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.

Experimental setup

The experiment aims to test the effect of nutrient recycling (excretion and egestion) by the invasive herbivorous fish Siganus rivulatus (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 S. rivulatus 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.

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 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 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.