Imprinting statistically sound conclusions for gut microbiota in comparative animal studies: A case study with diet and teleost fishes.

Despite the technical progress in high-throughput sequencing technologies, defining the sample size which is capable of yielding representative inferences in metabarcoding analysis still remains debatable. The present study addresses the influence of individual variability in assessing dietary effects on fish gut microbiota parameters and estimates the biological sample size that is sufficient to imprint a statistically secure outcome. European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) were fed three alternative animal protein diets and a fishmeal control diet. Gut microbiota data from 12 individuals per diet, derived from Illumina sequencing of the V3-V4 region of the 16S rRNA gene, were randomized in all possible combinations of n-1 individuals. Results in this study showcased that increasing the sample size can limit the prevalence of individuals with high microbial load on the outcome and can ensure the statistical confidence required for an accurate validation of dietary-induced microbe shifts. Inter-individual variability was evident in the four dietary treatments where consequently misleading inferences arose from insufficient biological replication. These findings have critical implications for the design of future metabarcoding studies and highlight the urgency in selecting an adequate sample size able to safely elucidate the dietary effects on fish gut microbial communities.