Internal taxonomy of the Byrcanus Group of Anopheles (Diptera: Culicidae) and its bearing on theincrimination of vectors of malaria in the west of the Palaearctic Region

Clement D. RamsdaleVamdean Lodge, London Road, Brighton BNI 6YA, United Kingdom. Email:

clem.ramsdale@claranet.co.uk

Abstract

Knowledge of the internal taxonomy and actual or potential importance as vectors of malaria of theAnopheles hyrcanus Group in the western Palaearctic is reviewed. Means of studying these neglecteddisease vectors in this area are discussed.

Introduction

The Hyrcanus Group (Genus Anopheles, Subgenus Anopheles) comprises an unknown number of species,some of which are vectors of malaria and other mosquito-borne diseases. The vectorial importance of someof these species in the Oriental Fmmal Region and contiguous parts of the fur eastern Palaearctic Regionbecame evident early in the last century, since when the group has been the subject of long series of studiesin this large area. In contrast, very little is known about the western Palaearctic taxa, where these more orless exophilic and exophagic mosquitoes were not generally considered to be important vectors. The adventofDDT during the 1940s, and of Malaria Eradication Projects relying heavily on house spraying, focusedattention on the vector species of the endophilic Maculipennis Complex with little or no interest in the moreexophilic Hyrcanus Group. Briefly re-awakened interest came in persistent or resurgent foci of malaria inthe later stages of the western Palaearctic Malaria Eradication Programmes, but only until abandonment ofthese programmes shortly afterwards. Despite evidence to the contrary, apart from a taxon described from aunique specimen collected near Tashkent, Uzbekistan, only a single polymorphic species was recognised inthe western Palaearctic until recently, when Anopheles pseudopictus was elevated to species status.

Mosquitoes of the Hyrcanus Group favour larval sites in marshes and similar situations, and proliferatewhere crops are grown under flood irrigation. Workers tending and harvesting crops grown under irrigationtend to sleep outside during the hot malaria season, making them wlnerable to attack by exophagicmosquitoes. Agricultural projects making use of impounded water for irrigation constitute areas ofcontinuing malaria transmission and the literature contains many references to An. hyrcanus s.L acting asvectors in rice and cotton growing areas. Impoundment of water for irrigation on a scale unprecedented inwestern Asia has begun. Importance of these mosquitoes as actual or potential malaria vectors must,therefore, be expected to increase.

It would seem prudent to study the systematics of the Hyrcanus Group in the western Palaearctic in order toestablish the vectorial importance of its constituent members before these planned, large irrigation projectscome on stream, allowing malaria control and/or prevention to be tackled in an informed manner.

Present state of knowledge of the Byrcanus Group of species

Anopheles hyrcanus (pallas), first recorded from the southern shores of the Caspian Sea, was shown tohave a wide distribution extending from Iberia in Europe to east and south-eastern Asia including some ofthe off-lying islands of the Indian and Pacific Oceans. It also became apparent that the taxon included anumber of morphologically separable forms, and that some were involved in the transmission of malariaand filariasis, particularly in the Oriental Region and contiguous parts of the eastern Palaearctic Region. Todate, the Hyrcanus Group has been shown to encompass a group of 29 currently recognised species (TableI). Of these, 26 have an Oriental or eastern Palaearctic distribution, with 4 being placed in the LesteriSubgroup and 4 in the Nigerrimus Subgroup, whilst insufficient data have yet been collected to enable theothers to be assigned to subgroups (Harbach, 1994; Nguyen et al., 2000). Only 3 currently recognisedspecies have a western Palaearctic distribution, here defined as occurring in the part of the PalaearcticRegion west of China and south of SOON.

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European Mosquito Bulletin, 10 (2001), 1-8. Journal of the European Mosquito Control Association ISSN1460-6127

TABLE 1. SPECIES, WITH SYNONYMs, CURRENTLY INCLUDED IN THE HYRCANUS GROUP OFANOPHELES.

species synonym

Oriental Region and Eastern Palaearetie Region

sinensis Wiedemann, 1828plumiger OOnitz, 1901jesoensis Tsuzuki, 1902

alhotoeniallls (Theobald, 1903)argyropus (SweUengrebel, 1914)sineroides Yamada, 1924pul/us Yamada, 1937kweiyangensis Yao & Wo, 1944yatsushiroensis Miyazaki, 1951anthropophagus Xn & Feng, 1975kiangsuensis Xu & Feng, 1975engarensis Kanda & Oguma, 1978changfus Ma, 1981dazhaius Ma, 1981heiheensis Ma, 1981xiaokuanus Ma, 1981liangshanensis Kang et aL, 1984kunmingensis Dong &Wang, 1985nimpe Nguyen et al., 2000vieJlUIII1ensisNguyen et al., 1993

Lesteri subgrouppeditaeniatus (Leicester, 19(8)lesteri Baisas & Ho, 1936crawfortli Reid, 1953paraliae Sandosham, 1959

Nigerrimus subgroupnigerrinuls GiIes, 1900

indiensis Theobald, 1901bentleyi Bentley, 1902minutus Theobald, 1903williamsoni Baisas & Hu, 1936venhuisi Bonne- Webster, 1951

pursati Laveran, 1902pseudosinensis Baisas, 1935nitidus Harrison et al., 1973

Western Palaearetie Region

type locality

Canton, ChinaHong KongHokkaido, JapanPerak, MalayaDeli, East SumatraBibai, Hokkaido, JapanKeiki-do and South Tyusei-do, KoreaKweiyang, Kweichow, ChinaYatsushiro City, Kyushu, JapanWukiang, Kiangsu ChinaWukiang, Kiangsu ChinaEngaru, Hokkaido, JapanEmai, Sichuan, ChinaSichuan, ChinaAihui, Heilungjiang, ChinaMudangjiang, Heihmgjiang, ChinaZhaojaio, Sichuan, ChinaKunming, Yunnan, ChinaCa Mau Province, VietnamSon La Province, Vietnam

MalayaRizal, Luzon, PhilippinesKoala Lumpur and Selangor, MalayaMalaya and Borneo

Calcutta, IndiaMadras, IndiaTezpur, Assam, IndiaLahore, PakistanPenang, MalayaJavaPursat, CambodiaCalauan, Luzon, PhilippinesSelangor, Malaya

hyrcanus (paUas, 1771) Caspian Seapictus Loew, 1845 Rhodes, Greecej1erowi Portschinsky, 1910 River Amu-Darya, Uzbekistanmesopotamiae Christophers & Chand, 1915 Lower Iraqmarzinovski Shingarev, 1926 Karayazi Steppe, South Caucasuspopovi Schingarev, 1928 Turkestanmahmuti Martini, 1930 Asia Minor

pseudopictus Grassi, 1899 Italychodukini Martini, 1929 Tashkent, Uzbekistan

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Studies in the Oriental Region and contiguous areas of the eastern Palaearctic Region

It was recognised at an early date that many forms of An. hyrcanus s.l. co-existed in east and south-eastAsia, which allowed malariologists a certain degree of confidence that they were working with particularspecies. However, results of vector studies with these sibling species still produced anomalous results.

Precipitin tests of bloodmeals of females of the taxa now known as An. nige"imus and An. sinensis,demonstrated that a variable, sometimes high, proportion of meals of both species may be of hlDllan blood(Boyd, 1949). Anopheles nigerrimus naturally infected with malaria occurred in Indochina, Malaya (partof present day Malaysia) and Indonesia though infected specimens were not found in Assam and thePhilippines (Boyd, 1949). Similarly, An. sinensis was a confirmed vector in Java, Sumatra, Celebes,Malaya, north Vietnam, north, central and south China, Taiwan, Korea, and central Japan (Boyd, 1949;Covell, 1949; Puri, 1949; Ho, 1965; Ma, 1981; Beales, 1984; Chow, 1991) but was of little importance insouthern Indo-China (Boyd, 1949). Anopheles hyrcanus s.l. was reported to be exophilic and eitherendophagic or exophagic in Myanmar (Burma) (Boyd, 1949), where it was an effective malaria vector inthe Irrawaddy Delta (Covell, 1949). It was also considered to be a vector in eastern SlDllatra, but not to beinvolved in malaria transmission in Java, Borneo, Macassar and Celebes (Boyd, 1949).

Further studies of these species in the Oriental and eastern Palaearctic Regions during the second half of thelast century (e.g. Reid, 1953, 1968; Harrison, 1972, 1973; Harrison et al., 1973, 1991; Harrison & Scanlon(1975); Takai & Kanda, 1986; Baimai et al., 1993; and others) did much to resolve these puzzling findings.Many named forms were found to refer to the same species, but at the same time other good species weredescribed in south eastern and eastern Asia.

This work enabled identification of further vectors within the Hyrcanus Group. In addition to An. sinensisand An. nige"imus, An. lesteri is considered a good vector of malaria in southern China (Ho, 1965; Ma,1981; Beales, 1984; Chow, 1991) and was suspected of being the principal vector in Japan (Tanaka et al.,1979). Other species, such as An. anthropophagus in southern China, An. dazhaius in Central China, An.xiao/cuanus in north China and An. nimpe in southern Vietnam are known to bite man and to be importantor probable malaria vectors (Ma, 1981; Chow, 1991; Nguyen et al., 2000).

Studies in the Palaearetie Region west of China

In his review of An. hyrcanus and related taxa in the Palaearctic Region, Martini (1929-31) regarded thedescribed forms, pictus, pseudopictus and sinensis as synonyms of hyrcanus. Anopheles chodukini wasdescribed from a unique female specimen sent to him from Taskent and, though he considered thepossibility that it may represent an extremely pale, desertic form of hyrcanus, he decided it more probablybelonged to a separate species. He regarded the other taxa described from west of China as geographicalvarieties of hyrcanus thus: hyrcanus type form (distribution: Spain to Far East), var. mesopotamiae (LowerIraq), var. marmuti (Asia Minor), var. marzinovski (Caucasus), and var. popovi (Turkestan). He provided akey (based on hind tarsal and wing scaling patterns) for differentiation between these forms but admittedthat differentiation could be difficult. He also provided a description of the adult morphology, distribution,and the involvement in malaria transmission of sinensis (which he regarded as a synonym of hyrcanus).

Because individual morphological variation within each taxon proved to preclude reliable separation bymeans available at that time, all came to be treated as populations of one species, An. hyrcanus, and otherindividual names were consigned to synonymy (Bates et al., 1949). Thus, An. hyrcanus was considered tobe a widely distributed polymorphic species with a range extending from the Iberian Peninsula, throughEurope south of the Alps and Asia south of about 50oN, to the Pacific. Gutsevich et al. (1971) andGutsevich (1976) regarded all Palaearctic forms, including An. chodukini, as varieties (aberrations) ofhyrcanus. Apart from a few more recent, fragmented, observations, this view has persisted, despite resultsof the work in the Oriental Region and the east ofthe Palaearctic Region.

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Reid (personal commooication, 1968) was of the opinion that ootil western Palaearctic forms of An.hyrcanus were shown to be conspecific with Oriental forms, they should be regarded as separate entitiesand suggested that it might better to label western Palaearctic forms 'hyrcanus' ootil the taxon had beenadequately studied.

Evidenee of speeiation within the Byreanus Group in the western Palaearetie Region

A limited number of crossing experiments (Ross Institute, 1976, 1977) between samples drawn frompopulatioos of An. hyrcanus s.1. from Camargue (France), <;OkOrova(Turkey), Osmancik (Turkey) andelsewhere, indicated that the Camargue and Osmancik populatioos were conspecific, but that the <;ukfirovapopulatioo belonged to a different species. Preliminary morphological studies during the same investigationindicated that a population from Jalalabad (Afghanistan) is probably conspecific with theCamarguelOsmancik form. in which hindtarsomere 4 (TIll-4) carries pale basal and apical white bandsseparated by a broad black median area. This segment is all white in the forms found at Kunduz.Afghanistan (Ward, 1972) and <;ilkiirova,Turkey (postiglione et al., 1973).

This work was discontinued after 1977, but more recently the taxon pseudopictus, with hindtarsomere 4(TllI-4) all white, was reinstated as a species following comparisoo with the type form of hyrcanus s.1. inwhich hindtarsomere 4 (mI-4) is dark except for a white apical band. In addition to these morphologicaldifferences, no evidence of natural hybridisation was found in south-west Asia where these forms aresympatric (Glick, 1992).

Byreanus Group as veetors of malaria in the western Palaearetie Region

Two hoodred and thirty years after it was first recorded at the southern end of the Caspian Sea (pallas,1771), ooderstanding of the status ofAn. hyrcanus s.1.in the western Palaearctic Region (west of China) isstill in an embryonic stage. This situatioo stems from an assumption that the taxoo was, at most, of onlyminor importance as a malaria vector.

Bates et al. (1949) did not regard An. hyrcanus s.1. as a primary vector, but thought it played a part intransmission in the presence of good vectors, and gave southern Ukraine and middle Asia as examples.Gutsevich et al. (1971) considered that its vectorial role varied between places, depending on localconditioos.

Precipitin tests of bloodmeals have given variable results, from complete zoophily in Romania (Bruce-Chwatt et al., 1966) to 25% positive for human blood in Greece (Barber & Rice 1935). However, there isample evidence from Greece (Waterstoo, 1918), Bulgaria and the Republic of Macedonia (Weyer, 1942),Turkey (postiglione et al., 1973; Ramsdale & Haas, 1978), Afghanistan (Zahar, 1974; Anofrieva et al.,1977) and the neighbouring coootries of Turkmenistan, Uzbekistan, Tadzhikistan, Kazakhstan and Russia(Sergiev et al., 1993) showing that this taxon may exhibit quite high degrees of anthropophily. It is notknown how fur it was involved in the devastating outbreaks of malaria affecting French and British forcesin Greek Macedonia during the 1914-1918World War. Nevertheless, Weyer (1942), working in the samearea during the 1939-1945 War, aptly observed that "An hyrcanus has high potential as a malaria vectorwhere it is abundant and OOOl>lesleep in the ooen". Quite recent observations in the rice growing area of thealluvial plain through which the River Axios flows found that An. hyrcanus s.1. is still one of the principalpest mosquitoes of northern Greece (Kaiser et al., 2(01).

Biting and baited net trap collections in Turkey (postiglione et al., 1973; personal observations) confirmedWeyer's observation. In human and animal baited net trap collections in a village of south-west Turkeywhere cattle were stalled amongst the houses at night, 980/0of 238 An hyrcanus s.1. caught were in theanimal baited net trap. Night biting collections 00 human bait in the same village failed to detect a singlespecimen attempting to bite, though many fed on the animals. However, when the same human bait were

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moved into open country away from this village, so losing the protection provided by cattle, they wereimmediately subject to mass attack by blood-seeking females. Further night biting collections in thec;iikiirova area of Turkey confirmed that large numbers viciously attack humans in the absence of herds oflarge domestic animals.

Rice fields, with their associated drainage systems, constitute prolific larval sites for this mosquito, andenormous populations of An. hyrcanus s.l. occur in some irrigated cotton and rice growing areas in Turkeyand elsewhere (postiglione et al., 1973; Cristescu et al., 1975; Onori et al., 1975; Cousserans et aI., 1976;Anofrieva et al., 1977; Ramsdale & Haas, 1978;Kaiser et al., 2(01).

Some of the most intractable problems of malaria transmission occur in small or large areas wheremonoculture crops, usually of rice and/or cotton, are produced under irrigation. Domestic animals areabsent or rare in these situations, where agricultural workers sleep out of doors during the hot summers,often around the edges of irrigated fields, particularly during the harvest (postiglione et al., 1973; Ramsdale& Haas, 1978).

After many years of developing in larval sites contaminated by crop spraying, both An. hyrcanus s.l. andAn. sacharovi had become highly resistant to insecticides (Ramsdale, 1975; Ramsdale et al., 1980)when aserious resurgence of malaria in the c;ukiirovaoccurred in the mid- to late-1970s. Hundreds of thousands ofpeople slept out of doors during the summer at this period, making it impossible to say with any degree ofcertainty which of these taxa played the dominant vectorial role. Although An. sacharovi, which readilyfeeds inside or outside buildings, was undoubtedly a good vector in these circumstances, the moreexophagic An. hyrcanus s.l. may have made a substantial contribution to malaria transmission.

In most irrigation schemes within the south-west Asian distribution of An. hyrcanus s.l. large numbers ofpeople sleep out of doors in situations where domestic stock are more or less absent. In these situationssome taxa within the Hyrcanus Group certainly transmit malaria and may well be primary vectors. Newareas are constantly being opened up to agriculture by the installation of irrigation systems. One of these,in the south-east Anatolian region of Turkey, will permit the irrigation of an additional 1.8million hectaresof agricultural land (Alten et al., 2000). Malaria transmitted by Hyrcanus Group mosquitoes in the westernPalaearctic is not then an unimportant historical curiosity. On the contrary, it is a growing and potentiallyserious problem in areas of the western Palaearctic Region where vivaxmalaria persists.

Malaria caused by Plasmodium vivax occurs in parts of Afghanistan, Kazakhstan, Kyrgyzstan, Russia,Tajikistan, Turkmenistan and Turkey, and P. falciparum is reported to have re-appeared in Tajikistan(Majori et al., 1999). Although mosquitoes of the Maculipennis Complex readily transmit Plasmodiumvivax, many studies have demonstrated refractoriness to infection with extant strains of the morepathogenic P. falciparum (James et al., 1932; Shute, 1940; Ramsdale & Coluzzi, 1975; Dashkova &Rosnicyn, 1982; Ribeiro et al., 1989), thereby reducing receptivity to malaria in Europe (Zulueta et al.,1975). There are no data on the susceptibility of the western Hyrcanus Group to infection with P.falciparum and receptivity of the southern part of the western Palaearctic may be higher than has beenassumed, especially in areas of rice and cotton production.

Resolution of the taxonomic and vector problems posed by the Byreanus Group in the westernPalaearctic

Species identification is clearly critical to any vector control programme that seeks to be efficient as well aseffective (COlliBSet al., 2(00).

Estimation of the possible role in malaria transmission of An. hyrcanus s.l. in the western part of thePalaearctic is complicated by the mct that existing data refer to an unknown Bumber of species, each ofwhich may have a different vector potential. It is known that behavioural differences between populationsexist, but the accumulated information on distribution, behaviour and other aspects pertinent to malariatransmission refers to an aggregate of species (Ramsdale& Snow, 2000).

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There is clearly a need for a thorough investigation of the Hyrcanus Group in the western Palaearctic.Persisting transmission of P. vivax in rice and cotton growing areas scattered through south central andsouth west Asia, and particularly the reported renewal of P. fa/ciparum transmission in Tajikistan, lendurgency to this investigation. Moreover, this problem is being compounded, especially in Turkey, by theplanning and construction of irrigation projects dwarfing the size of those already in existence.

Because of its wide distribution and possible inclusion of a large number of species, isolated localinvestigations of the Hyrcanus Group are of limited value. On the other hand, integrated taxonomic andbiological studies of Hyrcanus Group populations from different countries and habitats could unravel thislongstanding problem. This must aim to provide information on the relative vector importance of thecomponent taxa, both with regard to transmission of P. vivax and of P. fa/ciparum.

Mosquito systematics, in addition to comparative taxonomy, can call on many sophisticated tools forinvestigation of cryptic species, best suited to specialist laboratories. Clearly, institutions responsible fortaxonomic studies should have access to type specimens of the described taxa currently grouped under thename of Anopheles hyrcanus. DNA assays offer several advantages, as was demonstrated in the recentidentification of the newly recognised malaria vector, An nimpe (Nguyen et al., 2000). Not least of these isthe ability conferred by polymerase chain reaction (PCR) amplification and DNA profiling to identifypreserved material additionally used for other purposes, including the detection of malaria infections.Techniques for detection of malaria inrections in mosquitoes by immunoradiometric or enzyme-linkedimmunosorbent assays (IRMA or ELISA) are suitable for use in field studies. Electronic communicationfacilitates contact between workers in different disciplines, situations and countries, and is conducive tomultidisciplinary approaches to problems previously tackled piecemeal.

The integrated approach to resolution of taxonomic uncertainties in species groups containing malariavectors was successfully pioneered in the analysis the Anopheles quadrimaculatus complex in NorthAmerica (Reinert et al., 1997; Carlson et al., 1997). This approach provides the most effective means oftackling the problems outlined here and offers the best opportunity of resolving the inaeasingly urgentissues of vector inaimination. Such a project in the western Palaearctic would require close co-operationbetween laboratory and field workers in many countries.

The size of the parasite reservoir in human populations has an obvious bearing on malaria transmission.However, field studies should include assessment of the effect of human ecology on vector behaviour. Inthe case of the Hyrcanus Group, this aspect may be of major importance.

Aeknowlwdgements

I am grateful to Ralph Harbacl1, John Reinert and Bruce Harrison for their helpful advice and comments.

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