DOI: 10.5061/dryad.2280gb5r7
title: 16S sequences from mesocosms experiment testing the effect of Siganus rivulatus on marine microorganisms

description: Nutrient cycling is a key biogeochemical process underlying the
 functioning of marine ecosystems. Yet, the contribution of fishes is still
 poorly understood. This is problematic considering the current
 modifications of fish assemblages experienced in certain regions such as
 the Mediterranean Sea, and their potential consequences in terms of
 ecosystem functioning. In this study, we used a mesocosm experiment to
 test the effect of nutrient recycling by an invasive marine herbivorous
 fish (Siganus rivulatus) on planktonic and benthic microbial communities.
 The response of these communities was assessed using a variety of
 analytical approaches such as measures of nutrient concentration, flow
 cytometry, and metabarcoding of the 16S rRNA gene. Our results show that
 several microbial compartments of marine ecosystems respond to the
 nutrients released by fish through excretion and egestion. The nutrients
 contained in the macroalgae consumed by S. rivulatus were excreted in
 large amounts as dissolved nutrients, which resulted in higher
 concentrations of N-based nutrients in the water (NH4, NO2/NO3). This
 excess of N in the system was associated with higher abundances of
 planktonic microbes (phyto- and bacterioplankton), modifications of the
 structure of planktonic bacterial communities, and the tissue composition
 of the remaining macroalgae. Non-assimilated nutrients were released in
 the form of feces under the shelters where the fish spent most of their
 time and defecated during the night, leading to local increases in
 diversity and significant shifts in the structure of sediment bacterial
 communities. Overall, our results suggest that the impact of S. rivulatus
 on planktonic microbes was related to the indirect bottom-up effect
 induced by excreted nutrients while its effect on benthic microbes was due
 to a direct release of microbes from its gut microbiome. This study
 represents one of the first assessment of the effect of nutrient recycling
 by fishes on the microbial communities from several compartments of marine
 ecosystems and one of the first evidence of the invisible effect of
 invasive species on the microbial components of marine ecosystems.
description: <strong>Experimental setup</strong>
 The experiment aims to test the effect of nutrient recycling
 (excretion and egestion) by the invasive herbivorous fish
 <em>Siganus rivulatus</em> (Siganidae) on marine planktonic
 and benthic microbial communities. Eight 800L mesocosms were set up, which
 contained all the elements of natural marine ecosystems: sediment,
 macroalgae (4 species), rocks covered with turf and hiding places for
 fishes. Four mesocosm served as control and no fish were added while in
 the four treatment tanks we added three <em>S. rivulatus</em>
 individuals for a total biomass of 100g. The experiment lasted for eight
 days after which we sampled all the compartments of the system for
 analysis of their microbial communities (abundance, diversity) and their
 nutrient content. <strong>DNA
 extraction</strong> 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 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.
 <strong>PCR amplification</strong>
 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 extension at 72°C. The 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.
description: # 16S sequences from mesocosms experiment testing the effect of Siganus
 rivulatus on marine microorganisms
 The dataset contains 16s sequences describing the microbial communities from various compartments of mesocosms testing the effect of fishes on the environment. ## Description of the data and file structure The file entitled 16S_sequences_mesocosm.zip contains the raw sequences obtained from sequencing of the bacterial 16S rRNA gene amplified from various compartment of mesocosms mimicking the marine environment of the Levantine Sea. The file table_metadata.csv contains the metadata associated with the sequences. It contains the following columns: * sample_id_fastq: the id of the sample in the siganidae_microbiome_16S_sequences.zip file * barcoding_id: the id of the samples used for barcoding analyses * sample_id: the unique sample ID * taxonomy: the taxonomic identification of the sample * sample_type: the type of sample categorized in broad categories tank_id: the ID of the experimental tank * treatment: the treatment applied to mesocosm from which the sample was taken * treatment_tank: a vector combining the information between the treatment and the tank number * sampling_rank: the ID of the sampling event * sampling_day: the day of sampling from the begining of the experiment * sampling_date: the day of sampling * position_in_the_tank: the place in the tank where the samples was taken Missing data code: NA