Isolation and Characterization of Mosquito-Associated Spiroplasma cantharicola from Aedes japonicus Collected in Hokkaido, Japan

SIMPLE SUMMARY: Spiroplasma, which comprises a group of the smallest known bacteria, is commonly found in insects and plants. Some species of Spiroplasma have shown pathogenicity against mosquitoes and are expected to be useful vector control tools. In this study, we isolated S. cantharicola from the mosquito Aedes japonicus collected in Hokkaido, northern Japan. Field surveillance implied a relatively low prevalence of the bacteria in the local mosquitoes. Phylogenetic analysis showed that our isolate is closer to a strain of S. cantharicola isolated from mosquitoes in previous studies. Our isolate grew well in the R2 broth medium and grew into a visible colony on R2 broth agar. Co-culturing with mosquito-derived C6/36 cells resulted in slightly more bacterial growth than culturing without cells, showing the strong cytopathogenic effect of the cells. To our knowledge, this is the third report on the isolation of S. cantharicola from mosquitoes. ABSTRACT: Species of the genus Spiroplasma are common within arthropods and plants worldwide. Mosquito-associated Spiroplasma spp. have been reported to show pathogenicity toward mosquitoes, which serve as vectors of several infectious diseases that have detrimental effects on public health. Although Spiroplasma spp. are expected to have potential use as biological vector-control tools, characteristics such as their distribution, host species, and cytopathogenic effects (CPEs) are not well understood. In this study, we isolated a Spiroplasma sp. from a female Aedes japonicus collected in Hokkaido, northern Japan. Phylogenetic analysis based on the 16S rRNA gene sequence indicated our isolate was closely related to S. cantharicola. We screened 103 mosquito pools consisting of 3 genera and 9 species, but only detected S. cantharicola in the first isolation. In an in vitro assay, our isolate grew well at 28 °C, but no propagation was observed at 37 °C. Furthermore, the isolate showed strong CPE on a mosquito-derived cultured cell line (C6/36), and its propagation slightly increased when co-cultured with C6/36 cells. To our knowledge, this is the third report of the isolation of S. cantharicola from mosquitoes and the first case in Asia. Our findings provide epidemiological data on S. cantharicola distribution in the region.