Sex, age, and bacteria: How the intestinal Microbiota is modulated in a Protandrous Hermaphrodite fish

Abstract

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism and absorption, disease resistance and immune function. With the growth of the aquaculture industry, there has been a growing interest in the manipulation of fish gut microbiota to improve welfare and nutrition (Egerton et al., 2018). However, a long road lies ahead to establish the baseline parameters to guide this manipulation. Intestinal microbiota varies with many factors, including host species, genetics, developmental stage, diet, environment and sex. The aim of this study was to compare the intestinal microbiota of adult gilthead sea bream (GSB, Sparus aurata) from three groups of age maintained under the same conditions. One-, 2- and 4-year old GSB (Y+1, Y+2, Y+4) were kept in the same open-flow system and fed the same diet for more than 6 months. After 2-days of fasting, 10 fish per group were sacrificed and the anterior intestinal portion was dissected, opened and washed to remove non-adherent bacteria. Intestinal mucus was scrapped off and immediately used for DNA extraction. The V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. All Y+1 individuals were males, while the ones belonging to Y+2 and Y+4 age classes were females. A total of 686461 high quality reads (22882/sample) were assigned to 846 OTUs (97% identity). Almost 30% of the OTUs were classifiedup to the level of species. Microbiota diversity and richness did not differ among age groups; however bacterial composition did , highlighting the presence of Photobacterium and Vibrio only after 2 years of age and a higher presence of Staphylococcusand Corynebacterium in Y+1 animals. The core microbiota was defined by 14 OTUs and the groups that showed more OTUs in common were Y+2 and Y+4. PLS-DA analysis showed a clear separation by sex (component 1) and age (component 2), with bacteria belonging to the phyla Firmicutes, Proteobacteria and Actinobacteria driving the separation. Pathway analysis performed with the inferred metagenome showed significant differences between Y+1 and Y+4, with an increase with age of pathways related to cell death, motility, biofilm formation, environmental response, infection, metabolism of lipids, secondary metabolites, ansamycins biosynthesis and nitrotoluene degradation. Carbohydrate and lipoic acid metabolism and atrazine degradation were down-regulated with age. The effect of host age on the intestinal microbial diversity has been previously studied in other fish species finding an increase in diversity with age (Zhang et al., 2018). However, these studies mainly focused on early life stages of the fish.Nonetheless, it is clear that the intestinal ecological niche matures with age leading to changes in the associated microbiota (Stephens et al., 2016). The dominance of the phyla Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria has already been reported in several studies on carnivorous marine fish, including GSB (Estruch et al., 2015). The current results highlight a gradual increase of Proteobacteria abundance with age with the consequent decrease of the other phyla. Photobacterium and Vibrio were previously described as the dominant genera in GSB posterior intestine (Piazzon et al., 2017) and it is noteworthy that in the current study these genera are absent in the anterior intestine of Y+1 fish,becoming more dominant with age. These two generainclude many pathogenic and opportunistic species, but others are important symbionts, assisting in the breakdown of dietary components (Egerton et al., 2018). Overall, changes in microbial composition drive changes in metabolic pathways, as the up-regulation of infection-related pathways, nitrotoluene degradation and sphingolipid metabolism, and the down-regulation of carbohydrate metabolism pathways, which seem to indicate an inflammatory profile in the intestines of older fish.