# Influence of Macrophyte and Gut Microbiota on Mercury Contamination in Fish: A Microcosms Study
## 4 Discussion
### Involvement of Fish Gut Microbiota in Hg Methylation  
The relative abundance of _Deltaproteobacteria_ assessed using the molecular approach showed a low abundance and diversity in the fish gut (1%) with a high variability depending on the condition, whereas it is 5-fold higher in macrophye roots and 20-fold in sediment (Figure 4B). Strains isolated from the fish gut (the contaminated \"plant\" condition) belonged to _Desulfovibrionaceae_ and _Desulfobulaceae_ families. Some of the isolates obtained probably represent new taxa among the family _Desulfovibrionaceae_ and should be further characterized and described. The strain isolation overlooked the dominant _Deltaproteobacteria_ since the bacterial diversity seen through the cultural method is different and lower than that seen from using the molecular method. Indeed, members of _Desulfovibrionales_ were exclusively detected through the cultivation method in the fish gut from the \"plant\" condition. Some of them were detected in the fish gut from the \"plant+sediment\" condition by MISEQ, showing the potential influence of the external environment (plant roots and sediment) on the diversity of such a matrix. Their capacity to methylate Hg was assessed using a biosensor (the light intensity produced by the biosensor directly reflected the methylation potential of a strain) and seven were able to methylate Hg at variable rates (Figure 5). Few studies have demonstrated the ability of SRP and _Enterobacteria_ to methylate Hg in the digestive tract of terrestrial organisms [62,63] or fish [31] and the results are quite controversial [30,64]. IHg assimilation by intestinal epithelium is limited [65, 66, 67]. However, IHg can be methylated before passing the intestinal barrier into the blood. A mechanism of Hg assimilation was suggested in [68], involving both amiloride sensitive, and energy-dependent pathways to explain IHg absorption across the vertebrate gut. However, the mechanisms of Hg absorption through the intestinal cells have not yet been elucidated in fish or in mammals. In our work, a part of IHg could be methylated in the rhizosphere of aquatic plants before being ingested as MeHg by fish. Another part could be ingested as IHg, then methylated in the digestive tract, increasing the contamination risk. In addition, methylating strains also affiliated with _Desulfovibrio carbonicicus_ were isolated from _Ludwigia_ sp. roots from the Sanguinet Lake [8]. This indicates that SRP isolated from intestinal contents of fish are derived from the aquatic rhizosphere, which is a food resource for fish. These strains could be responsible for Hg methylation in the gut. Variability in bacterial diversity of gut microbiota could explain the variability of \({}^{199}\)MeHg production measured in fish muscle at the end of the experiment. This hypothesis remains to be checked and quantified since the methylation capacity of strains could be stimulated or inhibited within the intestine (due to the unfavorable physicochemical growth medium, competition with gut microbiota). It would be interesting to further investigate the gut microbiota of fish and confirm if Hg methylation is possible in vivo in the gut. Moreover, it is also possible that these strains are able to demethylate MeHg [47,69,70]. Another incubation experiment with Hgstable isotopes (\({}^{199}\)IHg and \({}^{201}\)MeHg) would be important to simultaneously and precisely quantify both processes.