Geographical distributions and host associations of larval parasitoids of frugivorous Drosophilidae in Japan

In Japan, dominant parasitoids attacking frugivorous Drosophilidae species were Asobara (Braconidae, Alysiinae), Leptopilina, and Ganaspis species (Figitidae, Eucoilinae). Asobara japonica was found throughout Japan, and its populations in the main islands of Japan were parthenogenetic whereas those in the subtropical islands were sexually reproducing. Other parasitoids showed rather restricted distributions; A.tabida, A. rossica, A. rufescens, and Leptopilina heterotoma occurred mainly in northern to central parts of the main islands, Ganaspis xanthopoda from central to southern parts of the main islands, A. leveri in a southern part of the main islands, and A. pleuralis, L. victoriae, and Ganaspis sp. mainly in the subtropical islands. Their major hosts were species of the D. melanogaster species group in the main islands, and species of the D. melanogaster, immigrans, and polychaeta species groups in the subtropical islands. Host use considerably varied among parasitoid species, especially in the subtropical islands.


Introduction
The range of geographic distribution and resource use are basic information in the study of ecology, environmental adaptations, and evolution of species. In Drosophilidae, intensive studies have so far been performed on their geographic distributions and resource use (reviewed in Ashburner et al. 1981Ashburner et al. , 1982Ashburner et al. , 1983. In contrast, only fragmentary information is available on their parasitoids except for some European species, e.g. Asobara tabida, Leptopilina heterotoma, and L. boulardi (Carton et al. 1986), and for some African Leptopilina species (Allemand et al. 2002). In this paper, we report geographic distributions and host associations of parasitoids attacking frugivorous Drosophilidae in Japan. From Japan, seven parasitoid species, Asobara japonica, A. tabida, A. rossica, Aphaereta sp., Phaenocarpa sp., Ganaspis xanthopoda, and Ganaspis sp. have so far been recorded, and all of them are reported to parasitize Drosophilidae larvae breeding in mushrooms (Yorozuya 2006;Ideo et al. 2008). However, major hosts of A. japonica, A. tabida, and G. xanthopoda are known to be Drosophilidae larvae breeding on fermenting fruits (Vet and Bakker 1985;Janssen et al. 1988;Ideo et al. 2008). Thus, parasitoids attacking Drosophilidae larvae often differ in habitats; some search larvae on mushrooms, some on fruits, and some on decayed leaves, although their habitat selection is not always rigid (Janssen et al. 1988;Ideo et al. 2008).
In Sapporo, collections were carried out in domestic areas (about 50 m above sea level) and forests at low (100 m) and high (600 m) altitudes 10 times from June to September in [2004][2005][2006]. In Tokyo, collections were carried out in domestic areas and lightly wooded areas at low altitudes (100-200 m) 29 times from early spring to late autumn in 2002-2005. At the remaining localities, collections were carried out in domestic, lightly wooded areas and/or forests at low altitudes (50-200 m) two or three times; i.e. in June (2004) and September (2003and 2004) in Sendai, in May (2004) and July (2006 in Kagoshima, in Figure 1. Collection localities. July (2006) andNovember (2005) in Amami-oshima, in June and November (2005) in Okinawa, and in March (2006), July (2003), and December (2003 in Iriomote-jima. Clumps of banana were placed in the collection sites, left for about a week, brought back to the laboratory, and placed in plastic containers with pieces of cloth or paper. Usually 30 g clumps of banana were used, and in most instances more than 10 such clumps were used in each collection. When smaller clumps (1, 2, 3 or 10 g) were used, the number of clumps was raised. When drosophilid larvae pupated on cloth or paper, they were collected, identified to species, placed on Petri dishes with wet filter paper, and examined for the emergence of flies or wasps. However, sibling species could not be discriminated by pupal morphology; i.e. D. melanogaster and D. simulans, a pair of D. lutescens and D. takahashii, and four species of the auraria species complex (D. auraria, D. biauraria, D. triauraria, and D. subauraria). In Tokyo, Kagoshima, and Iriomote-jima, some natural fruits were found. These fruits were collected to the laboratory, and drosophilid larvae in them were examined for parasitism as described above.
In the family Figitidae, Leptopilina heterotoma occurred in northern to central parts of the main islands (Sapporo, Sendai, and Tokyo), and an adult female of this species was collected in Amami-oshima. Ganaspis xanthopoda occurred in central to southern parts of the main islands (Sendai, Tokyo, and Kagoshima), and Leptopilina victoriae in Kagoshima and the subtropical islands (Amami-oshima, Okinawa, and Iriomote-jima). Two undetermined Ganaspis species were found; 171 individuals of sp. 1 from Iriomote-jima and 10 individuals of sp. 2 from Tokyo. In addition, an undetermined Leptopilina species was collected in Tokyo; an individual emerged from drosophilids and three individuals emerged from unidentified insects (probably drosophilids). Furthermore, seven individuals of undetermined Leptolamina species emerged from drosophilids collected in Sendai, and 57 individuals of this species emerged from unidentified insects (probably drosophilids) in Tokyo.
Trichopria specimens (Diapriidae) were widely found in Japan, and Pachycrepoideus vindemmiae (Pteromalidae) was found in Sapporo and Tokyo. It is uncertain whether the specimens of Trichopria from different localities are conspecific or not.

Host association
Tables II and III show host associations of parasitoid species in the main islands (Sapporo, Sendai, Tokyo, and Kagoshima) and the subtropical islands of Japan (Amami-oshima, Okinawa, and Iriomote-jima), respectively. In the main islands, drosophilids reared in large Table II. Numbers of drosophilid pupae collected and numbers of parasitoid individuals emerged from these pupae in the survey in the main islands of Japan (Sapporo, Sendai, Tokyo, and Kagoshima). The rate of parasitism is also presented.  triauraria) and D. immigrans. Among them, species of the D. melanogaster species group were used as major hosts by various parasitoids, but D. immigrans was rarely parasitized (Table II). Several species of the subgenera Drosophila (D. bizonata, D. sternopleuralis, and D. tsigana) and Dorsilopha (D. busckii) and the genus Scaptodrosophila (S. subtilis and S. coracina) were sometimes present in banana in the main islands, and they were often parasitized. In the subtropical islands, drosophilids reared in large numbers from banana were members of the D. melanogaster (D. takahashii, D. lacteicornis, D. ficusphila, and D. bipectinata), immigrans (D. albomicans), and polychaeta (D. daruma) species groups, and the rate of parasitism was often high (Table III). Host use varied considerably among parasitoid species, especially in the subtropical islands; A. japonica parasitized D. melanogaster and D. takahashii; A. pleuralis and L. victoriae attacked D. ficusphila, D. lacteicornis, D. bipectinata and D. albomicans, and Ganaspis sp. 1 parasitized D. albomicans and D. daruma.

Discussion
Asobara japonica was the most abundant species in the samples and was widely distributed in Japan. In this species, the sex ratio was much biased towards females in the specimens from the main islands of Japan but not in those from the subtropical islands. By our laboratory rearing, it was confirmed that individuals from Sapporo, Tokyo, and Kagoshima reproduce parthenogenetically, whereas those from Amami-oshima and Iriomote-jima reproduce sexually. The other Asobara species were limited to restricted districts; A. tabida, A. rufescens, and A. rossica were found in northern and central parts of the main islands, A. leveri in Kagoshima, and A. pleuralis in Iriomote-jima. The occurrence of A. tabida adults was also confirmed in Shiga-kogen (about 1500 m above sea level) located in central Japan (unpublished observation). Asobara tabida and A. rufescens have been previously recorded from Europe, A. rossica from Far East Russia, A. leveri from Fiji, and A. pleuralis from the Philippines (Carton et al. 1986;Belokobylskij 1998). Thus, the first three species are adapted to cool-temperate climates, and the last two are adapted to warm-temperate or subtropical climates. Table III. Numbers of drosophilid pupae collected and numbers of parasitoid individuals emerged from these pupae in the survey in the subtropical islands of Japan (Amami-oshima, Okinawa, Iriomote-jima). The rate of parasitism is also given. In the Figitidae, Leptopilina heterotoma and Ganaspis xanthopoda were mainly collected in the main islands of Japan (the former in northern to central parts and the latter in central to southern parts), and an individual of the former was collected in a subtropical island, Amami-oshima. These two species are also recorded from wide areas; the former from Southeast Asia, Europe, Africa, and North America, and the latter from these regions except for Europe (Nordlander 1980;Carton et al. 1986;Schilthuizen et al. 1998;Allemand et al. 2002;Fleury et al. 2004). In contrast to the above two species, L. victoriae and Ganaspis sp. 1 occurred mainly in the subtropical islands in Japan. L. victoriae is also recorded from the Seychelles, Thailand, and Africa (Nordlander 1980;Schilthuizen et al. 1998;Allemand et al. 2002), suggesting that it is adapted to subtropical and tropical climates.
Among the present parasitoid species, A. rufescens mainly parasitizes drosophilid larvae growing on decayed plant materials in Europe (Vet et al. 1984). In our unpublished study, this species was also observed to parasitize larvae of Scaptomyza pallida (Zetterstedt) breeding on decayed leaves. The remaining parasitoid species are assumed to use frugivorous drosophilids as major hosts, although A. japonica sometimes attacks drosophilid larvae feeding on mushrooms (Ideo et al. 2008).
Species of the D. melanogaster species group and D. immigrans have been reported from the main islands of Japan as dominant drosophilids breeding on fruits (Kimura et al. 1977;Nishiharu 1980;Kimura 2000a, 2000b;Mitsui et al. 2006). Among them, species of the D. melanogaster species group were frequently parasitized. In contrast, D. immigrans larvae were rarely parasitized. It has also been confirmed in laboratory experiments that D. immigrans larvae are not or seldom parasitized, at least by L. heterotoma, A. tabida, or A. japonica (van Lenteren and Bakker 1978;van Alphen and Janssen 1982;Ideo et al. 2008). Van Alphen and Janssen (1982) suggested that the thick cuticle of D. immigrans larvae prevents insertion of the ovipositor of A. tabida. In Europe, D. subobscura Collin of the D. obscura species group is reported as a major native host of A. tabida and L. heterotoma (Carton et al. 1986;Janssen et al. 1988;Kraaijeveld and van der Wel 1994;Kraaijeveld et al. 1995). In this study, D. bifasciata of the D. obscura species group was parasitized by A. japonica. In addition, this species was observed to be parasitized in our preliminary study in Shiga-kogen (central Japan). Although these parasitoids could not be raised to adults, they presumably were A. tabida since this was the only one of the species that was found at the collection sites. On the other hand, A. leveri has been reported to parasitize Tephritidae species (Belokobylskij 1998). In the subtropical islands of Japan, D. albomicans, D. daruma, and several species of the melanogaster species group were observed to breed on banana (also see Hirai et al. 2000), and they were often parasitized.
Host use differed considerably among parasitoid species, especially in the subtropical islands. Such a pattern arises if the parasitoid species occur in different environments, if they attack different hosts in the same environment, or if the host species differ in resistance to different parasitoids.
Among parasitoids found in this study, Trichopria sp. and P. vindemmiae are known as pupal parasitoids (Carton et al. 1986;personal observation). In the present study, however, these species emerged from drosophilid individuals that remained as larvae when collected in fields. In our laboratory rearing, at least one Trichopria species was confirmed to parasitize full-grown scrolling larvae of D. simulans, although at a low rate. Thus, these parasitoids are able to parasitize late instar larvae, although they predominantly attack the pupal stage. They are widely distributed in Japan and seemed to use a variety of Drosophilidae species as hosts.