Experimental Infection of the Biomphalaria glabrata Vector Snail by Schistosoma mansoni Parasites Drives Snail Microbiota Dysbiosis

Abstract

Host-parasite interaction can result in a strong alteration of the host-associated microbiota. This dysbiosis can affect the fitness of the host; can modify pathogen interaction and the outcome of diseases. Biomphalaria glabrata is the snail intermediate host of the trematode Schistosoma mansoni, the agent of human schistosomiasis, causing hundreds of thousands of deaths every year. Here, we present the first study of the snail bacterial microbiota in response to Schistosoma infection. We examined the interplay between B. glabrata, S. mansoni and host microbiota. Snails were infected and the microbiota composition was analysed by 16S rDNA amplicon sequencing approach. We demonstrated that the microbial composition of water did not affect the microbiota composition. Then, we characterised the Biomphalaria bacterial microbiota at the individual scale in both naive and infected snails. Sympatric and allopatric strains of parasites were used for infections and re-infections to analyse the modification or dysbiosis of snail microbiota in different host-parasite co-evolutionary contexts. Concomitantly, using RNAseq, we investigated the link between bacterial microbiota dysbiosis and snail anti-microbial peptide immune response. This work paves the way for a better understanding of snail/schistosome interaction and should have critical consequences in terms of snail control strategies for fighting schistosomiasis disease in the field.

Keywords: Biomphalaria snail; Schistosoma infection; bacteria; dysbiosis; immune response; microbiota.

Figure 1

Experimental protocol.

Figure 2

Biomphalaria glabrata microbiota characterization: Biomphalaria bacterial microbiota of six naive snails recovered at the starting of the experimentation (B0.1; B0.2; B0.3; B0.4; B0.5 and B0.6) and 6 naive snails recovered 25 days after the starting of the experimentation (B25.1; B25.2; B25.3; B25.4; B25.5 and B25.6) were analysed. (A). Phylum level composition of the 20 most abundant OTUs phyla among the 12 naive snails. (B). The Venn diagram represents the number of the 97 OTUs families, which shared between the 6 naive snails at B0 (left Venn diagram), and between the 6 naive snails at B25 (right Venn diagram).

Figure 3

Microbiota alpha Diversity: Boxplots of Chao1 and Shannon indices for all samples. For the Naive condition, B0 and B25 snails were pooled; BB: primary infection of BgBRE by SmBRE; BV: primary infection of BgBRE by SmVEN; BBB: primary infection of BgBRE by SmBRE and secondary challenge by SmBRE; BBV: primary infection of BgBRE by SmBRE and secondary challenge by SmVEN. The time point is mentioned with 1, 4 or 25 corresponding to the day after primary infection or secondary challenge. The differences between naive and infected conditions were tested with a Mann–Whitney U test and significant differences mentioned with *.

Figure 4

Beta diversity and bacterial communities following Biomphalaria infection: Dynamics of the bacterial microbiota of Biomphalaria glabrata following Schistosoma primary infection and secondary challenge. (A). Functional diversity comparisons of Biomphalaria microbiota along the infection. Principal coordinate analysis of pairwise Bray–Curtis distances across all infection type and time samples. Axes represent the two synthetic variables explaining the greatest proportion of variation in the data set. The sample name indicated in the figure corresponds to the centroid of all the biological replicates points of the respective experimental sample. (B). Phylum level composition of the 20 most abundant OTUs among all points of the kinetic. In this representation, the replicate naive snails were pooled for more readability.

Figure 5

Dynamic of Specific Phylum during Biomphalaria infection: Dynamic of the number of OTU for specific Phylum according to 2 parameters. The time of infections: Day 1 (1 DPPI (=Day Post Primary Infection)) vs. Day 4 (4 DPPI) Primary infection and Day 1 (1 DPC (= Day Post-Secondary challenge)) and Day 4 (4 DPC) Secondary challenge. The type of infections: Sympatric (BB) vs. Allopatric (BV) Primary infections and Homologous (BBB) vs. Heterologous (BBV) Secondary challenge. (A). Phylums influenced by the time of infections. (B). Phylum influenced by the type of infections. (C). Phylum influenced by both time and type of infections. The Mann–Whitney U test is used to test the significant difference between the time or the type in Primary infections and Secondary challenge and mentioned with *.

Figure 6

Differential gene expression of Biomphamacin antimicrobial peptides: Log2FC (fold change) of antimicrobial immune transcripts between naive and infected snails inferred from previous RNAseq analysis on the same experiment. A positive Log2 fold-change indicates over-expression in infected snails compared to the naive snails. Antimicrobial peptide families included 6 Biomphamacins (macin-like AMPs) consisting of 6 genes (shade of green).