Animals have been developing key associations with micro-organisms through evolutionary processes and ecological diversification. Hence, in some host clades, phylogenetic distance between hosts is correlated to dissimilarity in microbiomes, a pattern called phylosymbiosis. Teleost fishes, despite being the most diverse and ancient group of vertebrate, have received little attention from the microbiome perspective and our understanding of its determinants is currently limited. In this study, we assessed the gut microbiome of 12 co-occurring species of teleost representing a large breadth of ecological diversity and originating from a single family (i.e. the Sparidae). We tested how host evolutionary history, diet composition and morphological traits are related to fish gut microbiome. Despite fish species having different microbiomes, there is no phylosymbiosis signal in this fish family, but gut length and diet had a strong influence on the microbiome. We revealed that the only species with a specialized herbivorous diet, Sarpa salpa had a 3.3 times longer gut than carnivorous species and such a long gut favor the presence of anaerobic bacteria typical of herbivorous gut microbiomes. Hence, dietary uniqueness is paired with both unique gut anatomy and unique microbiome.

Samples collection

Fishes were collected in the shallow habitats (2-10 m) of the North-Western coast of Gulf of Lions (Hérault, France, Mediterranean Sea) between June 2018 and February 2019. Fishes were caught at night by artisanal fishermen at the end of the night using gillnets, stored on ice on the boat and were processed within 3 hours after being caught. Fish were measured (mm), weighted (g) and dissected using tools cleaned with 70° ethanol. The gut was stretched and a picture was taken for morphometric measurements. The last third of the gut was squeezed out on a piece of parafilm paper using a sterile pipette. If this gut content was made of digested food (i.e. not only mucus) it was then homogenized and collected in a 3 mL cryotube before storage at -80°C until DNA extraction. Gut microbiome from a total of 48 adult individuals from 12 Sparidae species were sampled.

DNA extraction

DNA extractions were performed in the molecular biology platforms of the MARBEC laboratory (UMR 9190, www.umr-marbec.fr) and at the Genseq platform (genseq.umontpellier.fr), using the Qiagen MagAttract PowerSoil DNA KF Kit, selected for its compliance with the Earth Microbiome Project (Marotz et al., 2017)⁠. Extractions were performed in 96 wells plates in which 3 wells were left empty to serve as negative controls and 3 wells were loaded using standard mock communities (ZymoBIOMICS Microbial Community DNA Standards II, Zymo Research). These standards were used to evaluate the quality of our sample processing pipeline and to identify potential contaminants from the reagents. Extraction wells were loaded with one spatula of homogenized gut content (~ 0.25 g). DNA extraction protocol included a bead beating steps and a chemical lysis. DNA recovery was based on magnetic beads and automated with a Kingfisher Flex robot. DNA was eluted in 200µL of elution buffer before quantification of DNA quantity and quality using a Nanodrop 8000 spectrometer. Extracted DNA was stored at 4°C until PCR amplification, which was done the next day.

PCR amplification

PCR amplification was done using primers selected for their compliance with the Earth Microbiome Project (Parada, Needham, & Fuhrman, 2016)⁠: 515F-Y (5′-GTGYCAGCMGCCGCGGTAA) and 926R (5′-CCGYCAATTYMTTTRAGTTT). The targeted sequence was 411 bp and corresponded to the V3-V4 regions of the prokaryotic 16S rRNA gene. PCR amplification was carried out in 96 well plates in triplicate for each DNA extract and was done in a 25 µL reaction volume. The PCR mix consisted of 9.75µL of water, 0.75µL of DMSO, 0.5 µL of each primer (concentration), 12.5 µL of Phusion ready-to-use Taq mix (Phusion High-Fidelity PCR Master Mix with GC Buffer) and 1µL of DNA. The PCR cycle consisted of 35 cycles of 10 sec denaturation at 98°C, 1 min annealing at 58°C and 1 min 30 sec of extansion at 72°C. Final extension was held for 10 min at 72°C before keeping the reaction at 4°C. The success of PCR amplification was checked on 2% agarose gel in TAE buffer and using a 100bp DNA ladder. The wells left empty during DNA extraction served as negative controls for contamination of the PCR reactions. PCR triplicates were pooled and stored at -20°C before sequencing. Amplicons library was constructed by the Genotoul platform (get.genotoul.fr) and sequencing was carried out using an Illumina MiSeq (2 × 250 bp) sequencer.