Blackfly vectors of zoonotic onchocerciasis in Japan

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1 Medical and Veterinary Entomology (2012) 26, doi: /j x REVIEW ARTICLE Blackfly vectors of zoonotic onchocerciasis in Japan H. TAKAOKA 1,2, M. FUKUDA 2,3,Y.OTSUKA 2, C. AOKI 2, S. UNI 1,4 and O. BAIN 5 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia, 2 Department of Infectious Disease Control, Faculty of Medicine, Oita University, Yufu City, Oita, Japan, 3 Research Promotion Project, Oita University, Yufu City, Oita, Japan, 4 Department of Medical Zoology, Osaka City University Medical School, Osaka, Japan and 5 Parasitologie Comparée, UMR 7205 Centre National de la Recherche Scientifique (CNRS), Museum National d Histoire Naturelle, Paris, France Abstract. Studies of blackfly vectors of Onchocerca dewittei japonica Uni, Bain & Takaoka (Spirurida: Onchocercidae), a parasite of wild boar implicated in the aetiology of zoonotic onchocerciasis in Japan, and six other zoonotic Onchocerca species of this country are reviewed. Molecular identification of infective larvae found in wild-caught female blackflies showed that Simulium bidentatum (Shiraki) (Diptera: Simuliidae) is a natural vector of O. dewittei japonica, and also Onchocerca sp. sensu Fukuda et al., another parasite of wild boar. Inoculation experiments demonstrated that Simulium arakawae Matsumura and four other Simulium species are putative vectors. Similarly, S. arakawae, S. bidentatum and (Shiraki) are putative vectors of Onchocerca eberhardi Uni & Bain and Onchocerca skrjabini Rukhlyadev, parasites of sika deer. Morphometric studies of infective larvae indicated that Onchocerca lienalis Stiles, a bovine species, is transmitted by S. arakawae, Simulium daisense (Takahasi) and Simulium kyushuense Takaoka, and that Onchocerca sp. sensu Takaoka & Bain, another bovine species, is transmitted by S. arakawae, S. bidentatum, S. daisense and S. oitanum. Prosimulium sp. (Diptera: Simuliidae) and Simulium japonicum Matsumura are suspected vectors of Onchocerca suzukii Yagi, Bain & Shoho and O. skrjabini [Twinnia japonensis Rubtsov (Diptera: Simuliidae) may also transmit the latter], parasites of Japanese serow, following detection of the parasites DNA genes in wild-caught blackflies. Key words. Onchocerca, blackfly, filariae, onchocerciasis, vector, zoonosis, Japan. Introduction Blackflies (Diptera: Simuliidae) represent one of the most important groups of blood-sucking insects. They have a worldwide distribution and are found in any location in which running freshwater streams or rivers suitable for the habitat of their aquatic stages are available. The blood-sucking habits of female blackflies are responsible for considerable deleterious effects on humans and their economic welfare (Crosskey, 1990). The medical and socioeconomic impacts associated with blackflies include reduced levels of tourism, the death of domesticated birds and mammals, and the transmission of viral, protozoan and filarial diseases (Adler et al., 2004). In particular, blackflies are well known as vectors of Onchocerca volvulus (Leuckart, 1893), the causative filarial species of human onchocerciasis or river blindness endemic in Africa and Central and South America (Crosskey, 1990). In the absence of such notorious blackfly-borne diseases, blackflies were poorly studied as disease vectors in Japan (Takaoka, 1999), as in other Asian countries, until 1989 when a case of zoonotic onchocerciasis was first reported in Oita in Correspondence: Hiroyuki Takaoka, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia. Tel.: ; Fax: ; takaoka@oita-u.ac.jp 372 Medical and Veterinary Entomology 2012 The Royal Entomological Society

2 Zoonotic onchocerciasis vectors in Japan 373 Kyushu, a southwestern island (one of the four main islands of Japan) (Beaver et al., 1989; Hashimoto et al., 1990). Zoonotic onchocerciasis, which is caused by Onchocerca species of animal origin, was considered to be very rare; only four cases had been reported prior to 1989, each from Canada (Ali-Khan, 1977), Switzerland (Siegenthaler & Gubler, 1965), Ukraine (Azarova et al., 1965) and the U.S.A. (Beaver et al., 1974). From 1990 to January 2012, 15 more cases of zoonotic onchocerciasis were reported. [Correction added on 17 August 2012, after first online publication: Number of cases of zoonotic onchocerciasis was changed from 16 to 15.] These include one from Albania (Pampiglione et al., 2001), one from Austria (Koehsler et al., 2007), one from Hungary (Sallo et al., 2005), one from Kuwait (Hira et al., 2008), eight (one unpublished and four published cases from Oita, one published case from Hiroshima, and two unpublished cases from each of Hiroshima and Shimane, in the southwest of Honshu) from Japan (Takaoka et al., 1996, 2001, 2004, 2005; Uni et al., 2010; Uni et al., 2012; unpublished data), one from Turkey (Otranto et al., 2011), and two from the U.S.A. (Burr et al., 1998; Wright et al., 2002). By contrast with human onchocerciasis, in which microfilariae produced from gravid female worms cause severe dermal and ocular lesions, zoonotic onchocerciasis is in general caused by a single immature adult female or male worm and thus no microfilariae are produced. However, despite the absence of the conspicuous clinical symptoms caused by microfilariae, conjunctivitis and other ocular lesions caused by an invading adult worm in the ocular or periocular tissue regions (ocular zoonotic onchocerciasis), or a subcutaneous nodule formed around the worm in various parts of the body, can be of clinical importance. The disease is diagnosed by detecting an Onchocerca worm or its parts in ocular tissue or a resected subcutaneous nodule. However, specific identification based on morphological characteristics is not easy because in most cases only small parts of a worm are available. Onchocerca gutturosa Neumann, 1910, a common filarial parasite of cattle, and Onchocerca cervicalis (Stiles, 1902), a common parasite of the horse, have been reported as suspected causes of cases identified in Europe and North America (Siegenthaler & Gubler, 1965; Beaver et al., 1974; Ali-Khan, 1977; Burr et al., 1998; Wright et al., 2002). Onchocerca jakutensis (Gubanow, 1964), a parasite of deer, was identified by DNA analysis in the case from Austria (Koehsler et al., 2007), and Onchocerca lupi Rodonaja, 1967, a parasite of canids, was suspected in the case from Albania (Sréter et al., 2002) and confirmed in the case from Turkey (Otranto et al., 2011). A recent study shows that O. lupi is responsible for ocular zoonotic onchocerciasis (Otranto et al., 2011). By contrast, the causative Onchocerca species for all nine cases found in Japan was demonstrated to be Onchocerca dewittei japonica Uni et al., 2001, a new subspecies from wild boar (Sus scrofa Linnaeus, 1758), although the aetiology of the first case was originally suspected to implicate O. cervicalis or O. gutturosa (Beaver et al., 1989) and that in the second case to implicate O. gutturosa (Takaoka et al., 1996). This review focuses on investigations of blackflies (and biting midges in part) carried out in order to determine the vector of O. dewittei japonica, and the vectors of other species of Onchocerca known or newly found in wild boar, cattle, sika deer (Cervus nippon Temminck, 1838) and Japanese serow [Capricornis crispus (Temminck, 1845)], all of which have the potential to act as causative agents of zoonotic onchocerciasis in Japan. Natural and putative vector blackfly species Onchocerca dewittei japonica, a parasite of wild boar and a causative agent of zoonotic onchocerciasis in Japan The third Japanese case of zoonotic onchocerciasis (Takaoka et al., 2001) directed our investigation for Onchocerca parasites from cattle to wild boar. In this case, the causative worm was a male, characterized by an outer cuticle bordered with numerous tiny crests in the sectioned specimen of the resected nodule, which were interpreted as minute longitudinal crests throughout its body. A similar characteristic was found in the male of Onchocerca dewittei Bain, Ramachandran, Petter & Mak, 1977 originally described from wild boar in Malaysia (Bain et al., 1977), when the Onchocerca male specimens preserved in Muséum National d Histoire Naturelle were re-examined (Uni et al., 2001). Shortly afterwards, an Onchocerca species was discovered from wild boar in Oita and described as a new subspecies, O. dewittei japonica (Uni et al., 2001). This new subspecies was identified as the causative species of the third zoonotic case and also in the aetiology of the second case (Takaoka et al., 2001), which was previously suspected to be O. gutturosa (Takaoka et al., 1996). This correction is based on certain morphological characteristics of the female worm of O. dewittei japonica, such as conspicuous outer ridges (triangular in transverse section), a relatively long distance between ridges and a lack of inner cuticular striae (Takaoka et al., 2001, 2004). The causative species of the fourth, fifth and ninth cases (female worms) found in Oita, the sixth and seventh cases (also female worms) from Hiroshima, and the eighth case (male worm) from Shimane, were all identified as O. dewittei japonica based on morphological characteristics and/or analysis of mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequences (Takaoka et al., 2004, 2005; Uni et al., 2010; Fukuda et al., 2011; Uni et al., 2012; unpublished data). The causative species of the first case (female worm) was corrected to O. dewittei japonica after reexamination of its morphological characteristics, such as the height of the outer ridges of the cuticle (Uni et al., 2010). As Table 1 shows, Simulium bidentatum (Shiraki, 1935), the predominant anthropophilic blackfly species in Kyushu, has emerged as a natural vector of O. dewittei japonica in Oita as evidenced by the detection of infective larvae of O. dewittei japonica in wild-caught female S. bidentatum (identified according to morphological criteria and subsequent analysis of mitochondrial COI and 12S ribosomal RNA genes of the infective larvae) (Fukuda et al., 2010a). Five other anthropophilic blackfly species, Simulium arakawae Matsumura, 1915, Simulium japonicum Matsumura, 1931, (Shiraki, 1935), Simulium quinquestriatum (Shiraki, 1935) and Simulium rufibasis Brunetti, 1911, have been revealed as putative vectors of O. dewittei japonica through intrathoracic inoculation experiments (Fukuda et al.,

3 374 H. Takaoka et al. Table 1. Vector status of blackfly species for seven species of Onchocerca in Japan. Vector status of blackfly species Onchocerca species* Host animals Natural vectors Putative vectors Suspected vectors O. dewittei japonica Sus scrofa Simulium bidentatum Simulium arakawae Simulium japonicum Simulium quinquestriatum Simulium rufibasis Onchocerca sp. sensu Fukuda et al., 2008 Sus scrofa Simulium bidentatum Simulium arakawae O. lienalis Bos taurus Simulium arakawae Simulium daisense Simulium kyushuense Onchocerca sp. sensu Takaoka & Bain, 1990 Bos taurus Simulium arakawae Simulium bidentatum Simulium daisense O. eberhardi Cervus nippon Simulium arakawae Simulium bidentatum O. skrjabini Cervus nippon Capricornis crispus Simulium arakawae Simulium bidentatum Simulium japonicum Prosimulium sp. Twinnia japonensis O. suzukii Capricornis crispus Simulium japonicum Prosimulium sp. *Onchocerca gibsoni and Onchocerca gutturosa, parasites of cattle, and Onchocerca cervicalis, a parasite of the horse, are not included because their vectors are unknown or dubious. Based on infective larvae found in wild-caught female blackflies. Based on complete development of microfilariae to infective larvae through intrathoracic inoculation infection experiments. Based on detection of parasite species-specific DNA sequences from wild-caught female blackflies. There is a possibility that this is Simulium kawamurae because females of Simulium japonicum and S. kawamurae are morphologically indistinguishable. 2008). The infective larvae of O. dewittei japonica (body length, μm; width, μm) are morphologically indistinguishable from the infective larvae tentatively identified as O. gutturosa found in female S. bidentatum caught in a cattle shed in Oita (Takaoka & Bain, 1990). Onchocerca sp. sensu Fukuda et al., 2008, another parasite of wild boar Onchocerca sp. sensu Fukuda et al., 2008, another species from wild boar, was first distinguished at the microfilarial stage by its body length, which was much greater than that of O. dewittei japonica, and its distinctness as a species was confirmed by DNA analysis (Fukuda et al., 2010b). The infective larvae of Onchocerca sp. sensu Fukuda et al., 2008, obtained through intrathoracic inoculation experiments, are morphologically indistinguishable from those of O. dewittei japonica and the infective larvae (tentatively identified as O. gutturosa) found in female S. bidentatum caught in a cattle shed in Oita (Takaoka & Bain, 1990). Their body measures μm by22μm (Fukuda et al., 2008). Adults of Onchocerca sp. sensu Fukuda et al., 2008 were recently discovered and their morphological characteristics are now under study (Uni et al., 2012; unpublished data). Simulium bidentatum has been shown to be a natural vector of this unnamed Onchocerca species in Oita because the identities of infective larvae found in wild-caught S. bidentatum females were confirmed by DNA analysis (Table 1) (Fukuda et al., 2010a). Simulium arakawae and S. oitanum were demonstrated to be putative vectors of Onchocerca sp. sensu Fukuda et al., 2008 in intrathoracic inoculation experiments (Fukuda et al., 2008). Onchocerca species of cattle In Japan, O. gutturosa was long thought to be the only species to affect cattle, and both its adult worms and microfilariae were known (Nimi & Kono, 1953; Sato et al., 1954). Two types of microfilariae were recognized in the skin of cattle in Japan, of which one had an uncoiled and the other a coiled posterior portion, but both of these were thought to be O. gutturosa by Nimi & Kono (1953). In dissections of female blackflies caught in cattle sheds in Oita, where the first case of zoonotic onchocerciasis occurred in Japan (Beaver et al., 1989; Hashimoto et al., 1990), two types of microfilariae (one with an uncoiled posterior portion, designated as type X, and the other with a coiled posterior portion, designated as type Y) were detected in ingested cattle blood in the flies midguts. The type X microfilariae were further classified into two species, based on differences in the

4 Zoonotic onchocerciasis vectors in Japan 375 shape of the anterior tip and body thickness, of which one was suspected to be O. gutturosa and the other Onchocerca lienalis Stiles, 1908 (Takaoka & Bain, 1990). In addition, three types of infective larvae were recognized in the head, thorax and/or abdomen of the blackflies: the first, designated as type I, was μm long and μm wide, with an oesophagus as long as the intestine; the second, designated as type II, was μm long and μm wide, with an oesophagus much longer than the intestine, and the third, designated as type III, was μm long and μmwide, with an oesophagus much longer than the intestine (Takaoka & Bain, 1990). The type I infective larva of Onchocerca sp. sensu Takaoka & Bain, 1990 was successfully associated with the type Y microfilaria by intrathoracic inoculation experiments using microfilariae recovered from cattle skin, indicating that the microfilaria with a coiled posterior portion, or type Y, was not O. gutturosa (Takaoka, 1990). Because it shared a similar type Y coiled microfilaria, Onchocerca sp. sensu Takaoka & Bain, 1990 was once thought to be Onchocerca suzukii Yagi, Bain & Shoho, 1994, a parasite of Japanese serows in the northern part of Honshu, Japan (Uni et al., 1998), but was eventually shown by DNA analysis to be a new species of Onchocerca distinct from O. suzukii and other known Onchocerca species (Fukuda et al., 2010a). Four blackfly species, S. arakawae, S. bidentatum [both in Oita and Kumamoto (in Kyushu)], and Simulium daisense (Takahasi, 1950) and S. oitanum [both in Iwate (in northeastern Honshu)], have been demonstrated to be natural vectors of Onchocerca sp. sensu Takaoka & Bain, 1990 (Table 1) (Takaoka et al., 1992; Takaoka, 1994). It is noteworthy that the infective larvae found in female Simulium nodosum Puri, 1933 collected in Chiang Mai, Thailand, are morphologically very similar to those (type I) of Onchocerca sp. sensu Takaoka & Bain, 1990 (Takaoka et al., 2003). The adult worm of Onchocerca sp. sensu Takaoka & Bain, 1990 remains unknown. The type II infective larva was thought to be O. gutturosa based on morphometric criteria, and S. bidentatum was regarded as its natural vector because it harboured type II infective larvae. However, as already noted, the infective larvae detected in female S. bidentatum collected from a different locality in Oita, which were morphologically indistinguishable from type II larvae, were not O. gutturosa but O. dewittei japonica and Onchocerca sp. sensu Fukuda et al., 2008, both parasites of wild boar, as revealed by DNA sequence analysis (Fukuda et al., 2010a). Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are likely to be a vector of O. gutturosa in Japan, as reported elsewhere (Bain, 1979; El-Sinnary & Hussein, 1980; Davies et al., 1989; Dohnal et al., 1990). However, dissections of 4868 female midges of eight Culicoides species (captured at the cattle shed in which the female S. bidentatum carrying type II infective larvae were collected) were all negative, although unknown, possibly avian, filarial larvae were detected from Culicoides arakawae (Arakawa, 1910) (Takaoka et al., 1995). At present, the vector(s) of O. gutturosa in Japan remains unconfirmed. The type III infective larva was tentatively identified as O. lienalis (Takaoka & Bain, 1990). A microfilaria of type X was shown to develop to a type III infective larva in S. arakawae by an intrathoracic inoculation experiment, thus supporting the suggestion that type X microfilariae include two species, O. gutturosa and O. lienalis (Takaoka, 1990). Three blackfly species, S. arakawae, Simulium kyushuense Takaoka, 1978 and S. daisense, were incriminated as natural vectors of O. lienalis by dissecting female blackflies caught in cattle sheds in Oita, Kumamoto and Iwate, respectively (Table 1) (Takaoka et al., 1992; Takaoka, 1994). Adults of O. lienalis have not yet been reported from cattle in Japan. This probably reflects the difficulty of recovering the tiny, slender adult worms from the gastrosplenic ligaments. In cattle slaughtered in Oita, microfilariae of O. lienalis, O. gutturosa and Onchocerca sp. sensu Takaoka & Bain, 1990 were found at prevalences of 17.2%, 3.0% and 6.7%, respectively (Takaoka, 1997). Another species, Onchocerca gibsoni (Cleland & Johnston, 1910), a Southeast Asian and Australasian species, was reported only once from slaughtered cattle in Tokyo (Itagaki, 1954). This species has not been reported from Japan since. Thus the possibility that the first finding represents a misidentification has not been ruled out. Onchocerca species of sika deer Two Onchocerca species were found in sika deer, of which one was identified as Onchocerca skrjabini Rukhlyadev, 1964 and the other was described as a new species, Onchocerca eberhardi Uni & Bain, 2007 (Uni et al., 2007). Intrathoracic inoculation experiments showed that the microfilariae of both Onchocerca species recovered from sika deer in Oita can develop to infective larvae in S. arakawae, S. bidentatum and S. oitanum, implicating these three blackfly species in the transmission of O. eberhardi and O. skrjabini (Table 1) (Fukuda et al., 2008). Infective larvae of O. eberhardi were μm in body length, the shortest of all examined, whereas those of O. skrjabini measured μm in body length, similar to those of O. lienalis (type III) (Fukuda et al., 2008). In Oita, O. skrjabini and O. eberhardi were found in 36% and 81%, respectively, of sika deer examined (Uni et al., 2007). Onchocerca species of Japanese serows Two Onchocerca species, O. skrjabini and O. suzukii, were reported from Japanese serows in the northern parts of Honshu (Yagi et al., 1994). Onchocerca suzukii is characterized in the adult female by a lack of distinct outer ridges, and in the microfilaria by the coiled posterior portion, as in Onchocerca sp. sensu Takaoka & Bain, 1990 from cattle. However, the microfilariae of O. suzukii are distinguishable from those of Onchocerca sp. sensu Takaoka & Bain, 1990 by their body size ( μm long and 7.5 μm wide in O. suzukii, vs μm long and 4 5 μm wide in Onchocerca sp. sensu Takaoka & Bain, 1990) (Fukuda et al., 2008). The infective larva of O. suzukii remains unknown.

5 376 H. Takaoka et al. The mitochondrial COI sequence of O. skrjabini was detected in wild-caught female Prosimulium sp. (hirtipes species-group), S. japonicum and Twinnia japonensis Rubtsov, 1960, and that of O. suzukii was also detected in the former two blackfly species, in central parts of Honshu, which suggests these blackfly species are involved in the transmission of O. skrjabini and O. suzukii (Table 1) (Iikawa et al., 2009; Sato et al., 2009a). Whether the development of microfilariae of O. skrjabini and O. suzukii to infective larvae takes place in these blackfly species remains to be confirmed. Onchocerca species of horses No studies were carried out on the abilities of blackflies and biting midges to transmit O. cervicalis, which is the only species parasitizing horses in Japan (Sato et al., 1954). Discussion Onchocerca dewittei japonica, a parasite of wild boar, has been demonstrated as the causative agent of all nine cases of zoonotic onchocerciasis found in Japan. However, definite identification of the causative species required the definition of the fauna of Onchocerca species in domestic and wild animals in the areas concerned. Thus, six other species of Onchocerca are candidate causative agents of potential zoonotic onchocerciasis transmitted by blackflies in Japan. Identification keys provided by morphometric studies of these Onchocerca species (Takaoka et al., 2004) will be useful in determining the aetiology of future cases of zoonotic onchocerciasis. Molecular techniques using analysis of DNA sequences will also be indispensable to accurately and rapidly identify the parasite, particularly when only limited material is available (e.g. tiny pieces of worm body in sectioned, slide-mounted tissue specimens obtained from subcutaneous nodules in patients), as already evidenced in the cases in Hiroshima and Shimane (Fukuda et al., 2011; Fukuda et al., 2012; unpublished data). To prevent zoonotic onchocerciasis caused by O. dewittei japonica, it is essential to understand its transmission cycle in nature. Among several anthropophilic blackfly species, only S. bidentatum has been reported as a natural vector of O. dewittei japonica in Oita, although its feeding on wild boar should be confirmed. There is a possibility that one or two of five putative vectors (experimentally infected by intrathoracic infection) and, indeed, other blackfly species not yet evaluated, may be vectors in other areas of Honshu, including Hiroshima and Shimane, where the vector species remain unknown. Further investigations of wild-caught female blackflies are needed because the identity of the predominant anthropophilic blackfly species in Honshu differs among localities (e.g. Takaoka et al., 1992). Intrathoracic inoculation experiments and subsequent morphometric characterizations of the infective larvae obtained reveal, to a certain degree, the limitation of morphological identification, as seen among type II from cattle, O. dewittei japonica and Onchocerca sp. sensu Fukuda et al., 2008 from wild boar, and between O. lienalis and O. skrjabini (Fukuda et al., 2008). When all specific DNA sequence data for all Onchocerca species have been accumulated, a molecular method will become a powerful tool to reliably identify infective larvae detected in wild-caught blackflies, as also demonstrated by Fukuda et al. (2010a, 2010b). Given the high diversity of Onchocerca species, the substantial infection rates of their host animals, and the wide variety of potential vector blackfly species throughout the country, cases of zoonotic onchocerciasis caused not only by O. dewittei japonica, but also by other Onchocerca species transmitted by various blackfly species, are likely to occur much more widely than was initially thought. To fully understand the complexities of blackfly-borne filarial disease in Japan, more data are required, such as rates of blood-feeding on host animals and humans by blackflies, and population densities of female blackflies and host animals during the transmission season, which differs in duration according to latitude and varies, for example, from late spring (May) to middle autumn (October) in central Kyushu (Takaoka, 1994), to summer only (July and August) in Iwate in northeastern Honshu (Takaoka et al., 1992). Other filarial and protozoan parasites and their blackfly vectors in Japan Loxodontofilaria caprini Uni & Bain, 2006 (Spirurida: Onchocercidae) was described from Japanese serows in the northeastern and central parts of Honshu and Oita in Kyushu (Uni et al., 2006). The mitochondrial COI sequence of this species was detected in females of two blackfly species, S. japonicum and T. japonensis, collected in central mountainous regions of Honshu (Iikawa et al., 2009). This was the first report to suggest that blackflies are involved in the transmission of L. caprini, although whether larval development of L. caprini actually occurs in these blackfly species remains to be studied. Dirofilaria immitis (Leidy, 1856) (Spirurida: Onchocercidae) is a canine parasite causing zoonotic dirofilariasis and is transmitted by mosquitoes. As many as 280 human cases were reported from 1964 to 2002 in Japan (Akao, 2005). Simulium takahasii (Rubtsov, 1962) has been experimentally shown to support the development of the microfilariae of D. immitis to the infective stage by inducing female blackflies reared from pupae collected in Oita to feed on blood from a dog infected with D. immitis (Takaoka & Baba, 1987). The larval development of D. immitis took place in the Malpighian tubules of S. takahasii, as reported in mosquito vectors (Takaoka & Baba, 1987). Simulium uchidai (Takahasi, 1950) has been shown to be a natural vector of an unknown filarial larva, which may be a parasite of a bird, by dissecting wild-caught blackflies in Oita, Japan (Fukuda et al., 2005). Simulium takahasii and S. uchidai are likely to be zoophilic and ornithophilic, respectively, in their host preference and are thus of little medical importance.

6 Zoonotic onchocerciasis vectors in Japan 377 Leucocytozoon lovati (Achromatorida: Leucocytozoidae) is a haematozoan parasite of an endangered rock ptarmigan (Lagopus mutus japonicus) in central alpine areas of Honshu; and three blackfly species, Prosimulium sp. (hirtipes speciesgroup), S. japonicum and S. uchidai, were reported as its probable vectors through the detection of DNA of L. lovati in wild-caught blackflies (Sato et al., 2009b). The identification of the latter two blackfly species should be confirmed by analysis of DNA sequences, as performed by Fukuda et al. (2005), because adult females of S. japonicum and S. uchidai are morphologically indistinguishable from those of Simulium kawamurae Matsumura, 1919 and Simulium koshikiense Takaoka, 1976, respectively, although they are easily separable in the pupal stage (Takaoka, 1976). Acknowledgements These investigations may not have been carried out without the kind collaboration of many other persons, including patients, clinical doctors, medical students and hunters, as well as current and former staff members of the Department of Infectious Disease Control, Oita University, Japan, to whom we are most grateful. Our appreciation goes to Dr Yukita Sato, Department of Veterinary Medicine, Nihon University, Kanagawa, Japan, who kindly provided valuable references. We also thank Dr Benjamin L. Makepeace, Institute of Infection and Global Health, University of Liverpool, Liverpool, U.K., for his kindness in proofreading the manuscript. Finally, the first author expresses his sincere gratitude to Dr Isao Tada, Professor Emeritus, Kyushu University, Fukuoka, Japan, for his excellent guidance and ceaseless encouragement. References Adler, P.H., Currie, D.C. & Wood, D.M. (2004) The Black Flies (Simuliidae) of North America. Cornell University Press, Ithaca, NY. Akao, N. 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