Fig 1.
Sites of Akoya pearl oyster collection (open circles).
Fig 2.
Strategy for analysis of shotgun metagenomic data.
Table 1.
Summary of sample characteristics for metagenomic analysis.
Fig 3.
Survivor adductor muscle a-value (bars) and cumulative mortality (plotted points) are shown for each group.
The groups are color coded as follows: positive control, black; negative control, white; sediment-inoculated groups, orange; and supernatant-inoculated groups, blue. Letters indicate statistically significant differences: a, cumulative mortality significantly different from the negative control (Fisher's exact test, p<0.05); b, cumulative mortality significantly different from the supernatant-injected group (Fisher's exact test, p<0.05); c, mean a-value significantly different from the supernatant control (Wilcoxon signed-rank test, p<0.05); and d, mean a-value significantly different from negative control (Wilcoxon signed-rank test, p<0.05).
Fig 4.
Relative abundance of identified reads in diseased oyster-specific sequences.
(A) Reads with significant similarity to sequences in the GenBank nr database. (B) Distribution of reads with similarities among major biological groups. (C) Distribution of reads among 10 major Bacteria phyla. (D) Distribution of reads mapping to viruses by host taxon. (E) Distribution of reads mapping to animal viruses by virus type. Parentheses indicate the type of virus genome.
Fig 5.
Effect of penicillin administration on cumulative mortality and a-value of the survivors.
The a-value of the survivors is represented with bars and cumulative mortality is plotted with open circles. Asterisks indicate significant differences between negative control groups (Wilcoxon signed-rank test, p-value<0.05).
Fig 6.
Bacterial diversity of the healthy and diseased pearl oysters.
Shannon alpha (A, B) and Bray-Curtis beta (C, D) diversity of the hemolymph (A, C) and mantle (B, D) microbes were evaluated using OTUs at the phylum level. Box plots show median, 25th, and 75th percentiles, and whiskers represent minimum and maximum values. Student’s t test; *p<0.05; **P<0.01.
Fig 7.
Normalized abundance distribution of each taxon at the phylum level from healthy and diseased pearl oyster in (A) hemolymph and (B) mantle.
Percentage of total sequence reads in samples from healthy and diseased pearl oyster of hemolymph and mantle is presented. The error bars show calculated standard deviation of the mean. Asterisks indicate significant difference between relative abundance for healthy and diseased oysters (Wilcoxon signed-rank test, P-value<0.05).
Fig 8.
Relative abundance of microbial communities in (A) hemolymph and (B) mantle of healthy and diseased individuals to phylum level.
Fig 9.
Phylogenetic tree for 16S rRNA gene sequences of putative causative agent and those of representative type species of phylum Spirochaetes.
Strain names and accession numbers of the 16S rRNA gene sequences follow the species name. The tree was constructed using the maximum-likelihood algorithm. Numbers at nodes represent the confidence limits estimated based on 1000 bootstrap replicates. The 16S rRNA gene sequence of Fusobacterium nucleatum subsp. nucleatum ATCC25586T was used to root the tree. Bar, 0.05 Knuc in nucleotide sequences.
Fig 10.
Fluorescence micrographs showing specific detection of bacteria cells from diseased pearl oyster hemolymph after (A) in situ hybridization and (B) fluorescence immunostaining.