Achutan with slide positivity ranging between 9.9 per (1956), to suggest that this species may have cent and 34.28 per cent throughout the year two biological races. Subbarao et al. (1994) (Oo, Storch and Becker, 2003). recognized An. fluviatilis as a species complex in India. In Myanmar, Bangladesh and north- eastern parts of India, only species D has been Evidence for recognition of sibling reported. An. dirus breeding in wells in species southern Myanmar calls for a detailed The evidence for the three sibling species examination of the population for sibling came from the absence of heterozygotes for species composition. Now that effective the three polytene chromosome molecular tools are available, a study should arrangements observed due to two fixed be initiated at the earliest.

Anopheline Species Complexes in South and South-East Asia 41 paracentric inversions q1 and r1 on Techniques available for chromosome arm 2. Chromosome arms 3, identification of sibling species 4 and 5 and X-chromosome were homosequential in all the three species. The Ploytene chromosomes three sibling species were designated as Paracentric inversions, q1 and r1, on species S, T and U (Subbarao et al., 1994). chromosome arm 2, are diagnostic: +q1+r1 is the standard arrangement diagnostic for A population differing from the other species S, q1+r1 for species T and +q1r1 three sibling species in polytene chromosome arrangement for species U (Subbarao et al., arrangement on chromosome arms 2 and 3 1994). Other chromosome arms are was recently recognized in district Hardwar, homosequential in all three species. A Uttaranchal state (a new state carved out of photomap of polytene chromosomes of the erstwhile Uttar Pradesh state in northern species S, with inversion break points, is India). In the same area species T and U presented by Subbarao et al. (1994). were found sympatric. The absence of Chromosome arm 2, marked with heterozygotes for the two new inversions was breakpoints q1 and r1, is shown in Figure 8. taken as evidence to designate this population Nanda et al. (unpublished) designated new as a new species (Nanda et al., unpublished). inversions as s1 on chromosome 2 and s on An. fluviatilis now is a comlex comprising four chromosome 3 and the new inversion sibling species, S, T, U and new species V. karyotype 2s1; 3s is diagnostic for species V. Recently, based on the homology of D3 domain of 28S rDNA, Harbach (2004), Mitotic karyotypes Garros, Harbach & Manguin (2005) and Chen No variations were observed in the mitotic et al. (2006) considered An. fluviatilis S karyotypes of any of these sibling species. conspecific with An. minimus C. Consequently, Harbach (2004) removed An. ASPCR assays fluviatilis S from the Fluviatilis Complex in his A PCR assay developed from rDNA ITS2 taxonomic update. Furthermore, all these region by Manonmani et al. (2001) was later authors said that the Fluviatilis Complex correlated with cytological identifications consists of only two sibling species, species T (Manonmani et al., 2003). This assay and U. However, recent analysis of Singh et accurately distinguished species S from al. (2006) on the sequences belonging to the species T in about 94 per cent of the internal transcribed spacer 2 (ITS-2) region specimens. Lack of 100 per cent correlation and D2-D3 domain of ribosomal DNA of An. was probably due to the presence of q1 fluviatilis S and An. minimus C showed that inversion polymorphism. This assay does not the two species are appreciably different with distinguish species T from species U pair-wise distance (Kimura-2-Paramtetre (Manonmani, personal communication). model) of 3.6 per cent and 0.7 per cent respectively for ITS-2 and 28SD2-D3 loci. An allele-specific PCR assay developed by Based on this analysis, Singh et al. (2006) Singh et al. (2004), based on differences in concluded that An. fluviatilis S and An. the D3 variable region of 28S ribosomal rDNA, minimus C were not conspecific. Further, differentiates all three species S, T and U. This from pair-wise and phylogenetic analysis, multiplex assay has four primers, two universal these authors showed that An. fluviatilis S was primers – D3A forward and D3B reverse and more closely related to other members of the two allele-specific primers – AFS and AFT – Fluviatilis Complex than to An. minimus C. which are specific for species S and T respectively. The specimens not reacting to

42 Anopheline Species Complexes in South and South-East Asia S- and T-specific primers were regarded as samples examined were very few. Allopatric species U. Cytological correlation was done populations of T were also observed (Figure with specimens collected from different parts 9). From Table 7, it can be seen that species of India, and in these areas the three species S is distinctly different from species T and U were found in different sympatric associations. in several biological characters. PCR identifications were in full agreement with A longitudinal study was carried out on the cytological identifications. In Mandya and the bionomics of An. fluviatilis in the foothills Gulbarga districts in Karnataka state (south of the Shivalik range of the Himalayas in India), q1 inversion is polymorphic with the Hardwar and Dehradun districts of karyotypes +q1/+q1, +q1/q1 and q1/q1 in Uttaranchal state (which is the newly Hardy-Weinberg equilibrium. The question demarcated state that was a part of the former that arose was whether these belonged to Uttar Pradesh state in north India) (Sharma species S or T. The allele-specific PCR assay et al., 1995). It was found throughout the identified all three karyotypes as species T year with high densities in October/ (Singh et al., 2004). November and low densities in May to Singh et al. (2006), by examining ITS-2 August, densities in peak months being sequences of species X reported by Manonmani recorded as more than 200 caught per man- et al. (2003) and of species S, conluded that hour of collection (“man-hour density”). An. species X was synonymous with species S. fluviatilis breeding in these villages was found in slow-running streams, irrigation channels The five sibling species of the Culicifacies and subsoil seepage water with grassy Complex which are sympatric with An. margins, preferably under some shade. No fluviatilis in many areas did not react with breeding was observed in fast-flowing steams species S or T primers. However, they reacted or in rice fields. In both the districts, species with the universal primers, giving an amplicon T and U were sympatric. In Dehradun, of 380 base pairs like species U. The universal which is at an altitude of 640 m, species T primers may react with other anophelines as was predominant (>95 per cent), while in well. Therefore, care must be taken to Hardwar (294.7 m altitude), species U was identify mosquitoes morphologically prior to predominant (>65 per cent). The biological the use of this assay. characteristics of the two species were as PCR-RFLP reported in Table 7. The new species found A slight modification of the above assay, by in a few villages in district Hardwar was including a restriction enzyme, distinguishes the sympatric with species U and T (Nanda et new species V (O. P. Singh et al., unpublished). al., unpublished). In Nainital district of Uttaranchal state, species T and U were also Distribution and biological sympatric (Shukla et al., 1998). characteristics In Malkangiri and Koraput districts in Following the discovery of sibling species Orissa state (in south-eastern part of India), within An. fluviatilis, studies were carried out where species S and T are prevalent to map the distribution of the sibling species (Manonmani et al., 2003), the breeding of and study their bionomics. The biological these species was observed in terraced paddy differences observed are summarized in Table fields, streams and stream channels (Sahu et 7. Species S was found either alone or in al., 1990). Species S was found to be highly sympatric association with species T, and anthropophagic (~91 per cent) while species species U was more frequently found with T and U were almost totally zoophagic species T. In Kamrup district, Assam state, (Nanda et al., 1996). From the district of only species U was found but the number 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. found and the EIR was 0.014 (Sharma et al., fluviatilis s.l. processed by immuno- 2006). In spite of continuous exposure to radiometric assay for sporozoite antigen were insecticides, An. fluviatilis still remains found not positive (Sharma et al., 1995). In susceptible to all insecticides—DDT, HCH, contrast, in the districts of Malkangiri and malathion and pyrethroids in Orissa (Sharma Koraput where species S is predominant, et al., 2004b) and also in other areas where earlier in 1989 An. fluviatilis s.l. was this species was tested in India (K. incriminated for sporozoites (Gunasekaran et Raghavendra, personal communication). al., 1989) and later species S was incriminated About 0.5% of An. fluviatilis s.l. from (MRC, 1995). In areas where species S has Afghanistan was found infective with both P. been found, malaria is hyperendemic, and falciparum and P. vivax (CSP 210) CSP antigens the prevalence of P. falciparum and deaths (Rowland et al., 2002). The villages where due to malaria are reported. this species was incriminated had river- In Sundergarh, a district in the north- irrigated rice fields. In the mountainous areas western part of Orissa, a longitudinal study of the Hormozgan province, south Iran, An. was carried out to examine the bionomics fluviatilis is considered an efficient vector of and to delineate the role of An. fluviatilis and malaria. It is exophilic and exophagic and An. culicifacies sibling species in malaria densities start to build up in September and transmission (Nanda et al., 2000). In Birkera two peaks, one in January and another in May, block in a village in the forest, mainly An. are observed. Using the PCR assay of fluviatilis species S and of the two An. Manonmani et al. (2001), An. fluviatilis in the culicifacies sympatric species B and C, species area was identified as sibling species T C maintained high transmission. The annual (Vatandoost et al., 2005). Specimens from parasite incidence (API) was 269 cases/1000 Iran were also cytologically identified as population with 83.5 per cent P. falciparum. species T (H. Vatandoost and N. Nanda, And in a deforested village, only An. personal communication). Species T in Iran, culicifacies was found with species B and C unlike in India, apprears to be a vector. sympatric (of these two, only species C is Recently, Adak et al. (2005) reported that vector). Malaria incidence in this village was sibling species T from India was susceptible relatively low (API 39 cases/1000 population) to P. vivax infection in laboratory feeding and P. falciparum cases accounted for 57.9 experiments. per cent; P. vivax was the other Plasmodium This suggests that species T in India is species in these villages. Another detailed genetically susceptible to Plasmodium longitudinal epidemiological study is being infection, but because of its preference to carried out in the same district in 13 villages, feed on animals and the environmental eight in forest and five in the plain areas. This conditions in addition may be making it a study is being conducted in order to develop non-vector. a field site for vaccine trial. The observations are similar to the above-mentioned study with Now that well-established cytotaxo- 347.9 API in the forest area and 61.0 in the nomic and molecular methods are available plain area (Sharma et al., 2004a). The for identification, populations from all the contribution of An. fluviatilis species S and countries where An. fluviatilis is reported need An. culicifacies species C to entomological to be examined for sibling species inoculation rate (EIR) in the forest area was composition and their bionomics need to be 0.395 and 0.009 infective bites per person studied to find intra- and inter-specific per night respectively, while in the plain area, variations, if any. only An. culicifacies species B and C were

44 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 Man-hour Feeding Sporozoite Prefered Ecotype Endemicity Species karyotypes ASPCR densities preference positives adult habitat S q1 r1 Yes Low Anthropophagic Found Human Hilly Hyper- (1-40) (>90%) dwellings forests & endemic foothills T q1+r1 Yes High Almost totally Not found Cattle Foothills & Hypo- (up to 200) zoophagic sheds plains endemic U +q1 r1 (~99%)

* 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 Sonapur Hardwar Nainital

Allahabad Alwar

Jabalpur

Kheda Purulia

Koenjahar Bahraich Mayurbhanj Sambalpur

Phulbani

Sundergarh Gulbarga Balangir Kalahandi Tumkur Koraput Malkangiri Kolar

Mandya Species S Species T

Species U

3.6 The Leucosphyrus Evidence for recognition of sibling species Complex Two isomorphic species, An. leucosphyrus A The Leucosphyrus Complex consists of and B, were identified following the Anopheles balabacensis Baisas, 1936, An. cytological examination of mitotic karyotypes introlatus Colless, 1957, and An.leutens and reproductive isolation observed in crosses (leucosphyrus A) and An. leucosphyrus s. s. between allopatric populations (Baimai, (leucosphyrus B) (see Table 5). 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,

46 Anopheline Species Complexes in South and South-East Asia near Muarabungo, Bungo Tebo regency, appears to be a character which distinguishes Jambi province); (ii) South Kalimantan these two species from An. balabacensis. This (Salaman, near Kintap, Tanah Laut regency); diagnostic character also distinguishes the and (iii) Southern Thailand (Pedang Besar, above-mentioned species A and B from the Songkla province). There was no evidence members of the Dirus Complex. Two types of reproductive isolation between the Thai of Xs due to size variation in the long arm of and South Kalimantan populations, as the F1 the X were observed in each species. A progeny were fertile from both reciprocal conspicuous pericentric heterochromatic crosses. In contrast, from both reciprocal segment was seen in both the autosomes. crosses between the Sumatra population and In the Sumatra population (species B), this the populations from South Kalimantan and was more prominent in autosome III. Thailand, F males were sterile, indicating 1 Hii (1985) reported, for the first time, reproductive isolation. Based on these results, that mosquitoes from Sabah, which he the authors concluded that An. leucosphyrus referred to as An. balabacensi s. s., were includes two allopatric species, the one distinct from An. dirus A and An. dirus B (Perlis inhabiting Borneo, west Malaysia, and form). An. balabacensis s.s.produced sterile southern Thailand designated as species A, hybrid males (partially-developed and the population confined to the island of reproductive organs) when crossed with An. Sumatra designated as species B. Species B dirus A and B. is An. leucosphyrus Doenitz, the nominotypical member of the widely distributed Peyton (1989) has assigned An. Leucosphyrus Group. balabacensis for the first time to the Leucosphyrus Complex (see Table 5) becuase Baimai, Harbach and Sukowati (1988) it is morphologically closely similar to An. presented photographs and diagrams of the leucosphyrus. A diagram of the mitotic mitotic karyotype of species A and B (see karyotype of An. balabacensis is presented Figure 7). The differences between the two by Baimai, Harbach and Kijchalao (1988) species were due to major blocks of (see Figure 7). This species has acrocentric heterochromatin. In species A, the X- and Y- X- and Y-chromosomes. The Y-chromosome chromosomes of specimens from South is totally heterochromatic and autosome III Kalimantan possess a very small segment of has a large block of heterochromatin. The extra heterochromatin at the centromeric authors also report that An. balabacensis, in region, which gives the chromosomes a crosses with species F of An. dirus, produced subtelocentric configuration. In the Thai F males with atrophied testes without sperm, population, because of the absence of this 1 and the polytene chromosomes were totally block, the sex chromosomes appear asynapsed in the F progeny. No information telocentric (as shown in Figure 7). The 1 on the fourth species, An. introlatus, has been Sumatra population of species B possesses found. submetacentric X- and Y-chromosomes. The short arm of the X and the whole of the Y- Formal designations chromosome is heterochromatic. The short Sallum, Peyton and Wilkerson (2005), after arm of the X has a secondary constriction in morphological examination of several the middle which is not found in any of the thousand specimens belonging to this members of the Dirus Complex. complex, formally designated An. On the X-chromosome of both the leucosphyrus species A as An. leutens and species, the presence of a distal block of species B as An. leucosphyrus s. s. heterochromatin is very conspicuous and

Anopheline Species Complexes in South and South-East Asia 47 Techniques available for Java, Indonesia, a sharp increase in malaria identification of sibling species occurred in the year 2000. Two important vectors, An. maculatus and An. balabacensis, Structural variations in mitotic karyotypes and which favour forested hill sides in Java, were the banding pattern associated with considered responsible for the transmission herochromatin variations are the only (Barcus et al., 2002). available methods for the identification of species. An. leutens (species A) is considered highly anthropophilic and an important Distribution and biological characters vector of human malaria, both in villages and An. leucosphyrus species A is widely forest areas of Sarawak, east Malaysia (Chang distributed in southern Thailand, west et al., 1995). This species is also considered Malaysia, and Sarawak, east Malaysia, and a vector of Wuchereria bancrofti to humans Kalimantan in Indonesia, while species B is in Sarawak (from Sallum, Peyton and probably confined to the island of Sumatra, Wilkerson, 2005). There is no report on the Indonesia (Baimai 1988; Baimai, Harbach incrimination of An. leucosphyrus species B. and Sukowati, 1988). An. balabacensis is confined to the locality from which the type specimen came in Balbac island and 3.7 The Maculatus neighbouring areas, i.e. Polawan island, Complex Sabah and north-east Kalimanatan (Peyton and Harrison, 1979; Hii, 1985; Peyton, Anopheles maculatus Theobald belongs to the 1989). All species of this complex breed in subgenus Cellia and the Maculatus Group in shaded temporary pools in forests. the Neocellia Series (Harbach, 2004). This species is recognized as an important vector An. balabacensis was found positive for of human malaria parasites in Thailand, P. falciparum sporozoite antigen by IRMA Indonesia and peninsular Malaysia. along with another species An. flavirostris on Banggi island, Sabah, Malaysia (Hii et al., To facilitate the understanding of the 1988). Based on the human landing rate and Maculatus Complex, information on eight sporozoite positives, the EIR/Year was nominal forms described in the literature calculated as 160. The vectorial capacity (Christophers, 1931; Rattanarithikul and calculated for March was 1.44-7.44 and for Green, 1986) is given below. Christophers November 9.97-19.7. On Banggi island, regarded maculatus as a single species based malaria is holoendemic. on studies of the morphological variation in adults and recognized two vectorial forms: In south Kalimantan where An. one with reduced abdominal scaling, the balabacensis and An. leucosphyrus species A nominotypical form (maculatus), and the are sympatric, these two species together other with heavy abdominal scaling, var. comprised 97.7% of the total anophelines willmori. He considered pseudowillmori, collected (Harbach, Baimai and Sukowati, dravidacus and hanabusai as synonyms of the 1987). Large numbers of these specimens nominotypical form and considered were collected from within the village than dudgeonii, indicus and maculopsa as in the forest. The P. falciparum sporozoite synonyms for var. willmori. Rattanarithikul antigen detection rate was 1.0% for An. and Green (1986) further state that in spite leucosphyrus species A and 1.3% for An. of extensive studies by Puri (1931), balabacensis. After more than 50 years of Christophers (1933), Crawford (1938), Reid, effective management of malaria, in the sub- Wattal and Peters (1966), and Reid (1968), districts of Menoreh Hills and Dieng Plateu, the morphological concept and formal

48 Anopheline Species Complexes in South and South-East Asia taxonomy of this group have remained chromosomes (X1, X2 and X3) and four types unchanged. of Y-chromosomes were observed. No heterozygotes were found of X either with The cytotaxonomy and morphological 1 X or X . And X was considered indicative studies together have now unequivocally 2 3 1 of species A in contrast to X and X in species identified eight biological species, and the 2 3 B, which were found in heterozygous DNA analysis has recently identified a new condition. Y-chromosome polymorphic species corresponding to the chromosomal forms were not found associated with any form K in this complex. Nine biological species are described and discussed below. particular species. The three species, A, B and C, were Evidence for recognition of sibling found sympatric in three localities, two near species Kanchanaburi, west of Bangkok, and one in All the rearrangements in An. maculatus are northern Thailand, near Chiang Mai and no referred to the An. stephensi map (Green, heterozygotes were found for the diagnostic 1982) which serves as an arbitrary standard. inversions (Green and Baimai, 1984). (This is in contrast to other species complexes Two allopatric populations that are close where chromosome maps prepared for an to species B but differ from it in the frequency unspecified member of the complex or one of two paracentric inversions on X- of the sibling species are used as standard). chromosome and four on chromosome arm The first evidence that An. maculatus is a 2 were identified as the E and F forms. The species complex came from two largely observation of heterozygotes for all the independent studies on chromosomes– inversions that distinguish between species polytene and mitotic chromosomes from B and the E form in Petchaburi (near natural populations of An. maculatus. Within Kanchanaburi, Thailand) suggested that the An. maculatus polytene chromosomes, no form E is a chromosomal race of species B variation has been seen in chromosome arms (Green and Baimai, 1984). The authors, 3, 4 or 5 while fixed and floating inversions however, caution that the sample size was were seen on the X and arm 2. A total of 18 small, hence the conclusions drawn are not paracentric inversions were observed and final. Furthermore, F1 progeny from both these inversions were observed in six distinct reciprocal crosses between the B and E forms chromosomal forms in Thailand. These were fertile, supporting the concept that chromosomal forms were found in different the form E may be a chromosomal race of sympatric associations in different areas. The species B. The status of form F could not be six chromosomal forms were given the species confirmed as B/E and F are separated by the status based on the population genetic data. Chao Phraya river basin from which An. The absence of heterozygotes for the alternate maculatus s.l. is absent (Green et al., 1985), arrangements of the inversions in a given area and larger samples of the F form are was taken as evidence for the reproductive required. isolation. Cuticular hydrocarbon analysis Species A, B and C (Green and Baimai, (Kittayapong et al., 1990) differentiated 1984, Green et al., 1985) species B from E form. There is also indirect evidence from cuticular hydrocarbon analysis In addition to the differences in paracentric that the two forms coexist in peninsular inversions (as mentioned above), Malaysia (Kittayapong et al., 1993). heterochromatin variation in X- and Y- Microsatellite marker analysis suggested that chromosomes was also observed (Green and there was restricted gene flow between the Baimai, 1984). Three types of X-

Anopheline Species Complexes in South and South-East Asia 49 northern populations of An. maculatus al., 1985). Another population was collected o o extending from latitude 11 to 16 N and the from Subic Bay, to the west of Manila o o southern populations from latitude 7 to 6 N (Philippines), and the cytological examination (Rongnoparut et al., 1999). Race B (species of this population revealed that this was o B) extends northwards from latitude 13 N, different from form D collected from the while race E extends southwards from latitude north-east of Manila. These two cytological o 12 N into peninsular Malaysia (Green et al., forms, though very different, could not be 1985). Based on microsatellite data analysis, designated as separate species because the the authors suggest that the southern two forms were identified from allopatric populations may become distinct species populations. (details in the next section). Rongnoparut et Rattanarithikul and Harbach (1990) al. (1999) report that unpublished data of R. correlated distinctive morphological traits to Rattanaritikul shows that inversion karyotypes each of the cytotypes. The observation of show little indication of hybridization between two morphotypes from single localities in the B and E. Philippines led them to state that there is Green et al. (1985) noted that B and E reproductive isolation between An. maculatus forms are either two distinct sibling species form D and the new (J) cytotype, and, hence, or they represent geographical variation these are distinct species. An. maculatus form within An. maculatus. Keeping in view that D described in Green et al. (1985) was given distinct cuticular hydrocarbon profiles were the formal name An. greeni, and the new observed for the two forms, Harbach (2004) cytotype, J form, the name An. dispar and Walton et al. (2005) suggest that they (Rattanarithikul and Harbach, 1990). This is may be distinct species. the first case where the morphological and cytological differences taken together Species G (Green and Baimai, 1984) demonstrated reproductive isolation. A population fixed for inversions x and y on chromosome arm 2, and for d and e on X- Species H and I (Green, Rattanarithikul chromosome, was found along with species and Charoensub, 1992) A and B in a locality near Petchaburi in the An. pseudowillmori and An. willmori were northern part of peninsular Thailand. No described morphologically by Rattanarithikul heterozygotes were observed for these and Green (1986) and they were also inversions and the inversions were unique for identified as distinct cytological forms (I and the population. This population was H respectively) (Green, Rattanarithikul and designated as species G. Charoensub, 1992). The evidence for their species status came from the cytotaxonomic The results from reciprocal crosses examination of populations from four between species A, B, C and G produced localities in north-western Thailand. In these sterile F males and fertile females, which 1 localities, An. willmori, cytologically provided support for the designation of designated as form H, and An. pseudowillmori separate species based on cytogenetic as form I, were found in sympatric association evidence. with species A and B, with a total absence of Species D and J (Rattanarithikul and heterozygotes for the inversions fixed in them. Harbach, 1990) Enzyme polymorphism at 6-Pgd (6- An. maculatus form D was identified on the phosphogluconate dehydrogenase) also basis of chromosomal differences noted supported the specific status of An. during the comparative cytological pseudowillmori. Three allozymes or examination of populations from the electromorphs were found associated with Philippines, Thailand and Malaysia (Green et

50 Anopheline Species Complexes in South and South-East Asia three species: An. pseudowillmori — 6- they have a unique sequence and is 3.7 per Pgd70, An. maculatus (species B) — 6- cent divergent from the next closely related Pgd100, and An. sawadwongporni (species A) taxon An. sawadwongporni in the group — 6-Pgd130, with a total absence of (Walton et al., 2007). The authors consider heterozygotes. that this corresponds to the chromosomal form K reported by Baimai (1989). Based on In Thailand, six species (A, B, C, G, H the negligible intra-specific variations and I) were found, while forms D and J were observed in the specimens analyzed, the confined to the Philippines. The six forms in authors designated the chromosomal form K Thailand were found in the following different as a new species in the complex. sympatric associations (Rattanarithikul and Green, 1986; Green, Rattanarithikul and The biological species that were Charoensub, 1992): provisionally designated with the letters of the A, B and C — from several widely English alphabet were formally recognized by separated localities in studying the morphological variations in western Thailand; progeny broods from wild-caught females that were identified by chromosomal A, B and I — from Mae Sariang, Mae rearrangements observed in their ovarian Hong Son province; polytene chromosomes. Papers by B, H and I — from Doi Inthanon, Rattanarithikul and Green (1986) and Chiang Mai province; Rattanarithikul and Harbach (1990) are strongly recommended for details regarding B and H — from Mae Sa, Chiang; the formal recognition of these species. The A, B, C and G — in a single locality near provisional letter designations and their Phet Chong, Nikhon species names are given in Table 8. Species Ratchasma province; K does not have a formal designation yet. A, B and G — in one locality near Phet Techniques available for Buri in the northern part identification of sibling species of peninsular Thailand; A and F — in a single locality near Polytene chromosomes Nokhorn Nayak, north- The members of the Maculatus Complex can east of Bangkok; be identified unequivocally from each other and from An. stephensi (which is used as an A, B, H and I — in four localities in north- arbitrary standard) by examining polytene western Thailand. chromosomes for paracentric inversions on In all these localities no heterozygotes autosomes. Sixteen inversions on were seen for the fixed inversions identified chromosome arm 2, one on arm 3 and one for these forms, which strongly supports the on arm 5 are unique to this complex and species status given to these cytologically hence diagnostic. Though differences in the distinct populations, and establishes the banding pattern in the X-chromosomes are presence of six species specific mate reported, the homologies have not been recognition systems in the An. maculatus worked out well. The diagnostic inversions Complex in Thailand. given in Table 8 are from Green, Rattanarithikul and Charoensub (1992). This New species, putative species K (Walton paper gives the photomap of An. stephensi et al., 2007) chromosome arm 2 with breakpoints of the The ITS2 sequence analysis of the specimens inversions marked, and the diagnostic collected from eastern Thailand revealed that inversions summarized in a figure. For

Anopheline Species Complexes in South and South-East Asia 51 photomaps of chromosome arms 3, 4 and 5, An. pseudowillmori, was developed (Ma, Li the readers are referred to Green (1982). and Xu, 2006). Another PCR-based diagnostic assay has been developed to facilitate field Morphological key research in northern Thailand, which A morphological key for the identification of distinguishes An. maculatus, An. dravidacus, all eight members of the Maculatus Complex An. pseudowillmori, An. sawadwongporni and is provided by Rattanarithikul and Green species K (Walton et al. 2007). (1986). Upatham et al. (1988) found a small percentage of errors in the identification of Microsatellite markers species A, F form of species B and species B, About 23 microsatellite markers were using a morphological key (identifications identified from An. maculatus s.s. (species B) were confirmed by cytological examination). (Rangnoparut et al., 1996). Seven of these microsatellites were used to study the genetic Electrophoretic variations/Cuticular variation in eight widely dispersed localities hydrocarbon profiles in the western and peninsular Thailand 6-Pgd allelic variations (Table 8) can also be (Rangnoparut et al., 1999). The data used to distinguish An. sawadwongporni suggested that the populations could be (species A), An. maculatus (species B) and An. grouped into two clusters: one including the pseudowillmori (species I) (Green, upper and lower northern populations Rattanarithikul and Charoensub, 1992). The (extending from latitude 11o to 16o N) and two forms of species B, E and F could be the other including the southern population distinguished by gas-liquid chromatographic (extending from latitude 7o to 6o N). Among analysis of cuticular lipids in association with the populations, within each cluster, extensive a multivariate principal component analysis gene flow was observed, while restricted gene (Kittayapong et al., 1990). flow was observed between the northern and southern populations. Geographical or PCR-RFLP genetic barriers could be limiting the gene An. greeni and An. dispar occur sympatrically flow between these populations. in the Philippinnes. In order to develop a simple and reliable method of identification, Distribution and biological characters Torres, Foley and Saul (2000) carried out an The distribution of the species given below is analysis of two regions of rDNA, ITS2 and from Rattanarithikul and Green (1986), D3 domain of the 28S gene of An. maculatus Baimai (1989), Ma, Li and Xu (2006) and s.l. specimens from localities throughout the Walton et al (2007): Philippines. Two distinct sequence groups An. sawadwongporni (species A) — Myanmar, were observed, one corresponding to An. China, Cambodia, Thailand and Viet Nam. greeni and the other to An. dispar. Digestion It is found at low and high altitudes in of the ITS2 amplicon with Hae II restriction association with all other members of the enzyme yielded distinct fragments, and the group two species could be identified with ease and accuracy. An. maculatus s.s. (species B) — Bangladesh, Myanmar, China, India, Indonesia, PCR assay Cambodia, Malaysia, Nepal, Pakistan, Sri Based on interspecific variation in the ITS2 Lanka, Taiwan, Thailand and Viet Nam region, a diagnostic PCR assay that An. dravidicus (species C) — Myanmar (Kaley distinguishes five members of the complex valley), India, China and Thailand found in China, An. sawadwongporni, An. maculatus, An. willmori, An. dravidacus and An. notanandai (species G)—Thailand

52 Anopheline Species Complexes in South and South-East Asia An. willmori (species H) — India, Nepal, during the first quarter of the night in all Pakistan, China and Thailand (Chiang seasons. In the Sadao district, only An. Mai) maculatus species B (form E) was detected with peak densities between February and An. pseudowillmori (species I)— China, India, June. The biting activities of this species Nepal, Thailand and Viet Nam varied according to the season. The Species K — Thailand prevalence of mosquitoes was influenced by An. greeni (species D) and An. dispar (species monthly rainfall, relative humidity and air J) are indigenous to the Philippines. temperature. The distribution of members of this complex All species identified in the study were in Thailand is given in Baimai (1989) and the found to be predominantly zoophagic and map is reproduced in this document as Figure preferred to bite humans outdoors rather 10. than indoors. The life expectancy recorded was the highest for species B (E form) — 0.7 An. greeni is widely distributed both in days to 21.2 days; the maximum recorded the low land and hilly areas of the Philippines for species A was 6.6 days and for the F form (Rattanarithikul and Harbach, 1990). An. 8.1 days. No sporozoite-positive glands were dispar appears to be more common, found in any species (out of 4472 dissected) particularly at higher elevations than An. but 0.23 per cent oocyst-positives were greeni (Rattanarithikul and Harbach, 1990). found. In both the areas in the same study, No sporozoite-positive specimens were found An. dirus were found positive for P. falciparum in An. greeni. However, Rattanrithikul and sporozoites. This indicates that species A, B Harbach consider that this may be the same (the E and F forms) and C do not play any species which Ejercito (1934) found infected role in the transmission of malaria in with oocysts and sporozoites of P. falciparum Thailand. The E form was incriminated as in the Bulacan province of Luzon island, the primary vector of human malaria in the Philippines. peninsular Malaysia. The first study conducted on the Another study was carried out to bionomics and vector potential of members determine the vector potential of the of the Maculatus Complex was by Upatham members of the Maculatus Complex at Mae- et al. (1988). An. maculatus s.l. were Tao-Kee near Maesod in north-western collected from a village in the Pakchong Thailand (Green et al., 1991). Four species, district, Nakhon Ratchasima province, central An. maculatus, An. dravidacus, An. Thailand, and another village in the Sadao sawadpongpornii and An. pseudowillmori, district, Songkhla province, southern were found sympatric. One specimen of An. Thailand. In the central Thailand, species pseudowillmori was found infected with P. A, B (form F) and C, and in southern vivax and one with P. falciparum sporozoites Thailand, only form E of species B, were (0.5 per cent sporozoite rate). These identified. specimens were collected on human baits. In Pakchong, An. maculatus species A The man-biting rate of An. pseudowillmori was the most dominant species, followed by was 4.41/night while that of An. maculatus species B (form F) and species C, which was and An. sawadwongporni were 0.89/night rare. The densities of species A and species and 0.41/night respectively. The man-biting B (form F) were high between July and rate of An. pseudowillmori was almost November, with their peaks in October. The equivalent to that of An. minimus species A biting activities of both the species occurred collected during the same study. No throughout the night, with a major peak 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 (chromosomal form E at present assigned to area had the sporozoite rates of 6.4 per cent species B) is a major vector of malaria (Green and 2 per cent respectively. These results et al., 1991). An. willmori from the higher clearly suggest that An. pseudowillmori is altitudes in Nepal is considered responsible capable of maintaining a low-grade for malaria transmission (Pradhan et al., transmission in the absence of efficient 1970). Since An. willmori is found at high vectors like An. dirus A and D. According to altitudes in Thailand, Green, Rattanarithikul Green, Rattanarithikul and Charoensub and Charoensub (1992) suggest that its role (1992), An. pseudowillmori is at the limit of should be studied. In river-irrigated, rice- its distribution in Thailand, and they growing districts of eastern Afghanistan, An. therefore suggest that its role in malaria may maculatus s.l. was incriminated for malaria be different in Myanmar and north-east transmission during a study carried out from India, which are the centre of its distribution. May 1995 to December 1996 (Rowland et Sporozoite-positive mosquitoes were earlier al., 2002). One specimen was found positive reported from Assam state in north-east for P. vivax CSP210 from the 30 tested. After India, Myanmar and Nepal (Rao, 1984). Two more than 50 years of effective management studies by Upatham et al. (1988) and Green of malaria, in sub-districts of Menoreh Hills et al. (1991) have shown that An. and Dieng Plateu, Java, Indonesia, a sharp sawadpongpornii (species A), An. maculatus increase in malaria occurred in the year s.s. (species B) and An. dravidacus (species 2000. Two important vectors, An. maculatus C) may not be playing a role in the and An. balabacensis, which favour forested transmission of malaria in Thailand. hill sides in Java, were considered responsible However, it should be noted that An. for the transmission (Barcus et al., 2002). maculatus s.s. in penninsular Malaysia

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

Cytotaxo- Diagnostic 6-Pgd PCR- ITS2-based Species name nomic inversion electro- RFLP PCR assay desig- genotypes morphs nation on arm 2*

An.sawadwongporni Rattanarithikul & A pt1u1v1w1 130 - ? Green 1986 An. maculatus s.s. Theobald 1901 B,E,F j 100 - ? An. dravidicus Christophers 1924 C x1y1z1 -- ? An. greeni Rattanarithikul & Harbach D q ? - An. notanandai Rattanarithikul & Green G xy -- - 1986 An. willmori James 1903 H - - - ? An. pseudowillmori Theobald 1910 I o1p1q1 70 - ? An. dispar Rattanarithikul & Harbach J r1 - ? - 1990 Putative species K ?** * 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))

95ooooo 97 99 101 103 105 o

VIETNAM

o 20 LAOS MYANMAR

18o

16o

THAILAND

14o

COMBODIA

12o

GULF OF THAILAND

10o

6o MALAYSIA

3.8 The Minimus Complex populations from Thailand. Suthas et al. (1986a) observed significant differences in Anopheles minimus Theobald belongs to the parous rates between females fed on bovids subgenus Cellia, the Minimus Subgroup, the and those on humans. Also, a significant Funestus Group in the Myzomyia Series tendency was observed in females to return (Harbach, 2004). This species has a wide to the type of host upon which they were distribution in the Oriental region, and first caught (Suthas et al., 1986b). To explain throughout the range of its distribution it is the genetic heterogeneity observed in An. considered an important vector of malaria. minimus populations in Thailand, the authors It is found in India, Nepal, Sri Lanka, suggest the possibility that the taxon Bangladesh, Myanmar, Thailand, Malaysia, comprises morphologically cryptic species. Indonesia, south China, Hong Kong, Taiwan and the Ryuku Archipelago, Japan (Rao, Yuan (1987) reported two morphological 1984). Harrison (1980) reported 12 forms, A and B, from the hilly regions of morphological variants in An. minimus China. Sucharit et al. (1988) identified three

Anopheline Species Complexes in South and South-East Asia 55 morphologically variant forms in the Thai among the five alleles observed at the Est-2 population: Typical minimus form (M), locus, Est-298 was predominant (frequency Varuna form (V) and Pampanai form (P). 0.833) in the Pu Toei population, while Est- Among the morphological differences 2100 allele was common in all other described, the characters that are distinct in populations (frequency 0.357 to 0.604). the three forms are: Based on these results, the authors, for the M form - wing with presector pale spot first time, concluded that An. minimus is a (PSP) on costa; species complex consisting of three species. Typical An. minimus (M form) with V form - wing with completely dark predominant Est-2100 or 102 electromorph was prehumeral and humeral bands designated as species A, the P form on costa; predominant in Pu Toei with Est-298 P form - with two pale spots, presectoral electromorph was designated as species C, (PSP) and humeral (HP), on costa. and the V form found in low frequency in Thailand was designated as species B. An. minimus has now been recognized as a complex of 5 sibling species, A, B, C, D Specis A and C (Green et al., 1990) and E. Though species B (Sucharit et al., The population genetic evidence 1988) and species D (Baimai, 1989) have demonstrating a lack of gene flow within An. been reported, no further information is minimus in Thailand came from the study of available on these species. wild populations for electrophoretic variations in six enzyme systems by Green et al. (1990). Evidence for recognition of sibling Sympatric occurrence of two electromorphs, species 134 and 100, at the Octanol dehydrogenase Species A, B and C (Sucharit et al., 1988) (Odh) locus, and the absence of heterozygotes in two localities, was taken as Sucharit et al. (1988) examined wild evidence that An. minimus is a complex populations in Thailand for electrophoretic comprising two sibling species. This variations in seven enzyme systems in relation conclusion was further supported by the to the morphological forms. The enzyme relative deficiency of heterozygotes observed systems studied were: esterases (EST), malic at both the Manose phosphate isomerase enzyme (ME), leucine aminopeptidase (LA), (Mpi) and the Glycerol dehydrogenase (Gcd) lactate dehydrogenase (LDH), malate loci, and the disappearance of the deficiency dehydrogenase (MDH), xanthine when the genotypes of these loci are classified dehydrogenase (XDH) and aldehyde oxidase into the two Odh classes. In the other enzyme (ALDOX). Of these, ALDOX and MDH were systems—phosphoglucomutase (Pgm), monomorphic. Although for such studies hydroxyacid dehydrogenase (Had), lactate more enzyme systems are required to draw dehydrogenase (Ldh) and malate reliable conclusions, a clear-cut relationship dehydrogenase (Mdh)—studied, the relative was nevertheless seen. In the Pu Toei frequencies of alleles were different in the population, the P form was predominant two populations. The authors ruled out (frequency 0.947), in other populations the possibilities such as immigration/emigration M form was predominant (frequency 0.963 and close linkage between loci which could to 1.00), and in Sattahip, the M form cause such associations. The evidence for frequency was 0.794, and that of the V form this was based on genetic analyses which was 0.206. This was the highest frequency established linkage relationship of these loci recorded for the V form; in other populations (Thanaphum et al., 1990) and consistent the frequency ranged from 0-0.033. absence of heterozygotes over a four-year Furthermore, the authors reported that,

56 Anopheline Species Complexes in South and South-East Asia study period at Ban Phu Rat. Following the C laboratory colony and provisionally nomenclature given by Sucharit et al. (1988) designated species E ISG strain, showed that to sibling species in this taxon, Green et al. species C and E are distinct species. In both 100 (1990) equated Odh to species A and the crosses, F1 males were sterile and polytene 134 Odh to species C. chromosomes of F1 hybrid larvae exhibited partial asynapsis. In all the chromosome Van Bortel (1999) studied An. minimus preparations an inversion heterozygote in 3R populations from Viet Nam using chromosomal arm was observed. electrophoretic variations in 14 enzyme systems. Significantly high positive Fis values Suspected new species (Sharpe, Harbach at the Odh locus gave a clear indication of and Butlin, 2000; Somboon et al., 2001) non-random mating within this species, and From the DNA sequences at a mitochondrial the deficiency of heterozygotes indicated locus (Cytochrome Oxidase II) and three reproductive isolation between two sympatric nuclear loci (ITS2 and D3 regions of rDNA populations, species A and C, as in Thailand. and Guanylate cyclase), Sharpe, Harbach and Species E (Somboon et al., 2001) Butlin (2000) confirmed the presence of species A and C within An. minimus from An. minimus specimens collected from Thailand and also reported the possible Ishigahi island, Ryuku Archipelago, Japan, presence of another species. The specimen were identified as species E by Somboon et suspected to be a new species (specimen no. al. (2001). Based on morphological 157) morphologically resembled species C characters and results from genetic crosses, (humeral pale spots on both wings), and had the population from Ishigahi island was unique D3 and ITS2 sequences. But the COII recognized as a new species. 99.5% of the sequence was identical to that of some specimens from Ishigahi island resembled specimens belonging to species C; thus, it species A in having a pale spot on the costa, resembled species C more than species A. but differed with species A and C in having a Since only one specimen was observed, a pale fringe spot at the tip of vein A (a character species status was not assigned to this rarely found in An. minimus populations from specimen. The authors also reported a other countries). In scanning micrographs, personal communication from C. A. Green cibarial armamature showed cone filaments which stated that allozyme data from the differing in shape from those of species A and same locality supported the presence of a C. In a cross between ISG males (strain new species. established from An. minimus collected from Ishigahi island) and species A (CM strain) Somboon et al. (2001) observed females, the hybrid males were sterile due to variations in D3 sequences in the specimens atrophied testes or presence of abnormal from Viet Nam. Sequences from two of the spermatozoa. The hybrid females, when back four specimens were similar to those of crossed to the parental males, laid very few species A and C, while two sequences eggs and there was low hatch. No F1 progeny differed from each other and also from those were produced from the reciprocal cross. of species A and C. These sequences also Metaphase karyotype of ISG differed from differed from that of species E. The authors CM strain (species A) in having different argue that novel sequences might represent polymorphic Y-chromosomes. Polytene new species because each specimen is chromosomes of F1 hybrids exhibited no homozygous for a particular sequence, and asynapsis. These data conclusively suggested suggest that this represents reproductive that species A and E are distinct species. isolation. Based on this evidence the authors Recently, Somboon et al., (2005), based on consider that four species are present in Viet results from genetic crosses between species Nam. Van Bortel and Coosemans (2003)

Anopheline Species Complexes in South and South-East Asia 57 argue that this evidence is unconvincing. Diagnostic Electrophoretic variations According to them, the two novel sequences Alleles at the Odh locus, allele 100 in species probably are due to intra-specific variation. A and 134 in species C, are diagnostic for And this has been insufficiently ruled out by the Thai populations (Green et al., 1990) Somboon et al. (2001) and considered that (Table 9). In the absence of known reference the sequences represent new species. standards, human AA haemoglobin has to be run on each gel to estimate the mobility of Techniques available for the bands of unknown specimens. The identification of sibling species relative mobility of haemoglobin AA was Mitotic karyotypes more useful in the TEB (Tris-boric acid EDTA buffer system) gels than in the TC (Tris-citric Sucharit et al. (1988) briefly described the acid buffer system) gels. Relative mobilities sex chromosomes and a fluorescent band in of a single human Had electromorph and the X-chromosome of the M and P forms. three intense bands of human Ldh were more Baimai, Kijchalao and Rattanarithikul (1996) useful on TC gels. Green et al. (1990) also described mitotic karyotypes of species A and reported Mdh-1 electromorphs to be C from Thailand. Species A can be diagnostic for distinguishing An. minimus s.l., distinguished from species C, as species C has An. aconitus and An. pampanai. Van Bortel prominent pericentric heterochromatin in the et al. (1999), following the Green et al. (1990) autosomes and the short arm of the X. technique, could distinguish An. minimus s. Furthermore, two types of X-chromosomes l. from the closely related species An. aconitus differing in the length of the long arm in and An. jeyporiensis at the Odh locus in species A and a third type of X in species C northern Viet Nam. The authors further were described. Both microphotographs and reported that in Viet Nam An. minimus A was karyotype diagrams are given in Baimai, monomorphic for Odh100 allele but in contrast Kijchalao and Rattanarithikul (1996). to Green et al. (1990) finding in Thailand, Polytene chromosomes An. minimus C in Viet Nam was polymorphic for the Odh locus. However, Van Bortel et al. Kanda et al. (1984) reported photmaps and (1999) in Viet Nam could not separate line-drawing maps of salivary gland polytene unambiguously An. minimus s.l. from An. chromosomes of An. minimus. The authors aconitus using Mdh-1 variation. Est-2 alleles concluded that the polytene chromosomes reported by Sucharit et al. (1988) should be of ISG strain and of two strains isolated from tested for their specificity against diagnostic Kanchanburi, Thailand, have homosequential Odh alleles. Yuan (1987) also reported that banding pattern as no asynapsis was observed their A form and B form exhibit distinct in F progeny from the crosses between the 1 esterase banding patterns. strains. Although the species status of the two strains from Kanchanburi is not known, the Molecular assays ISG strain has recently been identified by Now there are molecular assays available Somboon et al. (2001) as species E. Somboon which not only distinguish species A and C et al. (2005) reported that species A and (Table 9) but also other sympatric anophelines species E have homosequential banding found with An. minimus. Sucharit and pattern, while species C differs from species Komalamisra (1997) used RAPD-PCR to E by a fixed inversion in chromosome arm distinguish species A and C. Fifteen different 3L and one in arm 3R, and partial asynapsis commercially available primers (Operon was observed on chromosome arms 2R and oligonucleotide kit M from Operon 3L in F hybrid larvae. 1 Technologies, Inc.) were used. Six primers were found diagnostic on the basis of the

58 Anopheline Species Complexes in South and South-East Asia presence or absence of amplified fragments primer set for distinguishing species A and C in species A and C. However, these markers and one each for An. pampanai, An. varuna have not been validated on the field samples. and An. aconitus were combined. The method was validated on a large number of Sharpe et al. (1999) developed an allele- specimens collected from Viet Nam, specific amplification assay (ASPCR) from the Thailand, Cambodia and Laos. In northern D3 variable region of the 28S rDNA gene, Viet Nam and north-western Thailand where which distinguished species A and C. A single species A and C are sympatric, <1% of strand confirmation polymorphism (SSCP) hybrids were observed by the isoenzyme assay of the D3 amplified region was also technique (Van Bortel et al., 1999), the PCR- developed, which distinguished species A RFLP method (Van Bortel et al., 2000) and from C and also An. aconitus and An. varuna. by the multiplex PCR of Kengue et al. (2001). Van Bortel et al. (2000) developed a PCR- This suggests that all techniques are equally RFLP technique which distinguished species sensitive and specific. A and C and An. pampanai Buttiker and Beales, An. aconitus, An. varuna and An. Studies correlating the morphological jeyporiensis, which are sympatric. ITS2 rDNA characters with the electrophoretic variation amplification followed by digestion with BisZ by Van Bortel et al. (1999) and with molecular l enzyme accurately distinguished six species diagnostic markers by Sharpe (1999) and belonging to the Myzomyia Series (Van Bortel Chen, Harbach and Butlin (2002) have et al., 2000). The technique was evaluated established that the morphological characters on specimens collected from Viet Nam, mentioned by Sucharit et al. (1988) are Thailand, Cambodia and Laos. Garros et al. unreliable in distinguishing species A from (2004b) extended the above PCR-RFLP assay species C. Furthermore, it has been clearly by including more species from the Funestus shown that form A and form B described in Group and An. gambiae as outgroup in the China by Yuan (1987) should not be equated evaluation. The assay distinguished An. to species, and these forms have no minmus A and C, hybrids between A and C taxonomic status. The An. minimus described and 11 other species. by Harrison (1980) has been identified as species A. In an effort to provide a simple and robust technique for the identification of Another multiplex PCR assay has been members of this complex, Kengue et al. developed for the identification of An. (2001) developed a multiplex PCR method minimus and four other anopheline species based on RAPD markers. From the sequences belonging to the Minimus Group (Phuc et al., of the RAPD markers, sequence characterized 2003). Allele- specific primers were amplified regions (SCARs) specific primers of developed from the rDNA ITS2 region. 20-24mer were designed. In the assay, one Species A and C and An. varuna, An. aconitus,

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

Mitotic Electrophoretic RAPD- PCR- Species SEM SSCP ASPCR2 karyotypes Variation PCR RFLP

A Yes Yes Odh100 Yes Yes Yes Yes C Yes Yes Odh134 Yes Yes Yes Yes EYes------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 and D of the An. dirus complex had man- easily differentiated by this assay. The authors biting rates of only 1.26 and 0.61. Green et claim that their assay results can be al. (1991) suggest that An. minimus species A interpreted more readily than previous assays. in this area is not as efficient as members of Garros et al. (2004a) developed a multiplex the Dirus Complex in transmitting malaria. allele-specific PCR which not only An. minimus populations in India are highly distinguishes members of the Minimus anthropophagic (Rao, 1984). In Sonapur, Group, An. minimus A and C, An. aconitus, Assam state, this species has been found An. varuna, An. leesoni and An. pampanai, resting indoors, highly anthropophagic and but also four members of the Funestus Group, with sporozoite rates ranging between 2.3 An. funestus, An. vandeeni, An. rivulorum and and 3.35 (Wazihullah, Jana and Sharma, An. parensis, in a single step. The assay was 1992). It is from this area, based on Odh developed based on the variation seen in the electromorphs An.minimus specimens were ITS2 region. identified as species A (T. Adak, personal communication). Recently, from four north- Distribution and biological characters eastern states of India–Assam, Arunachal Baimai and Green (1988) give a map showing Pradesh, Meghalaya and Nagaland the distribution of the sibling species of An. An.minimus specimens were identified as minimus in Thailand (see Figure 11). Species species A using Phuc et al. (2003) PCR assay A is widespread in Thailand, in the Thai- (Prakash et al., 2006). In Orissa state, An. Myanmar border areas species C is present, minimus species A was collected from hilltop and in one locality species D is also seen. villages in Singhbhum hills of Koenjhar district Species A has now been reported from Viet after 42 years of its disappearance following Nam, Laos and Cambodia and is found DDT spraying (Jambulingam et al., 2005). sympatric with species C in Viet Nam (Van These findings suggest that there is only one Bortel et al., 2000, 2001; Kengne et al., 2001; member of the Minimus Complex, species A Garros et al., 2005), Laos (S. Manguin, in India. The introduction of insecticide- personal communication) and China (Zhou treated bednets in Assam, reduced the slide et al., 2002; Chen, Harbach and Butlin, positivity rate considerably (Jana-Kara et al., 2002). Though Sucharit et al. (1988) reported 1995). The An. minimus species A from Assam species B from Thailand and China, recent appears to be different from that found in molecular studies identified only species A Thailand with respect to host-feeding and C from these countries. preference and sporozoite positivity. Both species A and C from Thailand were In northern Viet Nam, Van Bortel et al. reported to be predominantly zoophilic, (1999) observed species C to be more feeding on humans more outdoors than exophagic and zoophilic than species A. indoors. At MaeTao-Kee near Masod in north- Species A was found to be highly endophagic western Thailand where members of the and five times more abundant in indoor Maculatus, Dirus and Minimus Complexes collections than species C. This behavioural were prevalent, An. pseudowillmori and difference is considered significant, because species A and D of the Dirus Complex were cattle are kept inside the houses. These found positive for sporozoites, but no observations suggest that An. minimus C may sporozoite-positive An. minimus species A not be a vector of importance in Viet Nam. were found, even though the man-biting rate/ In China this species is considered a vector night (4.41) of this species was almost (Chen, Harbach and Butlin, 2002). Van Bortel equivalent to that of An. pseudowillmori et al. (2001), using the PCR-RFLP method (Green et al., 1991). Furthermore, species A developed by Van Bortel et al. (2000) for the Minimus Group, correctly identified a

60 Anopheline Species Complexes in South and South-East Asia majority of the specimens from a village in dirus is the main vector in the area, several south-central Viet Nam as An. varuna, which specimens of An. dirus being found is predominantly a zoophagic species. Earlier, sporozoite-positive by ELISA (Van Bortel et following morphological identifications, a al., 2001). majority of the specimens from this area were A study by Van Bortel et al. (2003) found wrongly identified as An. minimus s. l. species A breeding along the banks of slow- Because it is considered an important vector, moving rural streams, while in the suburban the control strategies in the area were focused areas of Hanoi it was found breeding in water against two vectors, An. minimus and An. tanks. This demonstrates the adaptability of dirus. Now, it has been established that An.

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

VIETNAM

LAOS MYANMAR

THAILAND

COMBODIA

GULF OF THAILAND

MALAYSIA

Anopheline Species Complexes in South and South-East Asia 61 this species to different breeding philippinensis. Reid (1967) reported that environments. In a recent study carried out there is little or no overlap in distinguishing by Van Bortel et al. (2004), intra-specific these two species if the characters on the behavioural differences among the paddle at the pupal stage are used, and he populations of species A collected from six elevated nivipes to species level. An. different localities were observed. This philippinensis is reported to be distributed species was found more anthropophagic in from India to the Philippines, and northward central Viet Nam, Laos and Cambodia where to China. Because of the overlapping adult cattle were scarce, than in northern Viet Nam wing characters with An. nivipes, accurate where cattle were in good numbers. A similar distributions of these two species cannot be observation that the anthropophagy of species defined. Klein et al. (1984) reported that A was dependent on cattle was made by malaria workers in Thailand refused to accept Trung et al. (2005). These authors observed An. nivipes as a separate species from An. that species A is a late biter and it bites philippinensis because of the overlap in the throughout the night. These observations led adult morphological characters. Trung et al. (2005) to suggest that insecticide- In Bangladesh, An. philippinensis is treated bednets was a suitable strategy for the considered to be the principal vector of control of this species. Garros et al. (2005) malaria in the vast plains of its central, western reported that with the introduction of and northern regions. After the withdrawal permethrin-treated bednets from 1999 to of the large-scale DDT spraying in the late 2001 in central Viet Nam, species A 1970s, An. philippinensis reappeared in some population was drastically reduced, and of the areas (Elias et al., 1987). In India, An. during the same priod species C which was philippinensis was found abundantly in West found in low numbers in the area, increased Bengal, Assam and the neighbouring states significantly. In this study, molecular (Rao, 1984). For the first time, Nagpal and identifications were done with AS multiplex Sharma (1987) have reported An. nivipes, assay developed by Garros et al. (2004a). along with An. philippinensis, from Assam and Meghalaya states. In Thailand An. philippinensis/nivipes are considered 3.9 The Philippinensis- secondary vectors of malaria. Nivipes Complex Evidence for recognition of sibling An. philippinensis and An. nivipes belong to species the Annularis Group of mosquitoes in the Neocellia series. (Harbach, 2004) An. An. philippinensis and An. nivipes philippinensis and An. nivipes are (Klein et al., 1984) morphologically very similar to the only Two laboratory colonies were established, diagnostic wing character being: presector based on the distinguishing characters on the dark mark on vein 1 does not reach as far paddle at the pupal stage (Klein et al., 1982). back as the distal end of the humeral dark The genetic incompatibility, observed in mark on the costa in An. philippinensis, and reciprocal crosses between An. philippinensis the presector dark mark overlaps the humeral and An. nivipes laboratory colonies in the dark mark on the costa in An. nivipes. form of hybrid mortality at egg, larval and However, this character is equivocal (Reid, pupal stages, and hybrid sterility in the 1968). surviving males, established that these two are distinct species and justified the decision Because of the overlap in the of Reid (1967) to raise An. nivipes from the morphological characters, An. nivipes was synonym to species level (Klein et al., 1984). considered as a synonym for An.

62 Anopheline Species Complexes in South and South-East Asia An. nivipes species A and B (Green et al., break-points of inversions found in An. nivipes 1985) were presented by Green et al. (1985), and Two allopatric populations fixed for the the photomaps of An. nivipes are shown in alternative banding pattern on the X- Figure 12. chromosome due to a paracentric inversion, Molecular Assays b, were observed in Thailand (Green et al., 1985), which suggested that there was There are now two assays, an allele-specific another species in this complex. An. nivipes PCR assay of Walton et al., (2007) and a PCR- with X+b is designated as species A, and Xb RFLP assay of Alam et al., (2007), developed as species B. to distinguish members of the An. annularis Group which can be used to accurately Techniques available for identify An. nivipes and An. philippinensis. identification of sibling species The allele specific PCR assay was Morphological variations developed based on differences seen in ITS2 sequences of five species—An. annularis, An. The differences between An. nivipes and An. nivipes, An. philippinensis, An. pallidus and philippinensis are clear at the pupal stage on An. schueffneri—of the An. annularis Group, the paddle (Reid, 1967). In An. nivipes, the which are morphologically very close (Walton paddle has a longer refractile border with et al. al., 2006). While the An. schueffneri is distinctly numerous teeth and shorter paddle restricted to Java and Sumatra islands of hair. In An. philippinensis, the paddle has less Indonesia, An. pallidus to Sri Lanka, India and than ten fringe teeth on the refractile border Myanmar, the other species are widespread and has longer paddle hair, the in the region. The authors report that the unstraightened length being about one third assay developed will be a rapid and reliable that of the paddle. epidemiology tool, which should work from Polytene chromosomes northeastern India through Myanmar and Thialand to Laos and Cambodia. By examining polytene chromosomes of An. nivipes and An. philippinensis colonies (Klein Prakash et al. (2006) examined ITS2 et al., 1982), two autosomal inversions were sequences of morphologically confirmed An. identified that could unequivocally distinguish nivipes and An. philippinensis specimens from An. nivipes from An. philippinensis at the adult Assam and Nagaland states in the northeast stage by Green et al. (1985). The two India to assess the applicability of Walton et inversions were t on the chromosome arm 2, al. (2006) PCR assay. The sequences of An. and l on arm 5. The X-chromosome and phillipinensis shared 99.2% and An. nivipes chromosome arms 3 and 4 are shared 99.3% respective similarity to those homosequential in An. philippinensis and An. from the Thialand. nivipes. The b inversion on the X-chromosome The PCR-RFLP assay was developed based differentiates An. nivipes A from B. on endonuclease restriction sites in the D3 The diagnostic inversion genotypes of the sequence to differentiate four members in the species are: An. annularis Group—An. annularis, An. nivipes, An. philippinensis, and An. pallidus— An. philippinensis — X+b; 2+t; 5+l (Alam et al., 2007). Restriction digestion of D3 An. nivipes species A — X+b; 2t; 5l fragment individually with SmaI, ApaI and NcoI An. nivipes species B — Xb; 2t; 5l produced distinct pattern of all four species on 3. 5 per cent agarose-gel. D3-SmaI system The photomaps of the polytene can be used to distinguish An. nivipes from chromosomes of An. philippiensis with the An. philippinensis.

Anopheline Species Complexes in South and South-East Asia 63 Distribution and biological characters Collections were made from Kamrup district Subbarao, Vasantha and Sharma (1988) and in Assam, Umbling in Meghalaya, Bhalapur Subbarao et al. (2000) carried out extensive in Arunachal Pradesh, Imphal in Manipur and cytotaxonomic examinations of specimens Dimapur in Nagaland. All the specimens were collected from five states in north-east India found to be An. nivipes A. (All these following the report of Green et al. (1985). 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 + 2 t 3 4 5 l

t c

64 Anopheline Species Complexes in South and South-East Asia characters on the wing which distinguish An. philippinensis and An. nivipes in malaria nivipes and An. philippinensis). Some of these transmission in the north-eastern states of areas were the same as those surveyed by India. Nagpal and Sharma (1987) who reported the Following the discovery of the diagnostic presence of both the species based on their inversions, the Thai populations were wing characters. Thus, the wing character was examined for the presence of two species. not found diagnostic for Indian populations An. nivipes was found to be more common, as concluded by Reid (1967) for the Thai but An. philippinensis was found to be more populations. However, the presence of An. widespread than was earlier thought (Baimai philippinensis cannot be ruled out in these et al., 1984). In the light of the findings from five states. Prakash et al. (2000) reported An. India, the populations from Bangladesh need nivipes and An. philippinensis, based on larval to be examined. and pupal diagnostic characters, from the Changlang district of Arunachal Pradesh and recently (2004) from Dibrugarh district in 3.10 The Punctulatus Assam, and also from the neighbouring states, Arunachal Pradesh and Meghalaya. In Complex Dibrugarh, all the nine females from human- Anopheles punctulatus belongs to the landing collections were identified as An. subgenus Cellia, and the Punctulatus Group philippinensis, while all the 16 females from in the Neomyzomyia Series. This taxon has the resting collections made near cattle sheds a wide distribution from the Moluccas in the were An. nivipes. west through New Guinea and the Solomon An. philippinensis, once dominant in the Islands to Vanuatu in the east, and as far south deltaic regions of West Bengal and as northern Australia. Rozeboom and Knight responsible for the high incidence of malaria (1946) identified four species in the in this area, is almost absent today (Rao, Punctulatus Group, based on the 1984). The earlier dissection records showed morphological characters of the proboscis sporozoite-positive specimens from West (Bryan, 1973a; Foley et al., 1993). Bengal, but not from Assam, in spite of a large (i) An. farauti Laveran — totally dark number of dissections (Krishnan, 1961). proboscis, except for a pale subapical ring However, An. philippinensis, identified based (based on this character, An. moluccensis on its wing character, was incriminated as a from the Moluccas and Irian Jaya, vector of malaria in Burnihat, which is now Indonesia, was synonymized with An. in Meghalaya state (Rajgopal, 1976). The farauti). cytotaxonomic examination (Subbarao et al., 2000) has revealed only An. nivipes in (ii) An. punctulatus Doenitz — with the Meghalaya, and from pupal paddle characters apical third to half of the proboscis pale, recently by Prakash et al. (2004). Among An. the remainder dark. philippinensis-nivipes s. l. collected from CDC (iii) An. koliensis Owen — white scaling on light traps placed in human dwellings in the proboscis variable but more often districts Dibrugarh and Jorhat, Assam state, restricted to a small discrete patch on the specimens positive for P. vivax and P. apical third of the proboscis. falciparum sporozoite antigens were found by ELISA (Prakash et al., 2005). Extensive (iv) An. clowi (Rozeboom and Knight) — cytotaxonomic (Subbarao et al., 2000) or proboscis is similar to that of An. koliensis, molecular identifications (Prakash et al., and the legs are yellowish instead of 2006) along with ELISA tests are needed to having dark areas on at least some or all establish unequivocally the relative role of An. of the fore- and mid-tarsomers. This

Anopheline Species Complexes in South and South-East Asia 65 species was described from Irian Jaya, b). There was either reduced hatchability or Indonesia (Rozeboom and Knight, 1946). total sterility in crosses between An. koliensis This species was rediscovered by Cooper and An. farauti and An. punctulatus. The et al. (2000) in Papua New Guinea. adults which emerged had reduced or partially developed reproductive organs. Marked differences observed in the An. These results proved that An. koliensis is a farauti s.l. populations in peak biting times separate species. and breeding habitats suggested the presence of more than one species within this taxon. An. farauti No. 3 (Mahon and Miethke, Cross-mating experiments and cyto- and 1982) biochemical-taxonomic studies have A population found sympatric with An. farauti identified 11 species so far in the Punctulatus No. 1 and An. farauti No. 2 in Australia Complex. In the South-East Asia region, produced sterile progeny in reciprocal crosses members of this complex occur only in with An. farauti No. 1 and No. 2. A fixed Indonesia. Kondrashin and Rashid (1987) paracentric inversion was found in this report An. punctulatus, An. koliensis and An. population, and the population was farauti as important vectors in Irian Jaya/ considered a new species and was designated Maluku islands. as An. farauti No. 3. Evidence for identification of sibling An. farauti No. 4, No. 5 and No. 6 species Bryan et al. (1990) studied variations at 14 An. farauti No. 1 and An. farauti No. 2 enzyme loci in the populations from Papua An. farauti (Bryan, 1970; 1973a and b) from New Guinea and the colony material of An. two colonies established from (1) Rabraul, farauti No. 1, No. 2 and No. 3. The New Britain, Papua New Guinea, and (2) phenogram revealed nine clusters for which Innisfail, northern Queensland, Australia, more than 20 per cent loci were fixed for were crossed. Both female and male progeny different alleles. The authors considered that from the reciprocal crosses were sterile. This there may be nine species in this complex, was taken as evidence for the existence of as so many fixed differences could not be two species within An. farauti; the colony maintained within a population which shares from Papua New Guinea was designated as the same gene pool. An. farauti No. 1 and the colony from Australia Subsequently, Foley et al. (1993) as An. faruati No. 2. Bryan and Coluzzi (1971) analysed cellulose acetate gels of populations observed two paracentric inversions, one on from 19 localities in Papua New Guinea for the left arm and another on the right arm of electrophoretic variations at 35 loci. Based chromosome arm 2 in An. farauti No. 2. In on fixed allele differences, six species were hybrids between An. farauti No. 1 and An. identified. Of these, An. punctulatus, An. farauti No. 2, inversion heterozygotes were koliensis and An. farauti No. 1 had already observed in these regions. This suggests that been reported; three were new species, the two populations differ by fixed which were provisionally designated as An. paracentric inversions. farauti No. 4, No. 5 and No. 6. The population designated as An. farauti No. 4 An. koliensis (Bryan, 1973a and b) from Gonoa produced sterile F1 progeny Doubts about whether An. koliensis is a hybrid when crossed with An. farauti No. 1, and between An. punctulatus and An. farauti asynapsis in polytene chromosomes of F1 (because of the variable pale scales on the progeny (Bryan et al., 1990). proboscis) were removed by cross-mating experiments carried out by Bryan (1973a and

66 Anopheline Species Complexes in South and South-East Asia An. farauti No. 7 (Foley, Meek and Bryan, Formal names 1994) Schmidt et al. (2001) studied the The specimens collected either as larvae or morphological variations and identified adults from 33 localities in the Solomon Islands markers for three species. An. farauti No. 1, and six localities in Vanuatu were analysed for No. 2 and No. 3 in the adult female, IV instar electrophoretic variations. The same enzyme in larvae and pupae. On this basis formal systems that were used for the Papua New names were given: Guinea population (Foley et al., 1993) were used in this study, except that β-galactosidase An. farauti No. 1 (conspecific with An. was added and the Ak-1, Ao, Fdp-1 and Mpi farauti sensu stricto as described by Foley and loci were excluded because of unreliable Bryan, 1993, and Foley, Meek and Bryan, staining. Enzyme analysis revealed the three 1994) designated as An. farauti Laveran. already identified sibling species, An. farauti An. farauti No. 2 designated as An. No. 1 and No. 2 and An. punctulatus, and a hinesorum Schmidt sp. n. population which did not correspond with any of the earlier identified species was found in An. farauti No. 3 designated as An. two localities on northern Guadalcanal, torresiensis Schmidt sp. n. Solomon Islands. Fifteen specimens belonging Techniques available for to the new population were examined and all identification of sibling species had dark probosces as described for An. farauti s. l. This group of mosuqitoes was considered Crossing experiments as a new species and was designated as An. An. punctulatus, An. koliensis and An. farauti farauti No. 7. No. 1, No. 2, No. 3 and No. 4 produced An. sp. near punctulatus (Foley, Cooper progeny in reduced numbers, and with and Bryan, 1995) reduced reproductive organs in reciprocal crosses between the above-mentioned Within the specimens identified species (Bryan, 1973a and b; Mahon and morphologically as An. punctulatus, two Miethke, 1982; Foley et al., 1993). genetically distinct groups were found when examined for enzyme electrophoretic variations Polytene chromosomes and analysed for genetic distance. The new Polytene chromosomes from IV instar larval group differed from An. punctulatus populations salivary glands were used for the with 34-39 per cent fixed genetic differences identifications, since polytene chromosomes and Nei’s D ranging between 0.5 to 0.61. The from ovarian nurse cells were not readable maximum genetic divergence recorded within in contrast to those in other anopheline An. punctulatus collected from widely separated species. Bryan and Coluzzi (1971) identified localities in Papua New Guinea was 18 per cent paracentric inversions diagnostic for An. (Foley et al., 1993) and 6 per cent for specimens farauti No. 1, No. 2 and No. 3. collected in the Solomon Islands (Foley, Meek and Bryan, 1994). This was taken as evidence Salinity tolerance Test (STT) to show that in the western province of Papua An. farauti No. 1 showed a higher tolerance New Guinea there is a population conspecific of salinity than An. farauti No. 2 and No. 3 with An. punctulatus as described earlier and a (Sweeney, 1987). Tests carried out with 0- population that was not described earlier, i.e. a 3.4 per cent salinity showed complete new species designated as An. sp. near mortality of An. farauti No. 2 and An. farauti punctulatus. 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 DNA probes survival, while Port Moresby colony showed Three DNA probes, pAf1, pAf2 and pAf3, 73 per cent survival. An. farauti No. 7 was were selected by screening partial genomic thought to be an obligatory fresh-water libraries with radio-labelled total genomic species which would not tolerate salinity. DNA from each species (Booth, Mahon and However, Foley and Bryan (2000) found An. Sriprakash, 1991). pAf1 hybridized with both farauti No. 7 could survive the STT and thus An. farauti No. 1 and No. 2, pAf2 hybridized could not be efficiently differentiated from strongly to An. farauti No. 3, less to An. farauti An. farauti s.s. which is a vector on No. 1 and faintly to An. farauti No. 2, Guadalcanal, Solomon Islands. The authors, suggesting that there is cross-hybridization. therefore, recommend alternatives to the STT Hartas et al. (1992) sequenced these probes for the vector species on Guadalcanal. and identified oligonucleotides of 25-26 base pairs specific for each probe. Radio-labelled Biochemical taxonomy and oligonucleotides, oAf1, oAf2 and oAf2, electrophoretic keys hybridized respectively to An. farauti No. 1, Bryan et al. (1990) and Foley et al. (1993) No. 2 and No. 3. These oligonucleotide analysed populations from Papua New probes identified both dot-blots and squash- Guinea for electrophoretic variations and blots of larvae, pupae and adults very developed electrophoretic keys. Foley and efficiently. Bryan (1993) give the keys for six species, An. farauti No. 1, No. 4, No. 5 and No. 6, An. Cooper, Cooper and Burkot (1991) punctulatus and An. kolinesis, from Papua independently identified three DNA probes New Guinea, and for An. farauti No. 2 and specific for An. farauti No. 1, No. 2 and No. No. 3 from Australia. Alternative keys are 3. Total genomic libraries of the three species given depending on the availability of were screened by hybridizing with different species for standards. radiolabelled homologous and heterologous total genomic DNA of species. Three probes, Foley, Meek and Bryan (1994) presented 1/1, 9/2 and 5/3, specific for An. farauti No. a key for distinguishing An. farauti s.s., An. 1, No. 2 and No. 3 respectively, were farauti No. 2 and No. 7, and An. punctulatus identified. Probes labelled with P32 or horse- which are prevalent in the Solomon Islands radish peroxidase with enhanced and Vanuatu. These species can be chemiluminescence detection system or distinguished by staining for the enzyme LDH biotinylated streptavidin-conjugated alkaline only. The authors also discuss combinations phosphatase with the chromogenic detection of standards and diagnostic enzymes required system were all found equally sensitive in to identify the species. identifying the species both in dot-blots and Specimens of new species An. sp. near squash-blots. punctulatus were found homozygous for a Beebe et al. (1994) made DNA probes unique allele at Pep D-1 (Foley, Cooper and for the identification of five members of the Bryan, 1995). The authors provisionally Punctulatus Complex namely, An. reported this allele to be diagnostic for this punctulatus, An. koliensis and An. farauti No. species based on the small number of 4, No. 5 and No. 6 by differentially screening specimens examined. Additional alleles that genomic libraries with P32-labelled discriminate An. punctulatus and An. sp. near homologous and heterologous DNA. The punctulatus are at the Hk, Me-1 and Hbd II sizes of the probes that were selected ranged loci. between 273 and 630 bp. The probes identified specimens from DNA dot-blots and

68 Anopheline Species Complexes in South and South-East Asia squash-blots. These probes are sensitive (1994). In Gonoa, Papua New Guinea, An. enough to identify fragments of mosquitoes. farauti No. 4 and An. koliensis were caught on opposum, pig and human bait, while An. Beebe et al. (1996) developed two DNA punctulatus was caught on human and pig probes specific for An. farauti No. 7 and An. baits (Foley et al., 1993). On human bait sp. near punctulatus, by screening genomic four out of six An. farauti No. 4 were caught libraries. A probe which hybridizes to all before 2000 hours, suggesting early biting members of the Punctulatus Complex has also activity of this species. Now that eleven been developed. These probes have been members have been identified in this field-tested. complex and techniques are available for All members of this complex can be their identification, studies should be identified with DNA probes. undertaken to map their distribution and to establish their role in malaria transmission. PCR-RFLP Beebe and Saul (1995) differentiated 10 species, An. farauti No. 1 to No. 7, An. 3.11 The Sinensis Complex koliensis, An. punctulatus and An. sp near punctulatus, by restriction enzyme digestion Anopheles sinensis Wiedman, 1828 belongs of rDNA ITS2 product (750bp amplicon) with to the subgenus Anopheles, the Hyrcanus Msp-I. Group in the Myzorhynchus Series (Harbach, 2004). An. sinensis is an important vector of rDNA-ITS2 sequence analysis malaria throughout the Republic of Korea Beebe et al. (1999) analysed sequence (Chow, 1970). This species is considered to variations in the rDNA ITS2 region among be the main vector responsible for malaria in 10 species of this complex. The length of central China. Malaria in this area is unstable ITS2 ranged from 549 to 565bp. Sequence and outbreaks have regularly been reported variation among the 10 species studied since the 1950s. As recently as in 1995, ranged from 2.3 per cent to 2.4 per cent Honan province in China experienced an mainly due to indels of simple sequences. outbreak of P. vivax (Sleigh et al., 1998). This species is also considered to be a vector in Distribution and biological characters Japan, Indonesia and Thailand (O’Connor Information on the distribution of members 1980, Kanda et al., 1981). However, in of this complex in Papua New Guina, Thailand, it is not considered an important Solomon Islands and Australia is avilable from vector. recent investigations using allozymes (Bryan, Evidence for recognition of sibling Reardan and Spark, 1990; Foley et al., 1993 and Foley, Meek and Bryan, 1994). From Irian species Jaya Islands of Indonesia, An. punctulatus, An. Sibling species in China— An. sinensis koliensis and An. farauti s.l. have been and An. lesteri syn. anthropophagus reported (Kondrashin and Rashid, 1987), and Two morphological variants based on the size An. clowi (from an earlier report). of the deck of the egg were reported in the An. farauti No. 2 and No. 7 were found 1950s (cited in Reid, 1953). Later, it was to be zoophilic while An. farauti No.1 (now found that the narrow deck egg variety was An. farauti Levaran) was highly more anthropophilic, endophagic and anthropophilic. Differences in anthropophily endophilic than the wider deck variety which were observed on north Guadalcanal in the fed outdoors and predominantly on cattle. Solomon Islands by Foley, Meek and Bryan 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 be different physiologically from An. sinensis, subspecies of An. lesteri and was called An. in the mean frequencies of the clasper lesteri anthropophagus. Later, Ma (1981) (as movements during induced copulation mentioned in Gao, Beebe and Cooper, 2004) (Kanda and Oguma, 1978). Based on this raised anthropophagus to the species status evidence the Engaru strain was considered a and designated it as An. anthropophagus distinct sibling species, morphologically based on its morphology, distribution and similar to An. sinensis and designated as An. vectorial capacity. The wider deck variety is engarensis. designated as An. sinensis. An. Kanda et al. (1981) reported from the anthropophagus and An. sinensis are polytene chromosomal examination of about isomorphic species at the adult stage. 2000 specimens from Engaru, where both the Wilkerson et al. (2003), however, reported species exist sympatrically, that no that An. anthrophagus and An. lesteri are heterozygotes for 2RB inversion were found. identical in ITS2 sequences and are, The absence of heterozygotes further therefore, synonymous. This was based on supported the view that there are two distinct the examination of An. lesteri from the type species in the area, and also suggested that locality in Laguna province from the there is pre-mating isolation in addition to Phillippines and An. anthropophagus from the post-mating barrier already reported Jhangsu Province, China. Ma and Xu (2005) (Oguma, 1978). supported this conclusion and said that An. anthrophagus is a junior synonym of An. An. sineroides Yamada 1924 has a lesteri. Wilkerson et al. (2003) clarified the restricted distribution, being found from status of An. lesteri present in China by stating Kokkido to Kyushu in Japan, in China and in that it is another unknown species and is also Korea. An. sineroides and An. sinensis are found in Korea. This is supported by Gao, morphologically similar, hence hybridization Beebe and Cooper (2004) observation that it experiments were conducted to establish the is different from An. anthrophagus and An. taxonomic status (Kanda and Oguma, 1977). sinensis based on ITS2 sequences. In crosses between the strains of An. sinensis and An. sineroides collected from Japan, Sibling species in Japan — An. sinensis, heavy mortality in F1 larvae and pupae, and An. engarensis and An. sineroides asynapsis in polytene chromosomes of F1 Successful colonization of An. sinensis using progeny were observed. Based on these an induced copulation method devised by results, the authors concluded that An. Oguma and Kanda (1976) led to crossing sinensis and An. sineroides are two distinct studies. Strains collected from Koniya, sibling species. The X-chromosome of An. Kanoya, Karurizawa, Yomogita, Yakumo and sineroides was found similar to that of An. Engaru ranging from north to south in Japan koreicus rather than to that of An. sinensis were used in the crosses (Oguma, 1978). In (Oguma and Kanda, 1970). two crosses, Engaru with Kanoyo and Engaru Cytotypes of An. sinensis in Thailand and with Yakumo, F1 males were sterile and females were fertile. In the other crosses (of Republic of Korea the 36 possible crosses, 19 were carried out) Two forms designated as form A and form B where Engaru was not involved, the progeny were identified within An. sinensis, based on were fertile. Therefore, it was concluded that structural difference in the Y-chromosome the Engaru strain is distinct and was given (Baimai, Rattanarithikul and Kijchalao, 1993). species status, while all the other strains were Form A has a telocentric or acrocentric Y- considered to belong to one species, An. chromosome (short arm being very small) and sinensis. The Engaru strain was also found to form B has a sub-metacentric Y-chromosome.

70 Anopheline Species Complexes in South and South-East Asia Choochote et al. (1998) carried out crossing Molecular methods experiments between form A and form B Restriction fragment length differences of using induced copulation. In all crosses viable genomic repetitive DNA (Li et al., 1991 from progeny were observed and no genetic Abstract) incompatibility was observed in crosses with High molecular weight DNA of five species, the F1 progeny. Polytene chromosomes of An. sinensis, An. lesteri syn. anthropophagus, 4th instar larvae of F1 progeny showed An. liangshanensis, An. crawfordi and An. complete synapsis. In another study (Min et xiaokuanus, belonging to the Hyrcanus al., 2002), form A from Thailand was crossed Group, was cut with three restriction with form B isolines from Thailand and Korea. endonucleases. All three enzymes, Bgl II, Hae In the reciprocal crosses, and corresponding III and Pst I, produced diagnostic fragments back crosses, no genetic incompatibility was discernable on agarose gel for all species. observed affecting hatchability, viability of Thus, all five species could be distinguished immature stages, sex ratio or adult by this method. emergence. Thus, both cytotypes, form A and form B, were considered as intra-specific Y- An allele-specific PCR assay (Ma Qu and Xu, chromosome variations within An. sinensis. 1998 cited in Gao, Beebe and Cooper, 2004) This was further supported from sequence This assay was developed from ribosomal analysis of ITS2 and COII regions of form A DNA to differentiate An. sinensis from An. and B. The sequences were near identical lesteri syn. anthropophagus. With this assay with a sequence variability of 0.0-0.6% (Min only 78.9% of field-collected samples could et al., 2002). be identified, i.e. many remained Thus, it appears that An. sinensis is a unidentified because of failure to produce complex of at least four species, An. sinensis PCR products. According to the authors, this s.s., An. lesteri (syn. anthropophagus), An. was because unidentified samples belonged sineroides and An. engarensis. to another yet unknown cryptic species. Another possible reason as pointed out by Techniques for identification of Gao, Beebe and Cooper (2004) could be due sibling species to intra-specific sequence variation within the primer region which inhibited annealing. Morphological variations PCR-RFLP assay (Gao, Beebe and Cooper, 2004) At the adult stage all four species, An. sinensis, An. engarensis, An. sineroides and An. lesteri Universal primers from the rDNA ITS2 region syn. anthropophagus, are isomorphic. The were used to amplify, and the amplicons were size of the deck of the egg varies between digested with Hind I and Rsa I restriction the two species, i.e. it is narrow in An. lesteri enzymes. After digestion the patterns on syn. anthropophagus and is wider in An. acrylamide gel were distinct with both the sinensis. An. lesteri (an earlier designation enzymes. To validate the assay, field-collected and needs formal redesignation following specimens from seven areas in China and An. Wilkerson et al., 2003) also has a narrow deck sinensis specimens from an established and thus resembles An.lesteri syn. laboratory colony were used. All the anthropophagus. Both An. lesteri and An. specimens identified morphologically based yatsushiroensis (another species in the on egg deck size as An.lesteri syn. Hyrcanus Complex which is sympatric with anthropophagus were also identified by the these species) can be distinguished PCR as the same species except for 4 of 8 morphologically at the adult stage from An. specimens from Guangdong province which sinensis and An. lesteri syn. anthropophagus. 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 developing oocysts or sporozoites while both province mostly had narrow decks typical of exhibited low susceptibility to P. vivax with An.lesteri syn. anthropophagus, with a few 0.00– 0.571 per cent and 0.00-.588 per cent with wide decks typical of An. sinensis. But oocyst and sporozoite rates respectively. Both all the specimens were identified as An. lesteri the strains were refractory to P. yoelii when syn. anthropophagus by molecular assay. This fed directly on infected rodents (Rongstrriyam suggests that the egg deck size is not reliable et al., 1998). to distinguish these two species as it was Malaria is endemic in central China but thought earlier. it is unstable and with regular occurrence of Distribution and biological characters outbreaks (Li et al., 1995). Gao, Beebe and Cooper (2004) report An.lesteri syn. An. sinensis forms, A (XY ) and B (XY ), 1 2 anthropophagus to be widespread identified in Thailand by Baimai, throughout central China and it occurs Rattanaritikul and Kijchalao (1993) were also sympatrically with An. sinensis. However, reported to be found in Taiwan (China) and they report that the distribution of An.lesteri China. Recently, Ma and Xu (2005), based syn. anthropophagus is patchy compared to on ITS2 sequences reported that in China An. sinensis. An. sinensis is predominantly a only B form haplotype was found. cattle-feeder in China. Throughout central Throughout the Republic of Korea, An. China, An.lesteri syn. anthropophagus is sinensis has been considered for a long time considered a primary vector and An. sinensis to be an important vector. Recently, Coleman a secondary vector. Molecular identifications et al. (2002) collected anopheline mosquitoes (Gao, Beebe and Cooper, 2004) have from 29 locations distributed throughout the extended the distribution of An. lesteri syn. country. Three species, An. sinensis/lesteri (as anthropophagus northward by 1200 km (420 these two species cannot be identified N, 1200 E). This region is north of the Korean accurately, no effort was made to separate peninsula. A small sample examined using them), An. yatsushiroensis Miyazaki and An. molecular techniques by Ma et al. (2000) sineroides Yamada, were found. 89.7% were cited in Gao, Beebe and Cooper (2004) An. sinensis/lesteri, and of these two pools, identified only An. sinensis, An. lesteri and one of 10, and another of nine (screened by An. yatsushiroensis. Along the North/South ELISA) were found positive for P. vivax (CSP Korea border, Stricman et al. (2000) reported 247). that, based on morphological identifications, For An. sinensis, the anthropophilic index An. sinensis was the most abundant mosquito (AI) and average probability of daily survival biting humans, constituting more than 80 per rate respectively were 0.7 per cent and 0.89 cent of the anopheline human-landing in the Guonggi–do (Ree et al., 2001) and 0.8 collections. These mosquitoes were collected per cent and 0.79 per cent in the Paju (Lee during an outbreak of P. vivax malaria. Now et al., 2001) areas of the Republic of Korea. that a molecular assay is available (Gao, In both publications the investigators suggest Beebe and Cooper, 2004) for accurate that in spite of their tendency to zoophily and identification of An.lesteri syn. with moderate survival rate, this species is anthropophagus and An. sinensis, large responsible for malaria transmission because samples from the Republic of Korea are to of high population densities. In Thailand, An. be identified to establish whether only An. sinensis is not considered an important vector. sinensis is prevalent as is being reported. This In a laboratory-feeding experiment, two may resolve the anomalies reported in the cytologically different An. sinensis strains (form feeding behaviour of the mosquito hitherto described as An. sinensis. A-XY1, and form B-XY2) were found to be totally refractory to P. falciparum with none

72 Anopheline Species Complexes in South and South-East Asia 3.12 The Subpictus Complex together with species A in the Union Territory of Pondicherry in India (Suguna,1982). Anopheles subpictus Grassi belongs to the The four sibling species, provisionally subgenus Cellia, and the Pyretophorus Series designated as A, B, C and D, were identified (Harbach, 2004). An. subpictus is very widely by examining polytene chromosomes from distributed and found in abundance in the the ovaries of adult females collected from Oriental region. It is found to the west of the field and those from the salivary glands India in Pakistan, Afghanistan, and Iran, and of larvae collected from breeding sites. Two in the east as far as Papua New Guinea and inversions on the X-chromosome, a, a small the Marina island, Malaysia. It is also found inversion towards the tip of the chromosome, in Sri Lanka in the south and China in the and b, an inversion in the middle of the north. In India, it is found throughout the chromosome (this same inversion was mainland and in Lakshadweep islands but not designated as a in the earlier publication of in the Andaman and Nicobar islands (Rao, Suguna, 1982), and their combinations 1984). +a+b, ab, a+b and +ab were found with a In Indonesia, it is considered as a total absence of heterozygotes in the natural secondary vector (Van Hell, 1952; populations examined. This was taken as an Sundararaman, Soeroto and Siran, 1957). In evidence for the recognition of four species India, sporozoite-positive specimens were (Suguna et al., 1994). reported from a coastal village in the state of Tamil Nadu (Panicker et al., 1981) and in the Techniques available for district of Bastar in Madhya Pradesh (Kulkarni, identification of sibling species 1983). Amerasinghe et al. (1991; 1992) Inversions on polytene X-chromosome are reported this species to be a vector in Sri diagnostic characters in the identification of Lanka. sibling species. The four inversion Based on the morphological differences arrangements observed were: species A – in the eggs of An. subpictus, Reid (1966) X+a+b, species B – Xab, species C – Xa+b suggested that this might be a species and species D – X+ab. A photomap of complex, and Suguna (1982) for the first time polytene chromosomes complemented with provided clear evidence that this taxon is a break-points marked for inversions a and b complex of two sibling species, A and B. Later, are given in Figure 13. this taxon was found to be a complex of four Reuben and Suguna (1983) reported sibling species, A, B, C and D (Suguna et al., morphological differences in eggs, larvae, 1994). pupae and adults between sibling species A and B. Now that species C and D have also Evidence for recognition of sibling been reported within the fresh water- species breeding populations in Pondicherry (Suguna Differences in egg morphology and in the et al., 1994), the differences reported by banding pattern of polytene X-chromosome Suguna (1982), and Reuben and Suguna due to a paracentric inversion were the basis (1983) between species A and B, are not for the identification of species A and B. discussed here as they are no longer valid for Species A with the X+a karyotype (standard the identification of all the sibling species. arrangement) was predominant in inland Differences in egg, larval, pupal and adult villages while in the coastal villages species B morphological characters among the four with the Xa inversion arrangement was found 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 C of Suguna et al. (1994). Atrie (1994) An. subpictus population from villages around reported that in the Delhi population, the Delhi, India, were identified as species A apical pale band in adult females was longer (Subbarao, Vasantha and Sharma, 1988) by than the sub-apical dark band, which is the examining the ovarian polytene characteristic feature of species A (Table 10). chromosomes of adult females collected from Thus, it was felt that there was a need for a the field, following the report of Suguna detailed study to correctly identify the specific (1982). The ridge number on the egg float taxonomic status of the Delhi population. ranged between 21 to 30, and this did not Recently, Singh et al. (2004) conducted a correspond with the characters of species A detailed study on An. subpictus collected from which has, on an average, 33 ridges (Suguna, Sonepat district, Haryana (a state 1982), but it corresponds to those of species neighbouring Delhi). In this study, field- collected 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)

74 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)

Egg Larva Pupa Adult Breeding habitats

Species Inversions Mean Frill Seta 4M Seta 7-1 Length of (Salinity range %) on X- ridge No. apical pale chromo- (range) band on some female palpi

A +a+b 35 Opaque 2-branched Simple; as Longer than Paddy fields (31-36) (rarely 3) long as sub apical dark (0.0054-0.2636), hairs 6&9 band Riverine pools (0.0247-0.7827) and Back waters (0.5574-5.3554)

Bab 18 Transp- 2-branched Branched Shorter than Back waters (16-20) arent (rarely 3) 4-5; sub apical dark (0.5574-5.3554) shorter band than hairs 6&9

C a+b 27 Semi- 3-branched Branched - Equal to sub Same as A (25-29) transpa- (rarely 2) 2; shorter, apical dark rent but longer band than in sp. B

D +ab 22 Semi- 3-branched Branched - (21-24) transpa- 3; shorter rent isofemale lines, eggs, larvae, pupae and villages of Pondicherry by Panicker et al. adults, were examined for morphological (1981) suggests that species B may be a vector. characters following Suguna et al. (1994). Data of Kulkarni (1983) in Bastar district, Species A, C and D were found in almost Madhya Pradesh state in India, and equal proportions, and no variation was Amerasinghe et al. (1991 and 1992) in an observed in the proportion of the three sibling irrigation development area of the Mahaweli species from field-collected adults and project in Sri Lanka suggested that fresh water- isofemale lines. An. subpictus breeding in breeding sibling species may also be playing the villages was found in the river-bed pools. a role in malaria transmission. Fresh water- Thus, in northern India, all three fresh water- breeding An. subpictus from Delhi, when fed breeding species are sympatric. In Sri Lanka, on Plasmodium vivax-infected blood both species A and B were identified developed oocysts and sporozoites (Nanda (Abhayawardana, Wijesuriya and Dilrukshi, et al., 1987). This suggests that fresh water 1996) based on species-specific diagnostic breeding An. subpictus is genetically inversion genotypes reported by Suguna susceptible to plasmodial infection. Generally, (1982). In Sri Lanka, species A was found to the densities of this species are relatively high. be more endophilic and seasonally more The species may not be playing a role in abundant than species B (Abhayawardana, malaria transmission probably due to high Wijesuriya and Dilrukshi, 1996). zoophagy and poor longevity, which are relevant for the species to be a vector in the No studies have been reported so far on field. the biological characteristics of these four sibling species in India. Detection of Resistance to malathion and fenitrothion sporozoite-positive specimens in coastal was observed in An. subpictus predominant

Anopheline Species Complexes in South and South-East Asia 75 in inland regions, while in coastal areas those in which sea water and fresh water resistance to permethrin was observed in this mingle, but it was also found adapted to fresh species (Kelley-Hope et al., 2005). water in some areas. In Vishakapatnam, Abhayawardana, Wijesuriya and Dilrukshi Andhra Pradesh state, India, it was found (1996) reported species A’s predominance in exclusively in fresh water-breeding places inland regions and that of species B in coastal such as tanks, ponds and borrow pits. In Viet areas. Following this, Kelley-Hope et al. Nam and Indonesia, shrimp/fish ponds found (2005) suggested that different resistant along the coast or those irrigated by inland mechanisms may prevail in the two sibling sea water were reported as favourable species, with species A being more resistant breeding sites for this species (Collins et al., to organophosphates and species B to 1979). pyrethroids, with little evidence of Its populations were found both indoors introgression between the two sibling species. and outdoors in Malaysia (Reid, 1968), and Now that four sibling species are in Car Nicobar, Andaman and Nicobar identified, the distribution of the sibling Islands, India (Kumari and Sharma, 1994). In species should be mapped in all the areas Indonesia, in brackish water-breeding areas, wherever An. subpictus is prevalent and is this species is exophilic except in Kampund considered a vector, as in Indonesia and Sri Laut, Cilacap, where it is endophilic Lanka, and the biological characters of the (Kirnowardoyo and Gambiro, 1987). The sibling species should be studied. preference to feed on man varies in this species. In Vishakapatnam area, India, the anthropophilic index was found to be 5.6 per 3.13 The Sundaicus Complex cent (Rao, 1984). In Car Nicobar Island (Andaman and Nicobar islands), India, this Anopheles sundaicus belongs to the subgenus species was found to be predominantly Cellia and the Ludlowae Group in the zoophagic, with an anthropophilic index of Pyretophorus Series (Harbach, 2004). An. 2.5 per cent, but in human dwellings it was sundaicus is widely distributed in the Oriental 18 per cent (Kumari et al., 1993). However, region. The distribution extends from India, in Indonesia, it is primarily anthropophagic and eastward to China through Bangladesh, except in some places like Purworejo (Central Myanmar, Viet Nam, Cambodia, Thailand, Java) and Kulon Progo (Yogyakarta), where Malaysia, Singapore, Philippines and the human blood index was only 1.3 per cent Indonesia (Rao, 1984). In India it has now (Kirnowardoyo, 1988). Because of such disappeared from the mainland (Rao, 1984; distinct differences in the populations of An. Dash et al., 2000), except for a small focus in sundaicus, this species has long been the Kutch area of Gujarat state (Singh, Nagpal suspected to be a species complex (Kalra, and Sharma, 1985) and is now found 1978; Rao, 1984). abundantly and widely in the Andaman and Nicobar islands (Rao, 1984). This species is Evidence for recognition of sibling considered an important vector throughout species the areas of its distribution. Species A, B and C (Sukowati et al., 1999) An. sundaicus is generally a salt-water Sukowati and Baimai (1996), for the first time, breeder. The major breeding places are reported three cytological forms designated swamps and pits along bunds (embankments) as A, B and C differing in their polytene containing brackish water (Sundararaman, chromosome complement and with Soeroto and Siran, 1957). Rao (1984) reports heterochromatic variation in their Y- that the most suitable breeding places are chromosomes. The three cytological forms

76 Anopheline Species Complexes in South and South-East Asia were identified from the examination of variability in terms of mean heterozygosity, natural populations from different areas in which ranged between 0.101 and 0.179, and Thailand and in Java and Sumatra, Indonesia. polymorphism between 0.267 and 0.533, The differences observed between the three were compared with those observed for other forms are summarized in Table 11. A standard species complexes. No unique electromorphs photomap of the ovarian polytene were found fixed in any of the three chromosomes with a detailed description of cytological forms studied in these each chromosome is given by Sukowati and populations, but frequency differences and Baimai (1996). Wright’s F statistics for the Mpi enzyme distinguished the three cytological forms with In both Trat and Phangnga, two areas a high degree of probability. The data from surveyed in southern Thailand, only the A the Mpi locus strongly indicated a lack of form was found. In Asahan, in northern random mating. Therefore, the three Sumatra, all three forms were found while in cytological forms were considered as three south Tapanali, also in northern Sumatra, only isomorphic species and the Sundaicus as a the A and B forms were found in the two species complex. surveys conducted. In Lampung, situated in the most southern part of Sumatra, and in south Banten, Panganadaran and Cilacap in A new cytotype (Nanda et al., 2004) Java, only the A form was observed. In a single survey conducted in Purworejo in Java, Nanda et al. (2004) reported a new cytotype, both A and B were found with a D, from the Andaman and Nicobar Islands, predominance of the A form (90 per cent). India. In the polytene chromosomes complement, the X-chromosome resembled In spite of the fact that the cytological that of species A (Xa type), while the forms were found to be sympatric, the authors chromosome 2 had a prominent loosely could not give them the species status diffuse area in Zone 19 proximal to the because the forms were identified on the basis centromeric regions (2RB type) as in species of Y-chromosome variation, and differences C (Sukowati and Baimai, 1996). Thus, these in the polytene chromosomes were not as specimens did not completely resemble clear- cut as when there are inversions, for either species A or species C. In the mitotic which any heterozygotes are readily available. karyotype, both the X and Y chromosomes Sukowati et al. (1999) collected An. were telocentric and the Y-chromosome had sundaicus from bovine and human baits from two heterochromatic blocks as in species A six localities and the cytological identification and C (Table 12). The new cytotype could revealed A, B and C. Other Indonesian sites not be given a new species status, as it was with the forms found were as follows: not found sympatric with any of the other Purowerojo (forms A and B) and Cilacap (form forms. A) in Central Java; Panganduari (form A) in Evidence for another sibling species, An. west Java; Lampung (form A) in south Sumatra sundaicus s. s. (Dusfour et al., 2004) and Asahan (forms A, B and C), and south Tapanuli (form B) in north Sumatra. An. An. sundaicus specimens collected from two sundaicus breeds in fresh water in south sites, Lundu and Miri in Sarawak, Malaysia, Tapanuli while it breeds in brackish water in two in Thailand and two in southern Viet Nam the other five localities. F1 progeny were examined for differences, if any, in the cytologically identified for forms A, B and C sequences of partial regions of cytochrome b were analysed for 11 enzyme systems. Data and cytochrome oxidase I of the were analysed by BIOSYS-1 (release 1.7 mitochondrial DNA (Dusfour et al., 2004). University of Illinois programme). Genetic The specimens from Thailand and Vietnam

Anopheline Species Complexes in South and South-East Asia 77 were found distinctly different from the ones Techniques available for collected from Malaysia, and the genetic identification of sibling species divergence and cladistic analysis relationship established that they represent two sibling Mitotic karyotypes species: An. sundaicus s. s. from Malaysia and The three cytological forms have now been An. sundaicus species A from Viet Nam and given species status, and cytological Thailand. techniques can be used to identify these three species. Species B can be differentiated from Formal designation of An. sundaicus from species A and C by examining the Y- Malaysia and of species A chromosome in mitotic karyotypes. As Dusfour et al. (2004) designated specimens species A and C have similar Y-chromosomes, from the Sarawak region as An. sundaicus s. and if it is clearly known that only A and B or s., because Linton et al. (2001) reported An. only B and C exist in a given area, observation sundaicus from Lundu province as neotype of the Y-chromosome can give valid of this species (as no type specimens of this identifications. species exist). Linton et al. (2005) designated species A as An. epiroticus Linton & Harbach Polytene chromosomes based on ITS2, cytochrome b and All three species and new cytotype D can be cytochrome oxidase I sequences. Linton et differentiated by examining the X and al. (2005) also reported that no chromosome 2 in the polytene chromosome morphological characters were observed complements (Sukowati and Baimai, 1996). which could be used as diagnostic characters The differences observed in the Y-chromosome at adult, larval and pupal stages between An. in the mitotic karyotype and the X-chromosome sundaicus s. s. and An. epiroticus. and 2 (2R) are given in Table 11.

Table 11: Cytological descriptions of forms A, B, C and D in An. sundaicus Cytological forms/ Polytene chromosomes Mitotic Species X* 2R/2** Y-chromosome # # Species A Xa 2Ra Y1 (standard (standard Telocentric with 2 arrangement) arrangement) heterochromatin blocks # # Species B Xb 2Ra Y2 Telocentric but longer than

Y1 and has three heterochromatic blocks ? # # Species C Xb 2Rb Y1 # # Cytotype D Xa 2Rb 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 sundaicus was predominantly zoophagic Variations at the Mpi locus could be used to (Kumari et al., 1993). Cytotype D was found distinguish the three species (Sukowati et al., in indoor collections from human dwellings 1999) and cattle sheds and also in outdoor collections. It may be noted that An. Multiplex PCR assay sundaicus is the sole malaria vector in the Dusfour et al. (2004) developed a multiplex Andaman and Nicobar islands. Though only PCR assay consisting of four secies specific cytotype D was found in this study (Nanda et SCAR (sequence characterized amplified al., 2004), the authors did not rule out the region) primers derived from random possibility of another species existing in these amplified polymorphic region. This assay islands. Alam et al. (2005), however, found differentiated An. sundaicus s. s. from no differences in the D3 and ITS2 sequences Malaysia and species A from Thailand and of the specimens collected from four isolated Viet Nam. Species A specimens used in the islands of the Andaman and Nicobar group assay were neither cytogically nor of islands. Dusfour, Harbach and Manguin electrophoretically identified, but were from (2004) presented a consolidated account of the areas where, in earlier studies, only the bionomics of An. sundaicus s. l. and of species A was found. The assay needs to be the sibling species identified across the range validated with other sibling species in the of their distribution in the oriental region. An. complex. epiroticus was collected resting inside human habitations in Viet Nam (Linton et al., 2005). Distribution and biological characters This is an important species complex In southern Thailand only species A was responsible for the transmission of malaria in found, and in Sumatra, Indonesia, all three South-East Asian countries, hence, the species, A, B and C, were found in the bionomics of all the members of the complex northern part, but in the southern part there needs to be studied in detail to plan effective were only species A and B. Cytotype D was control strategies. Now that there are different found in laboratory colonies established from techniques, mitotic and polytene larvae collected from brackish and fresh chromosomal differences, electrophoretic water-breeding sites found in Andaman and variation, and PCR assay for the identification Nicobar Islands (Nanda et al., 2004). Salinity of sibling species, these techniques should be in the breeding sites in these islands was as validated for their specificity and sensitivity high as 14 gm/l (Das et al., 1998 and Sharma throughout the range of An. sundaicus et al., 1999). Recently, Alam et al. (2005), distribution. This is necessary to establish based on D3 and ITS2 sequnce analysis, also sibling species distribution and their host concluded that An. sundaicus breeding in feeding preferences, vectorial potential and fresh water and brackish water sites is responses to different control measures in identical. The three cytological forms reported different geographical regions. by Sukowati and Baimai (1996) were also found in both brackish and fresh water- breeding sites. Similarly, Dusfour et al. (2004) 3.14 The Anopheles found An. sundaicus s. s. in fresh water and saline water breeding sites in Malaysia. Thus, stephensi variants breeding in both fresh water and saline water Anopheles stephensi belongs to the subgenus does not appear to be a characterstic of any Cellia and the Neocellia Series (Harbach, one sibling species in the complex. In Car 2004). This species is found in all mainland Nicobar (Andaman and Nicobar Islands, zones of India but is rarely found at high India) where only cytotype D was found, An. altitudes in the Himalayas (Rao, 1984). The

Anopheline Species Complexes in South and South-East Asia 79 distribution of An. stephensi extends beyond variety mysorensis, on the basis of differences the west of India to Pakistan, Afghanistan, in the egg width and length and number of Iran, Iraq, Bahrain, Oman and Saudi Arabia, ridges on the egg float. and in the east to Bangladesh, south China Subbarao et al. (1987) studied several and Myanmar. It is not reported in the north laboratory colonies established from urban of India in Nepal nor in the south in Sri Lanka. and rural populations of An. stephensi for This species is considered an important variation in ridge numbers on egg float. In vector of malaria in Iran, Pakistan and India. addition to typical An. stephensi and var. Extensive studies have been carried out on mysorensis strains, some strains were found this species, but so far there is no evidence to have egg float ridge numbers falling which indicates that this is a species complex. between type form and var. mysorensis Sweet and Rao (1937) identified two values. The three categories of egg ridge populations, type form and variety mysorensis numbers were : 14 - 22, 12 - 17 and 9 -15. differing in egg morphometrics, which The mode number of ridges among the eggs inhabited urban and rural areas respectively. laid by individual females in these stocks were These populations were designated as races 16 - 19, 13 - 16 and 10 - 14 respectively. by Sweet, Rao and Subba Rao (1938), and as The categories with the highest ridge number subspecies by Puri (1949). Stone, Knight and corresponded with the type form, and the Starke (1959) accepted them as subspecies lowest with var. mysorensis of Rao, Sweet and and included them in the catalogue of Subba Rao (1938), and the new category with mosquitoes. Rutledge, Ward and Bickley intermediate numbers was designated as (1970) found the two forms sympatric and “intermediate”. The authors reported that considered them as variants and not areas where only mysorensis and subspecies. Subbarao et al. (1987) reported “intermediate” were found were the typical an additional type, intermediate, and rural villages and peri-urban localities. In the considered all three as “ecological variants”. heart of Delhi, in two rural areas surrounded In addition to egg morphometrics, studies on by high-income urban residential colonies, inversion polymorphism (Mahmood and all three forms were observed and the type Sakai, 1984; Subbarao, 1996) have also form was in the majority. The authors referred shown rural and urban populations to be to these areas as semi-urban localities. distinct.. Subbarao et al. (1987) referred to the three forms as ‘ecological variants’. Evidence for considering/not considering urban and rural Spiracular indices populations as distinct species Mosquitoes have two pairs of spiracles on the thorax and six pairs on the abdomen. The Egg morphometrics size of the spiracle is a key factor in the Sweet and Rao (1937) reported two races, adaptation of mosquitoes to different climatic An. stephensi type form and An. stephensi

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 copulatory barriers between the populations. that the anterior thoracic spiracular index can A detailed genetic analysis of the ‘ridge be used as a morphological indicator to number’ character was carried out following distinguish xerophilic, mesophilic and the likelihood method of Curtis, Curtis and hygrophilic mosquitoes. Nagpal et al. (2003) Barton (1985). The results indicated that the examined the spiracular index of type form variation in ridge numbers is controlled by and var. mysorensis collected from Jodhpur more than one genetic factor. The authors district, Rajasthan state, India. In type form, explained that intermediate-type strains the average spiracle length was 0.11 - 0.12 probably have an intermediate number of mm and the average spiracle index was 8.09 polygenes, and the strains maintain – 9.23, whereas in mysorensis, the intermediate-type ridge numbers in the corresponding figures were 0.09 - 0.1 mm laboratory strains because of the absence of and 6.82 - 7.60. These parameters showed any selection pressure. consistent differences between the populations of mosquitoes which emerged Polytene chromosomes and inversion during the monsoon and those in the summer polymorphism seasons. The spiracular indices correlated A photomap of polytene chromosomes of An. significantly with the number of ridges on the stephensi is presented by Mahmood and egg float. Thus, using this index the two Sakai (1985). In this species, six autosomal ecological races can be identified at the adult inversions were reported by Coluzzi, Di Deco stage. and Cancrini (1973a) and 16 polymorphic inversions (12 were new inversions) were Crossing experiments reported in Pakistan populations by Sweet, Rao and Subba Rao (1938) reported Mahmood and Sakai (1984). They observed failure of egg-laying by a majority of the 13 inversions in urban populations and, in females in reciprocal crosses between type contrast, only three in rural populations; the form and var. mysorensis, and of the eggs laid, three inversions observed in rural areas were a majority failed to hatch. not found in urban localities. Furthermore, the number of females observed with Rutledge, Ward and Bickley (1970) inversions was very low in rural areas (4/225 observed varied levels of fertility in reciprocal in Kasur district and 4/168 in Lahore district). crosses between three strains established from In Karachi city, 28 females out of 40 examined Iran, Iraq and India. It is not known whether were found with inversions. Similarly in India, these colonies were established from rural or 10 inversions were observed in urban urban populations. The characters exhibited populations of An. stephensi, while in rural by these colonies with reference to egg populations only the b inversion on morphology were of mixed type. They chromosome arm 2 in a low frequency and resembled mysorensis with respect to egg another inversion h1 were observed in the length but resembled the type form in egg heterozygous form in one specimen width and egg ridge number. (Subbarao, 1996). The h1 inversion was not Subbarao et al. (1987) made reciprocal observed in urban populations. Break-points crosses and back crosses between type form of inversions reported by Coluzzi, Di Deco and mysorensis colonies established, and Cancrini (1973a) and Mahmood and respectively, from urban and rural areas in Sakai (1984), and of those observed in Delhi, India. Only the ridge numbers on the egg India, are shown on photomaps of polytene float were studied in these crosses. The chromosomes by Subbarao (1996). crosses were fertile and the F hybrid progeny 1 Though rural and urban populations were also fully fertile, indicating no post- were found to be distinct with reference to

Anopheline Species Complexes in South and South-East Asia 81 inversion polymorphism, no pre-copulatory Y-chromosomes and the remainder were of barriers have been observed so far. One the metacentric type (Y1). However, in cannot, however, rule out the possibility of Pondicherry, Y1 was found in 31.9 per cent the existence of homosequential species and Y2 in 68.1 per cent of the An. stephensi within this taxon. male population.

Y-chromosome variation Distribution and biological characters Rishikesh (1959) and Aslamkhan (1973) In Pakistan, both the forms were reported, reported the mitotic karyotype of An. and in India, three forms were seen, including stephensi. The two autosomal pairs and the the new “intermediate” form (Subbarao et X-chromosome are metacentric, and the Y- al., 1987). Senior-White (1940) reported chromosome is submetacentric. Suguna both type form and mysorensis from Kolkata, (1992) reported two types of Y-chromosomes, India. Subbarao et al. (1987) did not find one submetacentric (as reported by earlier type form in rural localities but in semi-urban workers) and a new metacentric type from localities they found all three forms, with a two urban localities in Tamil Nadu, India. In majority of type form. These authors did not Cuddalore, 91.4 per cent of single female survey typical urban populations. cultures examined had submetacentric (Y2)

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 3 4 5

c a 1 f i1 p1

c p1

c g.d1 d a 1 1 i o q f1 c1 f1

1 1 a.b e .g d 1 r d e1.f1 n1

1 c c.c q a b q1 d1 m.n111.r o1

c.b1 dh1. 1 b q1 m1 b1 g1 h1 h1 b

g1 h1

82 Anopheline Species Complexes in South and South-East Asia Coluzzi, Di Deco and Cancrini (1973b) to be involved in the transmission of malaria reported that 2Rb inversion homozygotes had due to its predominantly animal feeding and “mysorensis type” of eggs. Suguna (1981) low parity during the transmission season. and Subbarao et al. (1987) observed no such However, in Iran, var. mysorensis is correlation in laboratory and field considered an important vector. Suguna populations. (1992) reports that in Cuddalore (Tamil Nadu, India) where the submetacentric Y- In rural areas where mysorensis is chromosome was found in about 91 per cent prevalent, a wide range of breeding sites, such of the male population, An. stephensi was as streams and channels, tanks and ponds, implicated in malaria transmission (Suguna, seepages, irrigation wells, etc., were found, 1981), but not in Pondicherry where the and in urban areas the breeding sites were submetacentric type was about 68 per cent. wells, overhead tanks, cisterns, fountains and In villages around Delhi, as well as in Delhi water collections at construction sites (Rao, city, An. stephensi was incriminated (Sharma 1984). et al., 1993). Recently, Rowland et al. (2002) Sweet and Rao (1937) and Rao, Sweet incriminated An. stephensi along with seven and Subba Rao (1938) reported that An. other anophelines in rural areas of eastern stephensi type form is an efficient vector, Afghanistan. Positive mosquitoes were found while var. mysorensis is a poor vector. with P. vivax (CSP210 and 247) and P. Recently, Nagpal et al. (2003) studied the falciparum CSP antigens, and the sporozoite bioecology of type form and var. mysorensis rate was 0.35%. Laboratory-feeding in an arid zone (Jodhpur, Rajasthan state) in experiments revealed that type form takes a India. The study was carried out during the shorter period for the development of pre-monsoon, monsoon and post-monsoon sporozoites than var. mysorensis (Vasanthi, seasons. The type form was found breeding 1996). indoors in domestic and peri-domestic There are 16 microsatellite markers containers and it was found inside, especially developed for this species (Verardi et al., on hanging objects, throughout the year. Var. 2002). Now, the taxonomic status of the two mysorensis was found breeding in the same ecological races in this species could again sites and resting indoors during the summer, be addressed using these markers. but it moved outside with the onset of rains. The authors do not consider var. mysorensis

Anopheline Species Complexes in South and South-East Asia 83 4. Prospects for the future

The importance of recognizing species The Culicifacies and Fluviatilis in India complexes and identifying their members and Dirus and Maculatus Complexes in became obvious as early as the 1930s with Thailand are the most thoroughly investigated the discovery of An. maculipennis as a species ones in this region. The data generated so complex in Europe. During the Second World far have clearly established the importance War, in Italy, G. Davidson (personal of identifying sibling species in malaria control communication from C. F. Curtis) had the job programmes. However, the information of locating the dangerous anthropophagic generated on these complexes so far is species An. labranchiae and the less insufficient for planning and implementing dangerous and generally zoophagic An. effective control strategies suitable for maculipennis s.s. so as to site military camps different situations. Since each species has a in safe locations. But soon after the war, different gene pool and hence may have malaria was eradicated from Europe. Malaria different behaviour and genetic potential, continues to be endemic in more than 100 data on the following aspects of all members countries and is a serious health problem in of each complex would be of interest: all the South and South- East Asian countries. (i) Breeding habitats With the escalating prices of insecticides and drugs, and beset with problems of resistance (ii) Resting behaviour (indoors/outdoors) of vectors to insecticides and of parasites to (iii) Feeding preference with reference to site drugs, almost all countries in this region are and host (human/animal; indoors/ experiencing challenges to contain and outdoors) control malaria. (iv) Biting rhythms (peak biting time) It is now increasingly being accepted by many control programmes that a single (v) Susceptibility to plasmodial infections in strategy for an entire country, and even for a the laboratory to establish whether the single province/district, is not applicable. low vectorial capacity is due to genetic/ Situation-specific, and at times even species- physiological factors or because of poor specific, strategies need to be employed. longevity, preference to feed on hosts Now that almost all important malaria vectors other than humans, etc. in South and South-East Asia have been (vi) Response to commonly-used insecticides identified as species complexes, it is important which could indicate the difference in to map the distribution of the sibling species the rate of development of resistance of the complexes and to establish the role of each sibling species in malaria transmission (vii) Molecular mechanisms responsible for and monitor their response to control resistance (if possible). measures. This requires the study of the In addition to these, studies should be biological characters of each sibling species. encouraged on more subtle, but equally

84 Anopheline Species Complexes in South and South-East Asia relevant and not so easily observed, techniques which are simple, accurate and differences such as (a) density regulation of affordable need to be developed. sympatric species in breeding places; (b) By definition, sibling species are factors responsible for distinct distribution morphologically very similar, but members of pattern; (c) physical and chemical properties a complex should nevertheless be examined of water in different breeding places to for any morphological variation. Members determine the factors influencing species of the Subpictus Complex can easily be preferences; (d) population structure, smallest distinguished by morphological characters, breeding unit, population bottlenecks, gene while for those of the Maculatus Complex, flow over the entire range of each species more skill and effort are needed. There are distribution; etc. other complexes, for which there are no Serious consideration is now being given reports of morphological variations. to genetic strategies as alternative vector Examination of polytene chromosomes for control tools. Efforts are under way to species-specific diagnostic inversion develop genetically manipulated/engineered genotypes is the cheapest technique now mosquitoes for use in vector control. Many available, but it requires specialized skill and research groups are working on different aptitude for microscopic examination. In spite aspects to contribute to the development of of certain limitations, this technique was anopheline strain(s) which will not be efficiently used for studying the Culicifacies susceptible to parasite development, and and Fluviatilis Complexes in India and consequently if these could be made to Maculatus in Thailand. In the 1980s, there replace the wild vector population to was more interest in the development of interrupt malaria transmission. For this final allozymes as diagnostic tools than there is achievement, other necessary aspects now. Advancements and advantages of DNA- essential for the release of such a desirable based techniques have shifted the interest of strain and spread it in nature are being researchers towards molecular methods. addressed with great vigour and care. With Initial establishment costs are high and also the completion of the sequencing of An. expertise is required to develop DNA-based gambiae genome (Holt et al., 2002), it is methods. However, once these techniques expected that more novel targets will be are standardized, the procedures are simple identified for anopheline vector control. and can routinely be used to identify the Volume 219 of Science (2002), in which this species and a large number of samples can sequencing data was published, also be screened on a single day. contained viewpoints of several distinguished An informal consultancy meeting on scientists on the implications of genome Malaria vector species complexes and intra- sequencing in developing vector control specific variations: Relevance for malaria strategies and for better understanding of the control and orientation for further research biology of vectors. For these and for other was held in Bangkok, Thailand, from 29 already existing vector control strategies, it is October to 3 November 1984. During this important to recognize all the species present meeting several recommendations were (whether they are morphologically made. More than two decades have since distinguishable or indistinguishable) and the passed. The author considers the re- biological features of each species (as commendations of the meeting are still valid mentioned above). and require attention. Therefore, the If identification of sibling species is to be recommendations are reproduced to done routinely in malaria control activities, 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 organized both these workshops. In the mapping and stratification at the country intercountry workshop, experts from level, as part of the programme planning Afghanistan, Bangladesh, India, Indonesia, process, attempts should be made to link Myanmar, Sri Lanka and Thailand genetically-determined sibling species and participated. Both these workshops provided associated variants with topographical and hands-on-experience to the participants on vegetational indicators.” the techniques used in the identification of sibling species. This is an indication of the “Species distribution does not recognize commitment of the organizing agencies and political boundaries, and it is strongly participating countries to study species recommended that collaboration between complexes. There is need for organizing more countries and regions be encouraged in such workshops, especially in view of the investigations involving species complexes of techniques (molecular methods) that have malaria vectors.” been developed recently. Thus, it becomes imperative that Another important requirement for any research findings on species complexes control programme, especially for genetic should find a place in the planning of national control strategies, is information on population vector control strategies and should not be structure, gene flow and geographical barriers, considered merely as academic achievements if any. Workshops covering these aspects and or pursuits. In order for this to be practical training on the tools that can be used accomplished, both researchers and for these studies are urgently required in this programme planners/managers have to make region. Training on genotyping of populations an effort to: using microsatellite markers should be (i) Establish close linkages between organized as markers are available for four organizations/universities where research important vectors, An. culicifacies, An. dirus, on species complexes is carried out and An. maculatus, and An. stephensi. Students authorities/directorates who plan control from developing countries will greatly benefit strategies; studying for their masters and doctoral degrees at universities in developed countries in the (ii) Create a database on species complexes specialized aspects of field biology related to to which national control programme can malaria vectors. have an access to (with the advancement in communication techniques, it would Lastly, it should be noted that sibling not be difficult to create such linkages); species are not a special kind of species and ought to be treated as any other species. This (iii) Organize intra-country courses to impart is an appeal to researchers that they should training to personnel in the national formally designate sibling species with formal programmes to learn methods to identify binomial nomenclature at the earliest sibling species and to utilize knowledge on opportunity and drop the provisional species complexes in malaria entomology. designations given to them. It can be assumed As a follow-up to this meeting, two that entomologists and programme planners/ workshops, one for Indian participants managers will not hesitate to accept sibling sponsored by the Indian Council of Medical species with formal designations. For many Research in 1994 and the other an inter- the concept of biological species/sibling country workshop sponsored by the WHO species is still alien and they continue to Regional Office for South-East Asia in 1997 consider them as races or variant taxa, which were held in New Delhi, India. The Malaria have been conclusively proved to be separate Research Centre (now renamed as National biological species that do not generally Institute of Malaria Research), Delhi exchange genes by mating in the field.

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102 Anopheline Species Complexes in South and South-East Asia SEARO Technical Publication No. 57

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