Probable arthropod vectors of avian blood protozoa, Leucocytozoon lovati, were collected in the alpine regions of Japan, the habitats of the host birds of Japanese rock ptarmigan (Lagopus mutus japonicus). Seven alpine regions of Japan, Asahidake, Chogatake, Tateyama, Jiigatake, Norikura, Kitadake, and Senjyogatake were investigated for black fly collection during 2004 to 2007. The collected 490 insects were morphologically identified as six species of female black flies, including Prosimulium hirtipes group (n = 59), Prosimulium mutata (n = 13), Prosimulium yezoense (n = 10), Similium japonicum (n = 359), Similium uchidai (n = 39), and Twinnia japonensis (n = 10). Extracted DNAs from individual black fly species were utilized for the amplification of the partial mitochondrial cytochrome b gene sequences of Leucocytozoon lovati previously reported. Four S. japonicum, two S. uchidai, and two P. hirtipes group studied were positive for the nested PCR among 490 black flies collected (1.6%; 8/490). All amplified sequences from the black flies were completely identical to those of L. lovati previously detected from Japanese rock ptarmigan. Our results suggest that at least three species of black flies, S. japonicum, S. uchidai, and P. hirtipes group, studied in this area could be regarded as potential vectors for L. lovati in the rock ptarmigan. This is the first detection case of Leucocytozoon from black flies of Japan.

ABSTRACT. In this study, we investigated the geographical distribution and seasonality of Leucocytozoon lovati infection in the Japanese rock ptarmigan (Lagopus mutus japonicus); this bird is one of the special natural monuments of Japan that inhabits the Japanese alpine regions. We examined blood samples from birds captured in the Kubiki, Hida, and Akaishi mountain ranges for three years from 2002 to 2005. Seventy-three blood samples from 42 males, 19 females, and 12 birds of unknown sex were used for this study. The rate of infection with L. lovati was 78.1% in the 73 birds examined. We demonstrated that the L. lovati infection was distributed across wide ranges of ptarmigan populations from the northern to the southern alpine zones. There was no sex bias in the prevalence ratio. The prevalence of L. lovati and the level of parasitization of the blood cells tended to increase from spring through summer; in contrast, a decrease was observed from summer through autumn. Although L. lovati infection was observed in a number of local populations inhabiting three mountainous regions, no infected birds were found in Mt. Johnen-dake and Mt. Maejohnen-dake. It is necessary to continue surveying the relationship between the population dynamics of the ptarmigan and the density of the arthropod vector from the perspective of in situ conservation of this endangered species.

One of vector-borne avian protozoa, Leucocytozoon lovati, has been found in the Japanese rock ptarmigans (Lagopus mutus japonicus), the endangered bird species distributed in the alpine regions in Japan. Vector arthropod species of L. lovati has also been estimated as Simuliidae black flies distributed in the same habitat of the host bird, however, possible blood meals of the black flies were not identified yet. To reveal host animals of black flies, we estimated the blood resources by using molecular techniques. Black flies were collected at Mt. Chogatake, one of the alpine regions of Japan in which Japanese rock ptarmigans live in June 2005. The analyzed 144 specimens were morphologically identified into five species including Simulium japonicum (n = 87), Prosimulium hirtipes (n = 48), Prosimulium yezoense (n = 3), Twinnia japonensis (n = 3), and Cnephia mutata (n = 3). Individually extracted DNA from the black flies was subjected to polymerase chain reaction (PCR) amplification targeting the partial mitochondrial cytochrome b gene of birds or mammals to identify the blood meals. Of 144 black flies examined, 34 specimens were PCR positive for avian hosts (23.6%). No mammalian-derived bloods were detected from the samples studied through. Sequences amplified from 11 black flies consist of S. japonicum, P. hirtipes, and C. mutata showed high similarity to that of the Japanese rock ptarmigan. Therefore, present results conclusively suggest that these three species of black flies might suck the bloods of Japanese rock ptarmigans and could be the vector for L. lovati infection among this endangered bird species of Japan.