In recent years, the study of the gut microbiota of cultured teleost fish has gained relevance as cornerstone studies that provide important information on the gut microbial status and their impact on the fish health and welfare. Considering the role of the microbiome in animal health, it is fundamental to explore the utility of microbiota studies using histological samples. These studies could provide valuable insights on how the gut microbiome is modulated upon dietary and environmental changes under which fish are being reared currently compared to rearing conditions in the past.
In this study, we analyzed 20 microbiomes targeting the V3-V4 hypervariable region of the 16S rRNA gene derived from Illumina® amplicon sequencing of the rainbow trout (Oncorhynchus mykiss) acclimated at two temperatures (16° and 22°C) for five weeks. At the end of the experiment trout were euthanized and the intestine was aseptically removed from each fish. Part of the intestine was flash frozen for molecular biology and the other section was fixed for histological processing. DNA was extracted from both samples, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE), and subjected to 16S rRNA amplicon sequencing.
Amplicon sequencing of the V3-V4 region of the 16S rRNA resulted in an average of 17,335 reads for the FF samples and 5,032 reads for the FFPE samples, corresponding to a total of 735 OTUs. The microbiome of the intestine of the rainbow trout acclimate at 16°C showed a higher diversity of species (α-diversity) (p-value = 0.04) than organism at 22°C when analyzed using FF samples of the intestine. The difference in the diversity was not captured when parallel but FFPE samples were used (p-value = 0.34). The O. mykiss microbiome was dominated by the bacterial taxonomic phylum Fusobacteria. This phylum constituted a high, but variable, sequence proportion at both temperatures and for both types of samples (FF and FFPE) (Fig. 1). This data reveals that microbiome can be successfully determined using FFPE tissue opening a new horizon in studying intestinal microbiota.