Microbiota Composition and Evenness Predict Survival Rate of Oysters Confronted to Pacific Oyster Mortality Syndrome

Abstract

Pacific Oyster Mortality Syndrome (POMS) affects Crassostrea gigas oysters worldwide and causes important economic losses. Disease dynamic was recently deciphered and revealed a multiple and progressive infection caused by the Ostreid herpesvirus OsHV-1 μVar, triggering an immunosuppression followed by microbiota destabilization and bacteraemia by opportunistic bacterial pathogens. However, it remains unknown if microbiota might participate to protect oysters against POMS, and if microbiota characteristics might be predictive of oyster mortalities. To tackle this issue, we transferred full-sib progenies of resistant and susceptible oyster families from hatchery to the field during a period in favor of POMS. After 5 days of transplantation, oysters from each family were either sampled for individual microbiota analyses using 16S rRNA gene-metabarcoding or transferred into facilities to record their survival using controlled condition. As expected, all oysters from susceptible families died, and all oysters from the resistant family survived. Quantification of OsHV-1 and bacteria showed that 5 days of transplantation were long enough to contaminate oysters by POMS, but not for entering the pathogenesis process. Thus, it was possible to compare microbiota characteristics between resistant and susceptible oysters families at the early steps of infection. Strikingly, we found that microbiota evenness and abundances of Cyanobacteria (Subsection III, family I), Mycoplasmataceae, Rhodobacteraceae, and Rhodospirillaceae were significantly different between resistant and susceptible oyster families. We concluded that these microbiota characteristics might predict oyster mortalities.

Keywords: bacteria; fitness; holobiont; metabarcoding; microbiome.

FIGURE 1

Experimental design. Three oyster full-sib families were produced in controlled condition (hatchery), and placed for 5 days in the environment (Atlantic Ocean, latitude: 48.335263; longitude: –4.317922) during an infectious period (July 2016). Then, oysters were flash frozen, DNA were extracted, and microbiota were sequenced using 16S rRNA gene-metabarcoding. Oysters deployed in the field during 5 days were followed during 8 days in the hatchery. Survival rates were recorded at endpoint.

FIGURE 2

OsHV-1 and bacterial colonization in oysters at day 5 post-transplantation in field condition during an infectious period. (A) The OsHV-1 load was quantified by qPCR and expressed as copies of DNA polymerase catalytic subunit of OsHV-1 per ng of total oyster DNA. Relative quantification of total bacteria (B), and total vibrios (C) abundance were measured by qPCR (n = 42).

FIGURE 3

Frequencies of known opportunistic bacterial genera associated with POMS. (A) Positive oysters for OsHV-1 detection (SF15.I.R14, SF32.I.R08, and SF32.I.R14). (B) Negative oysters for OsHV-1 detection for each family. These opportunistic genera were identified in a previous experimental infection (de Lorgeril et al., 2018). Frequencies above and below 4% are displayed in red and blue, respectively (n = 42).

FIGURE 4

Comparison of alpha diversity indices between oyster families in controlled (hatchery) and infectious (field) conditions. P-values correspond to global tests. Horizontal lines above boxplots indicate significant pairwise comparisons between group levels (post hoc analyses) (n = 80).

FIGURE 5

Microbiota compositions between oyster families in controlled (hatchery) and infectious (field) conditions. (A) Principal coordinate analysis (Bray–Curtis dissimilarities between samples). Each dot corresponds to one individual. Colors correspond to oyster families in controlled (C) and infectious (I) conditions. Labels are displayed at the barycenter of dots. (B) Multivariate homogeneity of group dispersions. P-value corresponds to the global test. Horizontal lines above boxplots indicate significant pairwise comparisons between group levels (post hoc analyses) (n = 80).

FIGURE 6

Heatmap of bacterial families that were significantly different between resistant and susceptible oyster families in the controlled condition (hatchery). Only bacterial families with a frequency above 4% in at least one sample are shown. Frequencies above and below 4% are displayed in red and blue, respectively (n = 41).

FIGURE 7

Heatmap of bacterial families that were significantly different between resistant and susceptible oyster families in the infectious condition (field). Only bacterial families with a frequency above 4% in at least one sample are shown. Frequencies above and below 4% are displayed in red and blue, respectively (n = 39).