Microbiome Associated with <i>Polypedilum</i> sp. (Diptera; Chironomidae), a Midge Adapted to an Extremely Acidic Environment

Chironomids (Diptera; Chironomidae), non-biting midges, are a highly diverse family of holometabolous insects, many of which are known for their tolerance to extreme environmental conditions, such as desiccation, pollution, and high acidity. The contribution of microbial symbionts to these adaptations was recently suggested. Therefore, we herein exami-ned the microbiome associated with the larvae of the undescribed acid-tolerant chironomid species, Polypedilum sp., which inhabits the Yukawa River (Gunma, Japan), an environment that is characterized by an extremely low pH (≤2) and high concentrations of heavy metal ions (including arsenic). Amplicon sequencing of the 16S rRNA gene revealed a distinct larval microbiome with a lower alpha diversity value and more enriched and specific bacterial taxa than the surrounding river water and detritus. Full-length 16S rRNA gene sequencing using nanopore long-read technology identified several previously undescribed operational taxonomic units (OTUs), among which OTU_Bacillaceae_Yukawa was consistently present in larvae reared in the laboratory for more than 4 months, suggesting persistent, possibly vertically transmitted, symbiosis. An inferred pathway ana-lysis suggested the contribution of the larval microbiome to host nutritional physiology. The possibly acid-sensitive OTU_Bacillaceae_Yukawa localized to midgut segments, indicating internal pH-buffered niches for microbial survival. These results provide novel insights into the ecology of acid-tolerant chironomids and lay the groundwork for further examinations of holobiont-based stress tolerance.