Host Defense Effectors Expressed by Hemocytes Shape the Bacterial Microbiota From the Scallop Hemolymph

Abstract

The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.

Keywords: Vibrio; bactericidal/permeability-increasing protein; big defensin; host defense effectors; host-microbiota interactions; invertebrate immunity; mollusk.

Figure 1

Characterization of bacterial communities present in hemolymph from single scallops determined by 16S rDNA deep amplicon sequencing. Upper panel: Venn diagrams indicate the number of common and distinct bacterial groups at phylum (A), class (C), and genus (E) levels between five non-stimulated individuals. Lower panel: Relative abundances of common bacterial groups at phylum (B), class (D), and genus (F) levels. Bacterial groups with relative abundances higher or closer to 1% are shown for each taxonomic level.

Figure 2

Relative abundances of bacterial groups present in scallop hemolymph after immune stimulation determined by 16S rDNA deep amplicon sequencing. Stack bar graphs indicate the average relative abundance of major bacterial groups found in Vibrio-injected scallops (VS) and seawater-injected scallops (SW) after 48 h and 168 h, at class (A) and genus (B) taxonomic levels. Bacterial groups with relative abundances higher or closer to 1% are shown for each taxonomic level.

Figure 3

Transcript expression of ApBD1, ApLBP/BPI1, ApLBP/BPI2, and ApGlys in scallop hemocytes during immune response. Bar graphs indicate the relative expression of antimicrobial effectors genes in Vibrio-injected scallops (VS) and seawater-injected scallops (SW) after 12, 24, 48, 72, and 168 h. SW-injected scallops were considered as injury control condition. Relative expression was calculated using non-stimulated scallops as control group, where gene expression values were considered 1. Graphed data are represented as the mean ± ES (n = 7). Asterisks indicate significant differences compared to SW-injected scallops (*P < 0.05).

Figure 4

Relative abundances of target bacterial groups present in scallop hemolymph after immune stimulation determined by qPCR and absolute quantification. Stack bar graphs indicate the average relative abundance of target bacterial groups in Vibrio-injected scallops (VS) and seawater-injected scallops (SW) at 24 h (A) and 48 h (B) after challenge. Number of total copies of specific bacterial 16S rDNA gene for Vibrio spp. at 24 h (C) and 48 h (D) after challenge are represented as the mean ± SE, and asterisks indicate significant differences compared to SW-injected scallops (***P < 0.005; *P < 0.05).

Figure 5

Effect of the silencing of ApBD1 and ApLBP/BPI1 expression in scallop by RNAi on the relative abundances of target bacterial groups. (A) Transcript expression of ApBD1 and ApLBP/BPI1 in scallop hemocytes injected with ApBD1-dsRNA (red bar) and ApLBP/BPI1dsRNA (blue bar) compared to GFP-dsRNA (gray bar), considered as control of specific gene silencing. Values are represented as the mean ± SE, considering the gene expression in GFP-dsRNA-injected animals as 100%. (B) Relative abundances of target bacterial groups present in the hemolymph of dsRNA injected scallops determined by qPCR and absolute quantification. (C) Number of total copies of specific bacterial 16S rDNA gene for Vibrio spp. present in dsRNA injected scallops. Values are represented as the mean ± SE and asterisks indicate significant differences compared with GFP-dsRNA-injected scallops (*P < 0.05, ***P < 0.005). (D) cultivable Vibrio spp. present in hemolymph from dsRNA injected scallops. Values are represented as the mean number of colony forming units/ml of hemolymph ± SE.