Introduction
It is generally accepted that plastics, consisting mainly of micro-sized (1 μm–1 mm) and nano-sized (<1 μm) particles, have become a serious threat to marine ecosystems, not only because they can physically damage the organisms, but also due to the toxicity of the associated chemical pollutants (Jambeck et al., 2015). Microplastics (MPs), which are consumed by marine organisms that confuse them with food sources, can causes various adverse effects. In fish, environmental chemicals can effectively induce changes in gut microbiota. Therefore, a dysbiosis might develop in the host’s intestine after consuming MPs due to the ingestion of foreign and potentially pathogenic bacteria or to chemicals that make up or adhere to MPs (Fackelmann and Sommer, 2019). The consequences of exposure to MPs and the combined effects of MPs and adsorbed pollutants, on the microbiota community has not been exhaustively investigated. Accordingly, we exposed European sea bass juveniles to chemical pollutants and to MPs contaminated or not with a blend of chemical pollutants (dichlorodiphenyldichloroethylene (p,p’-DDE), chlorpyrifos (CPF), and benzophenone-3 (BP-3)), by adding them to the fish diet. The aim of the study was to determine the individual and combined effects of dietary MPs and chemical pollutants on the fish gut microbiota population.
Materials and Methods
The feeding trial was set in a flow-through marine water system with three hundred European sea bass (80.91 ± 13.28 g), randomly distributed in twelve tanks (500 l). Four different diets were formulated. Commercial diet with no additives was considered as the control diet, whereas diet MP, diet P and diet P+MP were manufactured adding to the commercial diet: MPs alone, or a mix of three chemical pollutants, or MPs contaminated by the same mix of chemical pollutants, respectively. The level of inclusion of MPs in the diets was 10% (w/w), while, for diets P and P+MP the blend of the three chemical pollutants was composed by p,p’-DDE, CPF, and BP-3 in concentration of 1000, 100, and 300 ng/g, respectively. At the end of the trial, two fish per replicate tank (6 fish/diet) were sacrificed to collect the fecal matter and the autochthonous intestinal bacteria for gut microbiome analysis. The sequencing was carried out using Illumina MiSeq platform and processed using QIIME™ 2 (v. 2018.4) then, the representative sequences from each operational taxonomic units (OTUs) were aligned to Greengenes database v. 13.8 down to genus taxonomical level.
Results and Discussion
In line with previous studies (Wan et al., 2019), the present metagenomic analysis showed an alteration in sea bass gut microbiome composition and in bacterial species richness following the ingestion of pollutants or polluted MPs. Furthermore, the PCoA analysis revealed that pollutant treatments significantly altered gut microbiome community structures that clearly separated them from the control group. Irrespective of the diet, the gut microbiota of European sea bass was dominated by Firmicutes, Proteobacteria, and Actinobacteria phyla, as already reported in previous microbiome studies in this species (Rimoldi et al., 2020). However, their relative abundances were significantly changed by ingesting pollutants. In particular, compared to the control group, although the dietary inclusion of virgin MPs did not cause relevant changes in microbiome, the polluted diets increased the abundance of the phylum Proteobacteria, widely considered a microbial signature of gut inflammation (Shin et al., 2015), while reduced the Firmicutes content, which instead are generally considered beneficial and associated with a healthy intestine. These bacteria in fact, are known for having anti-inflammatory potential and a crucial role in regulating the host immune system (Terova et al., 2016). These results are in line with those reported for yellow croaker (Larimichthys crocea) and adult zebrafish (Danio rerio) exposed to PS-MPs (Jin et al., 2018). In agreement with the reduction in beneficial bacterial taxa belonging to Fimicutes phylum, PICRUSt analysis predicted a decrease in starch and carbohydrate metabolism as well as in cysteine, methionine, and purine metabolism. On the contrary the two-component and secretion system pathways were instead greatly upregulated. This would agree with the gut inflammatory signs and microbiome profiles observed in fish fed diets containing chemical pollutants. Likewise, an increase in lipopolysaccharide biosynthesis could be related to the inflammatory status of intestine and to dysbiosis.
Conclusion
In summary, our results clearly demonstrate a significant perturbation in gut microbial communities, including the reduction in beneficial bacteria genera and the increase in potentially pathogenic microorganisms. The results obtained in this study provide, therefore, new insights into the potential risks of ingesting MPs as pollutant carriers in marine fish.
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