Figure 2.

Impact of anti-microbiota vaccine and P. relictum infection on bird antibody response and mosquitoes’ microbial diversity (A). Levels of IgY specific to E. coli proteins were measured by semiquantitative ELISA in sera of canaries before immunization (day 0) and in sera of canaries immunized with a mock vaccine, E. coli BL21 and E. coli O86:B7 alone or challenged with P. relictum (day 36 after first vaccination) (****p < 0.00001; ns, not significant). (B) Faith’s phylogenetic diversity and (C) Pielou’s evenness indexes were used to measure the richness and evenness, respectively, of microbiota of mosquitoes fed on canaries immunized with a mock vaccine, E. coli BL21 and E. coli O86:B7 alone or challenged with P. relictum. The asterisk indicates significative differences between groups (pairwise Kruskal-wallis, p = 0.03). (D) Beta diversity of mosquito microbiota in the different experimental conditions represented in PCoA plot obtained by Betadisper function. There were no differences in the intragroup dispersions (variances) (ANOVA, p = 0.426), whereas differences were found in the community composition between groups (PERMANOVA, p = 0.001), specifically the group infected with P. relictum alone was different from the other groups. Taxonomic profiles at the level of ASVs, used to measure the alpha and beta diversity, were obtained from 16S rRNA sequences from midgut of mosquitoes fed on mock-immunized (n = 5 mosquitoes midgut pool), E. coli O86:B7-immunized (n = 5 mosquitoes midgut pool), E. coli O86:B7-immunized and challenged with P. relictum (n = 8 mosquitoes midgut pool) and challenged only with P. relictum (n = 8 mosquitoes midgut pool).