Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
DNA oligo nucleotides used in this study.
Fig 1.
Pathology of Pacific oyster larvae before and after infection with V. coralliilyticus strain OCN014.
(A) A sedentary, uninfected control larva with internal structures visible through the transparent shell and (B) actively swimming, uninfected larvae with smooth velum and ordered cilia. (C) A diseased larva with disfigured or clumped velum (CV) and disorganized cilia (DC). (D) Diseased larvae with release of the velar cells (VC) as the velum disassociates. (E) A diseased larva with substantially degraded velum and disorganized cilia. Most of the velar cells have been released into the seawater. (F) Dead larva without visible internal structures. The black scale bar represents 50 μm and applies to all images.
Fig 2.
Protruding velum of oyster larvae maintained at elevated temperature.
An unusual protruding velum (PV) in Pacific oyster larvae was occasionally noted after infection with all four V. coralliilyticus strains at 27°C. Pictured here are representative larvae infected with (A) RE98 and (B) OCN008 24 h post-inoculation. Scale bar is 50 μm and applies to both images.
Fig 3.
Percent mortality of oyster larvae 72 h post-inoculation with V. coralliilyticus strains.
Larvae exposed to varying concentrations of V. coralliilyticus strain (A) RE98, (B) OCN008, (C) OCN014, (D) BAA-450, (E) Vibrio sp. strain HMSC5, and (F) V. tasmaniensis strain LGP32. Light gray bars represent the mean counts from larvae incubated at 23°C; dark gray bars represent larvae at 27°C. Control wells were inoculated with sterilized seawater. An asterisk (*) indicates that larval counts are significantly different from the controls (2-way ANOVA, p < 0.05, n = 12). A dagger (†) indicates the larval counts are significantly different for experiments at 23°C versus 27°C (2-way ANOVA, p < 0.05, n = 12).
Fig 4.
Percent mortality of C. gigas larvae exposed to OCN008 mutants not virulent to coral.
Larvae were exposed to OCN008, the OCN008 ΔMSHA strain, the OCN008 ΔtoxR strain, or the OCN008 ΔompU strain. Control larvae were inoculated with (A) sterile seawater while test larvae were inoculated with strains of V. coralliilyticus to a final bacterial concentration of (B) 102, (C) 103, (D) 104, (E) 105, or (F) 106 CFU/ml of seawater. Light gray bars represent the mean counts from larvae incubated at 23°C; dark gray bars represent larvae at 27°C. An asterisk (*) indicates larval counts significantly different from the uninoculated control averages (2-way ANOVA, p < 0.05, n = 12). A dagger (†) indicates the larval counts are significantly different for experiments at 23°C versus 27°C (2-way ANOVA, p < 0.05, n = 12).
Fig 5.
Percent mortality of oyster larvae exposed to genetically complemented OCN008 mutants.
Larvae were exposed to 104 CFU/ml of OCN008, OCN008 with the empty expression vector (pBU246), the ΔtoxR strain, the ΔtoxR strain with pBU246, the ΔtoxR strain with the toxR complementation vector (pBU248), the ΔompU strain, the ΔompU strain with pBU246, or the ΔompU strain with the ompU complementation vector (pBU249) for 72 h. Control larvae were inoculated with sterile seawater while test larvae were inoculated with strains of V. coralliilyticus to a final bacterial concentration of 105 CFU/ml of seawater. Light gray bars represent the mean counts from larvae incubated at 23°C; dark gray bars represent larvae at 27°C. An asterisk (*) indicates larval counts significantly different from the uninoculated control averages (2-way ANOVA, p < 0.05, n = 12). A dagger (†) indicates the larval counts are significantly different for experiments at 23°C versus 27°C (2-way ANOVA, p < 0.05, n = 8).
Fig 6.
Representative photographs of M. capitata fragments used in infection experiments.
(A) A coral fragment before exposure to OCN008, and (B) the same coral fragment 24 h post-exposure to OCN008 exhibiting extensive tissue loss/lysis. (C) A fragment before exposure to the ΔompU strain of OCN008, and (D) the same fragment 240 h post-exposure to the ΔompU strain. (E) A fragment before exposure to the control bacterium HMSC5, and (F) the same fragment 240 h post-exposure to HMSC5. The white square grating measures 1 x 1 cm.