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

Clement D. Ramsdale

Varndean Lodge, London Road, Brighton BN1 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 the Anopheles hyrcanus Group in the western Palaearctic is reviewed. Means of studying these neglected disease 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 some of these species in the Oriental Faunal Region and contiguous parts of the far eastern Palaearctic Region became evident early in the last century, since when the group has been the subject of long series of studies in this large area. In contrast, very little is known about the western Palaearctic taxa, where these more or less exophilic and exophagic mosquitoes were not generally considered to be important vectors. The advent of DDT during the 1940s, and of Malaria Eradication Projects relying heavily on house spraying, focused attention on the vector species of the endophilic Maculipennis Complex with little or no interest in the more exophilic Hyrcanus Group. Briefly re-awakened interest came in persistent or resurgent foci of malaria in the later stages of the western Palaearctic Malaria Eradication Programmes, but only until abandonment of these programmes shortly afterwards. Despite evidence to the contrary, apart from a taxon described from a unique specimen collected near Tashkent, Uzbekistan, only a single polymorphic species was recognised in the 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 proliferate where crops are grown under flood irrigation. Workers tending and harvesting crops grown under irrigation tend to sleep outside during the hot malaria season, making them vulnerable to attack by exophagic mosquitoes. Agricultural projects making use of impounded water for irrigation constitute areas of continuing malaria transmission and the literature contains many references to An. hyrcanus s.l. acting as vectors in rice and cotton growing areas. Impoundment of water for irrigation on a scale unprecedented in western 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 to establish the vectorial importance of its constituent members before these planned, large irrigation projects come on stream, allowing malaria control and/or prevention to be tackled in an informed manner.

Present state of knowledge of the Hyrcanus Group of species

Anopheles hyrcanus (Pallas), first recorded from the southern shores of the Caspian Sea, was shown to have a wide distribution extending from Iberia in Europe to east and south-eastern Asia including some of the off-lying islands of the Indian and Pacific Oceans. It also became apparent that the taxon included a number of morphologically separable forms, and that some were involved in the transmission of malaria and filariasis, particularly in the Oriental Region and contiguous parts of the eastern Palaearctic Region. To date, the Hyrcanus Group has been shown to encompass a group of 29 currently recognised species (Table 1). Of these, 26 have an Oriental or eastern Palaearctic distribution, with 4 being placed in the Lesteri Subgroup and 4 in the Nigerrimus Subgroup, whilst insufficient data have yet been collected to enable the others to be assigned to subgroups (Harbach, 1994; Nguyen et al., 2000). Only 3 currently recognised species have a western Palaearctic distribution, here defined as occurring in the part of the Palaearctic Region west of China and south of 50°N.

TABLE 1. Species, with synonyms, currently included in the Hyrcanus Group of Anopheles.

species synonym type locality

Oriental Region and Eastern Palaearctic Region

sinensis Wiedemann, 1828 Canton, China

plumiger Dönitz, 1901 Hong Kong

jesoensis Tsuzuki, 1902 Hokkaido, Japan

albotaeniatus (Theobald, 1903) Perak, Malaya

argyropus (Swellengrebel, 1914) Deli, East Sumatra

sineroides Yamada, 1924 Bibai, Hokkaido, Japan

pullus Yamada, 1937 Keiki-do and South Tyusei-do, Korea

kweiyangensis Yao & Wu, 1944 Kweiyang, Kweichow, China

yatsushiroensis Miyazaki, 1951 Yatsushiro City, Kyushu, Japan

anthropophagus Xu & Feng, 1975 Wukiang, Kiangsu China

kiangsuensis Xu & Feng, 1975 Wukiang, Kiangsu China

engarensis Kanda & Oguma, 1978 Engaru, Hokkaido, Japan

changfus Ma, 1981 Emai, Sichuan, China

dazhaius Ma, 1981 Sichuan, China

heiheensis Ma, 1981 Aihui, Heilungjiang, China

xiaokuanus Ma, 1981 Mudangjiang, Heilungjiang, China

liangshanensis Kang et al., 1984 Zhaojaio, Sichuan, China

kunmingensis Dong & Wang, 1985 Kunming, Yunnan, China

nimpe Nguyen et al., 2000 Cà Mau Province, Vietnam

vietnamensis Nguyen et al., 1993 Son La Province, Vietnam

Lesteri subgroup

peditaeniatus (Leicester, 1908) Malaya

lesteri Baisas & Hu, 1936 Rizal, Luzon, Philippines

crawfordi Reid, 1953 Kuala Lumpur and Selangor, Malaya

paraliae Sandosham, 1959 Malaya and Borneo

Nigerrimus subgroup

nigerrimus Giles, 1900 Calcutta, India

indiensis Theobald, 1901 Madras, India

bentleyi Bentley, 1902 Tezpur, Assam, India

minutus Theobald, 1903 Lahore, Pakistan

williamsoni Baisas & Hu, 1936 Penang, Malaya

venhuisi Bonne-Webster, 1951 Java

pursati Laveran, 1902 Pursat, Cambodia

pseudosinensis Baisas, 1935 Calauan, Luzon, Philippines

nitidus Harrison et al., 1973 Selangor, Malaya

Western Palaearctic Region

hyrcanus (Pallas, 1771) Caspian Sea

pictus Loew, 1845 Rhodes, Greece

flerowi Portschinsky, 1910 River Amu-Darya, Uzbekistan

mesopotamiae Christophers & Chand, 1915 Lower Iraq

marzinovski Shingarev, 1926 Karayazi Steppe, South Caucasus

popovi Schingarev, 1928 Turkestan

mahmuti Martini, 1930 Asia Minor

pseudopictus Grassi, 1899 Italy

chodukini Martini, 1929 Tashkent, Uzbekistan

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-east Asia, which allowed malariologists a certain degree of confidence that they were working with particular species. 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. nigerrimus and An. sinensis, demonstrated that a variable, sometimes high, proportion of meals of both species may be of human blood (Boyd, 1949). Anopheles nigerrimus naturally infected with malaria occurred in Indochina, Malaya (part of present day Malaysia) and Indonesia though infected specimens were not found in Assam and the Philippines (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 in southern Indo-China (Boyd, 1949). Anopheles hyrcanus s.l. was reported to be exophilic and either endophagic or exophagic in Myanmar (Burma) (Boyd, 1949), where it was an effective malaria vector in the Irrawaddy Delta (Covell, 1949). It was also considered to be a vector in eastern Sumatra, but not to be involved 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 the last 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 were described in south eastern and eastern Asia.

This work enabled identification of further vectors within the Hyrcanus Group. In addition to An. sinensis and An. nigerrimus, 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. xiaokuanus in north China and An. nimpe in southern Vietnam are known to bite man and to be important or probable malaria vectors (Ma, 1981; Chow, 1991; Nguyen et al., 2000).

Studies in the Palaearctic Region west of China

In his review of An. hyrcanus and related taxa in the Palaearctic Region, Martini (1929-31) regarded the described forms, pictus, pseudopictus and sinensis as synonyms of hyrcanus. Anopheles chodukini was described from a unique female specimen sent to him from Taskent and, though he considered the possibility that it may represent an extremely pale, desertic form of hyrcanus, he decided it more probably belonged to a separate species. He regarded the other taxa described from west of China as geographical varieties of hyrcanus thus: hyrcanus type form (distribution: Spain to Far East), var. mesopotamiae (Lower Iraq), var. marmuti (Asia Minor), var. marzinovski (Caucasus), and var. popovi (Turkestan). He provided a key (based on hind tarsal and wing scaling patterns) for differentiation between these forms but admitted that 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 by means available at that time, all came to be treated as populations of one species, An. hyrcanus, and other individual names were consigned to synonymy (Bates et al., 1949). Thus, An. hyrcanus was considered to be a widely distributed polymorphic species with a range extending from the Iberian Peninsula, through Europe south of the Alps and Asia south of about 50°N, to the Pacific. Gutsevich et al. (1971) and Gutsevich (1976) regarded all Palaearctic forms, including An. chodukini, as varieties (aberrations) of hyrcanus. Apart from a few more recent, fragmented, observations, this view has persisted, despite results of the work in the Oriental Region and the east of the Palaearctic Region.

Reid (personal communication, 1968) was of the opinion that until western Palaearctic forms of An. hyrcanus were shown to be conspecific with Oriental forms, they should be regarded as separate entities and suggested that it might better to label western Palaearctic forms ‘hyrcanus’ until the taxon had been adequately studied.

Evidence of speciation within the Hyrcanus Group in the western Palaearctic Region

A limited number of crossing experiments (Ross Institute, 1976, 1977) between samples drawn from populations of An. hyrcanus s.l. from Camargue (France), Çükürova (Turkey), Osmancik (Turkey) and elsewhere, indicated that the Camargue and Osmancik populations were conspecific, but that the Çükürova population belonged to a different species. Preliminary morphological studies during the same investigation indicated that a population from Jalalabad (Afghanistan) is probably conspecific with the Camargue/Osmancik form, in which hindtarsomere 4 (TIII-4) carries pale basal and apical white bands separated by a broad black median area. This segment is all white in the forms found at Kunduz, Afghanistan (Ward, 1972) and Çükürova, Turkey (Postiglione et al., 1973).

This work was discontinued after 1977, but more recently the taxon pseudopictus, with hindtarsomere 4 (TIII-4) all white, was reinstated as a species following comparison with the type form of hyrcanus s.l. in which hindtarsomere 4 (TIII-4) is dark except for a white apical band. In addition to these morphological differences, no evidence of natural hybridisation was found in south-west Asia where these forms are sympatric (Glick, 1992).

Hyrcanus Group as vectors of malaria in the western Palaearctic Region

Two hundred and thirty years after it was first recorded at the southern end of the Caspian Sea (Pallas, 1771), understanding of the status of An. hyrcanus s.l. in the western Palaearctic Region (west of China) is still in an embryonic stage. This situation stems from an assumption that the taxon was, at most, of only minor importance as a malaria vector.

Bates et al. (1949) did not regard An. hyrcanus s.l. as a primary vector, but thought it played a part in transmission 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 local conditions.

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 is ample evidence from Greece (Waterston, 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 countries of Turkmenistan, Uzbekistan, Tadzhikistan, Kazakhstan and Russia (Sergiev et al., 1993) showing that this taxon may exhibit quite high degrees of anthropophily. It is not known how far it was involved in the devastating outbreaks of malaria affecting French and British forces in Greek Macedonia during the 1914-1918 World War. Nevertheless, Weyer (1942), working in the same area during the 1939-1945 War, aptly observed that “An. hyrcanus has high potential as a malaria vector where it is abundant and people sleep in the open”. Quite recent observations in the rice growing area of the alluvial plain through which the River Axios flows found that An. hyrcanus s.l. is still one of the principal pest mosquitoes of northern Greece (Kaiser et al., 2001).

Biting and baited net trap collections in Turkey (Postiglione et al., 1973; personal observations) confirmed Weyer’s observation. In human and animal baited net trap collections in a village of south-west Turkey where cattle were stalled amongst the houses at night, 98% of 238 An. hyrcanus s.l. caught were in the animal baited net trap. Night biting collections on human bait in the same village failed to detect a single specimen attempting to bite, though many fed on the animals. However, when the same human bait were moved into open country away from this village, so losing the protection provided by cattle, they were immediately subject to mass attack by blood-seeking females. Further night biting collections in the Çükürova area of Turkey confirmed that large numbers viciously attack humans in the absence of herds of large domestic animals.

Rice fields, with their associated drainage systems, constitute prolific larval sites for this mosquito, and enormous populations of An. hyrcanus s.l. occur in some irrigated cotton and rice growing areas in Turkey and elsewhere (Postiglione et al., 1973; Cristescu et al., 1975; Onori et al., 1975; Cousserans et al., 1976; Anofrieva et al., 1977; Ramsdale & Haas, 1978; Kaiser et al., 2001).

Some of the most intractable problems of malaria transmission occur in small or large areas where monoculture crops, usually of rice and/or cotton, are produced under irrigation. Domestic animals are absent 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. and An. sacharovi had become highly resistant to insecticides (Ramsdale, 1975; Ramsdale et al., 1980) when a serious resurgence of malaria in the Çükürova occurred in the mid- to late-1970s. Hundreds of thousands of people slept out of doors during the summer at this period, making it impossible to say with any degree of certainty which of these taxa played the dominant vectorial role. Although An. sacharovi, which readily feeds inside or outside buildings, was undoubtedly a good vector in these circumstances, the more exophagic 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 of people sleep out of doors in situations where domestic stock are more or less absent. In these situations some taxa within the Hyrcanus Group certainly transmit malaria and may well be primary vectors. New areas 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 1.8 million hectares of agricultural land (Alten et al., 2000). Malaria transmitted by Hyrcanus Group mosquitoes in the western Palaearctic is not then an unimportant historical curiosity. On the contrary, it is a growing and potentially serious problem in areas of the western Palaearctic Region where vivax malaria 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 Plasmodium vivax, many studies have demonstrated refractoriness to infection with extant strains of the more pathogenic 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 been assumed, especially in areas of rice and cotton production.

Resolution of the taxonomic and vector problems posed by the Hyrcanus Group in the western Palaearctic

Species identification is clearly critical to any vector control programme that seeks to be efficient as well as effective (Collins et al., 2000).

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

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 and south west Asia, and particularly the reported renewal of P. falciparum transmission in Tajikistan, lend urgency to this investigation. Moreover, this problem is being compounded, especially in Turkey, by the planning 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 local investigations of the Hyrcanus Group are of limited value. On the other hand, integrated taxonomic and biological studies of Hyrcanus Group populations from different countries and habitats could unravel this longstanding problem. This must aim to provide information on the relative vector importance of the component taxa, both with regard to transmission of P. vivax and of P. falciparum.

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

The integrated approach to resolution of taxonomic uncertainties in species groups containing malaria vectors was successfully pioneered in the analysis the Anopheles quadrimaculatus complex in North America (Reinert et al., 1997; Carlson et al., 1997). This approach provides the most effective means of tackling the problems outlined here and offers the best opportunity of resolving the increasingly urgent issues of vector incrimination. Such a project in the western Palaearctic would require close co-operation between 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. In the case of the Hyrcanus Group, this aspect may be of major importance.

Acknowlwdgements

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

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