Introduction: Microsporidia constitute the most common black fl y pathogens, although the species’ diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization. Methods: Larvae were collected at a single point in a stream in a rural area of the city and were kept under artifi cial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 x 5.0 ± 0.7μm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 x 3.5μm in sporophorous vesicles. Results: The molecular analysis confi rmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon. Conclusions: Samples identifi ed as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fl y-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group
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