Virulence of Vibrio alginolyticus Accentuates Apoptosis and Immune Rigor in the Oyster Crassostrea hongkongensis

Vibrio species are ubiquitously distributed in marine environments, with important implications for emerging infectious diseases. However, relatively little is known about defensive strategies deployed by hosts against Vibrio pathogens of distinct virulence traits. Being an ecologically relevant host, the oyster Crassostrea hongkongensis can serve as an excellent model for elucidating mechanisms underlying host-Vibrio interactions. We generated a Vibrio alginolyticus mutant strain (V. alginolyticus△vscC) with attenuated virulence by knocking out the vscC encoding gene, a core component of type III secretion system (T3SS), which led to starkly reduced apoptotic rates in hemocyte hosts compared to the V. alginolyticusWT control. In comparative proteomics, it was revealed that distinct immune responses arose upon encounter with V. alginolyticus strains of different virulence. Quite strikingly, the peroxisomal and apoptotic pathways are activated by V. alginolyticusWT infection, whereas phagocytosis and cell adhesion were enhanced in V. alginolyticus△vscC infection. Results for functional studies further show that V. alginolyticusWT strain stimulated respiratory bursts to produce excess superoxide (O2•−) and hydrogen peroxide (H2O2) in oysters, which induced apoptosis regulated by p53 target protein (p53tp). Simultaneously, a drop in sGC content balanced off cGMP accumulation in hemocytes and repressed the occurrence of apoptosis to a certain extent during V. alginolyticus△vscC infection. We have thus provided the first direct evidence for a mechanistic link between virulence of Vibrio spp. and its immunomodulation effects on apoptosis in the oyster. Collectively, we conclude that adaptive responses in host defenses are partially determined by pathogen virulence, in order to safeguard efficiency and timeliness in bacterial clearance.

Hemocyte Responses of the Oyster Crassostrea hongkongensis Exposed to Diel-Cycling Hypoxia and Salinity Change

Marine hypoxia caused by nutrient enrichment in coastal waters has become a global problem for decades, especially diel-cycling hypoxia that occurs frequently in the summer season. On the contrary, sudden rainstorms, and freshwater discharge make salinity in estuarine and coastal ecosystems variable, which often occurs with hypoxia. We found mass mortality of the Hong Kong oyster Crassostrea hongkongensis in the field where hypoxia and salinity fluctuation co-occur in the summer season during the past several years. To investigate the effects of diel-cycling hypoxia and salinity changes on the hemocyte immune function of C. hongkongensis, oysters were exposed to a combined effect of two dissolved oxygen (DO) concentrations (24 h normal oxygen 6 mg/L, 12 h normal oxygen 6 mg/L, and 12 h hypoxia 2 mg/L) and three salinities (10, 25, and 35‰) for 14 days. Subsequently, all treatments were restored to constant normal oxygen (6 mg/L) and salinity under 25‰ for 3 days to study the recovery of hemocyte immune function from the combined stress. Hemocyte parameters were analyzed by flow cytometry, including hemocyte mortality (HM), total hemocyte count (THC), phagocytosis (PHA), esterase (EST) activity, reactive oxygen species (ROS), lysosomal content (LYSO), and mitochondrial number (MN). The experimental results showed that diel-cycling hypoxia and salinity changes have obvious interactive effects on various immune parameters. In detail, diel-cycling hypoxia and decreases in salinity led to increased HM, and low salinity caused heavier impacts. In addition, low salinity, and diel-cycling hypoxia also led to decreases in LYSO, EST, and THC, while the decrease of PHA only occurs in the early stage. On the contrary, ROS production increased significantly under low salinity and hypoxic conditions. After 3-day recovery, THC, PHA, EST, LYSO, and MN were basically restored to normal, while HM and ROS were still significantly affected by diel-cycling hypoxia and salinity change, indicating that the combined stress of diel-cycling hypoxia and salinity changes had latent effects on the immune function of C. hongkongensis. Our results highlight that diel-cycling hypoxia and salinity change may impair the health and survival of the Hong Kong oyster C. hongkongensis and may be the key factors for the mass mortality of this oyster in the field.

Gender Differences in Hemocyte Immune Parameters of Hong Kong Oyster Crassostrea hongkongensis During Immune Stress

Gender differences in individual immune responses to external stimuli have been elucidated in many invertebrates. However, it is unclear if gender differences do exist in the Hong Kong oyster Crassostrea hongkongensis, one of the most valuable marine species cultivated along the coast of South China. To clarify this, we stimulated post-spawning adult C. hongkongensis with Vibrio harveyi and lipopolysaccharide (LPS). Gender-based differences in some essential functional parameters of hemocytes were studied via flow cytometry. Obvious gender-, subpopulation-, and immune-specific alterations were found in the hemocyte immune parameters of C. hongkongensis. Three hemocyte subpopulations were identified: granulocytes, semi-granulocytes, and agranulocytes. Granulocytes, the chief phagocytes and major producers of esterase, reactive oxygen species, and nitric oxide, were the main immunocompetent hemocytes. Immune parameter alterations were notable in the accumulation of granulocyte esterase activities, lysosomal masses, nitric oxide levels, and granulocyte numbers in male oysters. These results suggest that post-spawning-phase male oysters possess a more powerful immune response than females. Gender and subpopulation differences in bivalve immune parameters should be considered in the future analysis of immune parameters when studying the impact of pathogenic or environmental factors.