Revision of simulium quinquestriatum (diptera: simuliidae) in thailand and the study of filarial infections of black fly vectors in Chiang Mai Province, Northern Thailand

Abstract

Black flies (Diptera: Simuliidae) are small blood-sucking insects of medical, veterinary and ecological importance. Some species are known to be vectors of the filarial parasite, Onchocerca volvulus, which is responsible for human diseases such as onchocerciasis or river blindness. The bites of infected female black flies with the filarial worm of animal origin also can cause a rare disease, i.e., human zoonotic onchocerciasis. Up until now, at least 38 human cases of zoonotic onchocerciasis have been reported throughout the world. In Thailand, at least 134 black fly species belonging to the six subgenera in the genus Simulium Latreille s.l., have been recorded so far. Although no human onchocerciasis or zoonotic onchocerciasis has been reported in Thailand, three anthropophilic black fly species, including S. nodosum, S. asakoae s.l. and S. nigrogilvum, were incriminated as the natural vectors of three different types of filarial worms in Chiang Mai province, northern Thailand. However, previous studies on natural filarial infections in black flies in Chiang Mai province were based solely on morphological characteristic analysis of the recovered larvae, from which molecular analysis has never been used to clarify species status. Therefore, this study explored the filarial infections in adult female black flies collected in Chiang Mai province, northern Thailand, and clarified the species status of the recovered filarial parasites and their black fly vectors by using the combined method of morpho-taxonomic and molecular analyses [mitochondrial cox1 and 12S rRNA, and nuclear 18S rRNA (SSU HVR-I)]. Female black flies were collected from two study sites (Ban Lek and Ban Pang Dang) in Chiang Mai province from March 2018 to January 2019 using humans and swine as baits. At Ban Lek, a total of 805 adult female black flies belonging to seven taxa were captured, and four of 309 adult female S. nigrogilvum were positive for Onchocerca sp. type I in the hot and rainy seasons. At Ban Pang Dang, a total of 4,597 adult female flies belonging to nine taxa were collected. Among them, five unknown filarial larvae (possibly new species) were detected in Simulium sp. in the S. varicorne species-group and three species (S. asakoae, S. myanmarense and S. monglaense/S. nanthaburiense) in the S. asakoae species-group in all seasons. Furthermore, three non-filarial larvae of three different taxa also were found in three females (S. asakoae, S. monglaense/S. nanthaburiense and S. pitasawatae) of the S. asakoae species-group. The results obtained in this study provided a new body of knowledge for a better understanding of zoonotic filarial parasites and black fly vectors in Thailand, especially in Chiang Mai province. The Simulium striatum species-group is one of 33 species-groups of the subgenus Simulium (Simulium) in the Oriental region and includes 31 named, and one unnamed species. In Thailand, five species, namely S. chiangmaiense, S. nakhonense, S. quinquestriatum, S. thailandicum and S. phraense have been documented. A few previous studies attempted to differentiate members of this species-group molecularly. DNA barcoding using cox1 gene failed to separate S. chiangmaiense, S. nakhonense and S. quinquestriatum, and the nuclear elongation complex protein 1 (ECP1) gene also could not separate S. chiangmaiense and S. nakhonense. Subsequently, the big zinc finger (BZF) gene could differentiate S. phraense, S. chiangmaiense and S. nakhonense clearly. When focusing on S. quinquestriatum, preliminary phylogenetic analysis, based on cox1 sequences, suggested that the morphologically named S. quinquestriatum may consist of at least two species. Thus, in order to clarify whether S. quinquestriatum from various locations in Thailand represents only one single species or species complex, this black fly species was reexamined morphologically. In addition, the phylogenetic relationships of S. quinquestriatum were elucidated by molecular analysis using the mitochondrial (cox2) and nuclear (BZF) genes. Adults reared from pupae were re-examined, with pupal exuviae and cocoons of morphologically identified S. quinquestriatum collected from 16 locations in 10 provinces of Thailand. Three new species, namely S. wangkwaiense Takaoka, Srisuka & Saeung, sp. nov.; S. tadtonense Takaoka, Srisuka & Saeung, sp. nov.; and S. maeklongkeense Takaoka, Srisuka & Saeung, sp. nov., were discovered and morphologically identified, one of which being known formerly as S. quinquestriatum. Phylogenetic analyses, based on BZF gene sequences, could separate all six Thai members (except for S. thailandicum) of the S. striatum species-group clearly from one another. Furthermore, two possibly new species were found based on molecular analyses. However, more specimens in all stages need to be re-examined in order to describe possible new species. The results obtained from this study further supported the effectiveness of the BZF gene, which is not only useful in differentiating members of the S. striatum species-group, but also revealing more cryptic or new species in this species group.