DOI: 10.5061/dryad.kprr4xh35
title: 16S rRNA sequences from Mediterranean Sparidae gut microbiome

description: 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.
description: <b>Samples
 collection</b> 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>i.e.</i> 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. <b>DNA
 extraction</b> 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. <b>PCR
 amplification</b> 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.