Reveal of Gut Microbiota of Nymphs Ameletus inopinatus (Eaton), 1887 (Ephemeroptera: Ameletidae) with Molecular and Microbiological Analyzes

Abstract

Mayflies, recognized for their hemimetabolous life cycle and ephemeral adult phase, exhibit diverse larval forms categorized as burrowing, flattened, swimming, and creeping. Ameletus inopinatus Eaton, 1887, a member of the Ameletidae family within the order Ephemeroptera, undergoes aquatic development and is commonly found in the tranquil stretches of small rivers or streams, particularly among stones and boulders. This study focused on A. inopinatus nymphs collected from Yarımca village in Eskişehir, Turkiye. Employing molecular techniques and culture-independent methods, rRNA-based analyses were conducted to characterize the archaeal and bacterial communities within the nymphs’ gut. Microbial DNA was extracted from the gut, and PCR amplification targeted 16S rRNA genes for both Archaea and Bacteria domains. Denaturing gradient gel electrophoresis (DGGE) separated the amplified products, yielding six distinct bands with Bacteria-specific primers. The subsequent analysis of 16S rRNA sequencing from DGGE bands revealed a predominance of the Proteobacteria phylum within the gut lumen. Complementary investigations into prokaryotic diversity employed fluorescence in situ hybridization (FISH) and 4',6-diamidino-2-phenylindole (DAPI) staining. Total DAPI cell counts indicated a microbial cell population of approximately 10⁶ ± 0.18 in the samples. The overarching conclusion derived from these findings is that the microbial community within the Ameletus inopinatus gut is predominantly associated with the Bacteria domain. Recognizing the ecological importance of this symbiotic relationship, the coexistence of mayflies with specific microbial communities is emphasized, underscoring the potential mutualistic roles played by these microorganisms in the life cycle and ecological dynamics of Ameletus inopinatus. This research not only contributes valuable insights into the microbial ecology of mayfly nymphs but also underscores the significance of understanding the intricate relationships between insects and their associated microorganisms for broader ecological perspectives and potential applications in environmental management.