Pathological analysis of hemolymphs of Charybdis japonica infected with Vibrio alginolyticus

• Microscopic and ultrastructural analysis of hemocyte pathology of Charybdis japonica infected with Vibrio alginolyticus.
• V. alginolyticus induces innate immune enzyme activities in C. japonica.
• Polysaccharides confer immunoprotection against V. alginolyticus in C. japonica.

We explored the pathogenic mechanism of Vibrio alginolyticus in the stone crab Charybdis japonica by studying the hemolymph of C. japonica artificially infected by V. alginolyticus. To this end, Wright-Geimsa staining and electron microscopy were used, and phenoloxidase (PO) activity and the immune protection rate of C. japonica injected with immune polysaccharide during infection were analyzed. The results indicated that the total hemocyte and hyaline hemocyte (HH) counts in diseased crabs were significantly lower than those in healthy crabs (P < 0.05), whereas the large granule hemocytes (LGHs) were significantly higher in diseased crabs than in healthy crabs (P < 0.05). The cellular sizes of HHs and LGHs showed an increasing trend after V. alginolyticus infection, while the nuclear/cytoplasmic ratio (NP) of these cells showed a sharp decline after V. alginolyticus infection (P < 0.05). Micro-pathological analysis of hemocytes revealed fewer hemocytes in the hemolymph of diseased crabs and the presence of disintegrated cells. Ultrastructural and micro-pathological analyses showed damage in all types of hemocytes. The mitochondria were damaged and incomplete in structure, parts of the nuclear membrane were anamorphic and parts of the nuclei had shrunk, hematocyte nuclei exhibited heterochromatinization, hemocyte granules were increased in the polysaccharide-treated group infected with V. alginolyticus, and the numbers of mitochondria and rough endoplasmic reticulum were also increased. PO activity in the two Vibrio-infected groups peaked at 6 h and 24 h after infection, respectively, and PO activity increased in the hemolymph of infected crabs but sharply decreased with prolonged infection. Finally, the PO activities in the two Vibrio-infected groups were significantly lower than controls at 120 h post-infection (P < 0.05). Interestingly, PO activity was higher in polysaccharide-treated crabs than non-polysaccharide-challenged infected crabs, resulting in an immunoprotective rate of 69.64% at 7 days post-infection. This phenomenon suggests that polysaccharides could enhance the organism's antibacterial defenses by improving immune-related enzyme activity.