The study of the eukaryotic fraction of the microbiota using a metabarcoding approach is usually hindered by the high host to eukaryotic microbiota DNA ratio in samples. Indeed, the 18S rRNA gene is very similar for both the host and its eukaryotic communities, leading to a preferential amplification of the predominant host DNA when using universal primers. Multiple approaches have been developed to reduce host DNA amplification. One method is based on elongation arrest blocking primers, oligonucleotides modified with a C3 Spacer that stops the advancement of the DNA polymerase at non-conserved regions of a target gene. In this paper, we successfully developed and tested species-specific elongation arrest blocking primers to block the Flag cichlid, Mesonauta festivus, 18S rRNA SSU. Our elongation arrest blocking primers significantly reduced the amount of host DNA in samples by 66 %. In addition to reducing the amount of sequencing wasted, the blocking primers increased the detectability of potentially dangerous parasitic taxa in fish gut, highlighting the potential of the method for parasitic screening. For instance, we discovered a case of infection by the parasitic ciliate Nyctotherus sp. Also, we detected the presence of a parasitic Trematode and an Amoebae, collected compelling data on the species feeding habits and obtained data on the commensal eukaryotic diversity present in M. festivus gut. While our data support the possibility of achieving a complete inhibition of host DNA amplification using elongation arrest blocking primers, more research is still required. Still, there is a need for the development and additional testing of protocols to study the eukaryotic diversity present in fish gut, a slow-growing field of study in comparison to its prokaryotic counterpart.
Lowland central Amazonia is characterized by heterogeneous riverscapes dominated by two chemically divergent water types: black (ion-poor, rich in dissolved organic carbonate and acidic) and white (rich in nutrient and turbid) waters. Recent phylogeographic and genomic studies have associated the ecotone formed by these environments to an ecologically driven genetic divergence between fish present in both water types. With the objective of better understanding the evolutionary forces behind the central Amazonian Teleostean diversification, we sampled 240 Mesonauta festivus from 12 sites on a wide area of the Amazonian basin. These sites included three confluences of black and white water environments to seek for repeated evidences of ecological divergence at the junction of these ecotones. Results obtained through our genetic assessment based on 41,268 SNPs contrast with previous findings and support a low influence of diverging water physicochemical characteristics on the genetic structuration of M. festivus populations. Conversely, we detected patterns of isolation by downstream water current and evidence of past events of vicariance potentially linked to the Amazon River formation. Using a combination of population genetics, phylogeographic analysis and environmental association models, we decomposed the spatial and environmental genetic variances to assess which evolutionary forces shaped inter-population differences in M. festivus’ genome. Our sampling design, comprising three confluences of black and white water rivers, supports the main role of evolution by allopatry. While an ecologically driven evolution admittedly played a role in Amazonian fish diversification, we argue that neutral evolutionary processes explain most of the divergence between M. festivus populations.
Associations between host genotype and the microbiome of holobionts have been shown in a variety of animal clades, but studies on teleosts mostly show weak associations. Our study aimed to explore these relationships in four sympatric Serrasalmidae (i.e. piranha) teleosts from an Amazonian lake, using datasets from the hosts genomes (SNPs from GBS), skin and gut microbiomes (16S rRNA metataxonomics), and diets (COI metabarcoding) from the same fish individuals. Firstly, we investigated whether there were significant covariations of microbiome and fish genotypes at the inter and intraspecific scales. We also assessed the extent of co-variation between Serrasalmidae diet and microbiome, to isolate genotypic differences from dietary effects on community structure. We observed a significant covariation of skin microbiomes and host genotypes at interspecific (R2=24.4%) and intraspecific (R2=6.2%) scales, whereas gut microbiomes correlated poorly with host genotypes. Serrasalmidae diet composition was significantly correlated to fish genotype only at the interspecific scale (R2=5.4%), but did not covary with gut microbiome composition (Mantel R=-0.04; only 6 microbiome taxa involved). Secondly, we tested whether microbial taxa represent reliable host traits to complement host genotypic variations in these species. By using an NMDS ordination-based approach, we observed that subsets of the skin and gut microbiomes selected by a machine-learning Random Forest algorithm can complement host genotypic variations by increasing significantly the average interspecific differentiation. The complementarity of genome and microbiome variations suggests that combining both markers could potentially benefit our understanding of the evolution of Serrasalmidae in future studies.