Evaluation of 16S rRNA gene sequencing for species level microbiome analysis in Channa striata

Abstract

Microbiota plays a crucial role in a host organism due to its phylogenetic diversity and wild distribution. Though microbiome study of various fresh water fishes was conducted, so far, no such studies focused on Channa striata. In this study we evaluated the microbiota of Channa Striata gut (stomach, intestine and rectum) using 16S rRNA amplicon sequencing.Live fishes were sampled and dissected to obtain the respective regions of the gut. These regions were incubated in a nutrient medium and the grown broth cultures were then subjected to serial dilution followed by spread plating. Isolated colonies obtained from the spread plates were subjected to colony PCR using 16S V3-V4 region primers. PCR amplicons were visualized and amplified products were sequenced both in forward and reverse directions. The resulting sequences were analyzed phylogenetically using index metrics such as Shannon and ChaoI. Concurrently, an antibiotic resistance test was carried out using the same procedure to identify antibiotic-resistant varieties in the microbiota. Fish were fed with antibiotic-mixed food for 14 days before dissection and colony isolation.Our analysis of 16S rRNA amplicon sequencing revealed a diverse array of flora in the gut microbiome of Channa striata. Bacteria belonging to Pseudomonas, Staphylococcus, Microbacterium, E. coli, Microcystis and three unknown species were identified in the gut of the organism. Those belonging to Microbacterium and Pseudomonas were considered to be predominant in the stomach of the fish. Phylogenetic tree construction revealed that Microcystis flos-aquae formed a separate clade along with three unknown species. Some of the microbiota persisted in the gut even after feeding with antibiotics, indicating that these species are resistant to the fed antibiotic.Results obtained indicate a strong influence of the host environment in the microbiota composition. Knowledge of the microbiota is the first step in exploring proper management strategies for this freshwater fish.