Charybdis japonica is an important marine crab, that is widely distributed in the China sea area. Enzymatic and histological methods were used to analyze changes in the activities of immune-related enzymes in the blood serum, and in the histology of major organs of this crab exposed to different levels of ammonia-N stress. The primary objective of the study was to provide a theoretical basis for research into ammonia-N toxicity in relation to the control of water quality in artificial cultures of this crab.
The crabs were exposed to ammonia-N concentrations of 0, 2, 5, 10, 15, and 20 mg/L for up to 15 days. After times of 0, 1, 3, 5, 10, and 15 days, blood samples were taken for measurement of changes in immunity-related indicators, including the density of hemocytes (DHC); hemocyanin concentration; the activities of phenoloxidase (PO), lysozyme (LSZ), superoxide dismutase (SOD) and catalase (CAT); and the malondialdehyde (MDA) concentration. On day 15, samples were taken of the gills, hepatopancreas and stomach tissue of the crabs in the 20 mg/L of ammonia-N medium, for observations of histological changes.
Many of the measured indices showed an initial increase in response to ammonia-N stress, which was followed by a decreasing trend over time. This was observed in the values of DHC and hemocyanin concentration, in the activities of the SOD and CAT in all treatments, and in the activities of the PO and LSZ in the experimental groups at low ammonia-N concentrations. In contrast, the LSZ activity declined continuously in crabs at high concentration of ammonia. At low ammonia-N concentrations, the MDA content initially increased, and then decreased, and finally rose again. At higher concentrations, MDA continuously increased. After exposure to ammonia-N for 15 days, most of the measured indices were lower in the ammonia-N treatments than in the control group, with the exception of DHC, hemocyanin concentration, and PO activity, which were slightly higher. There were significant negative correlations between ammonia-N concentration and DHC, and ammonia concentration and the activities of PO, LSZ, SOD, and CAT. In contrast, the MDA content was positively correlated with the ammonia-N concentration (P < 0.01). Noticeable changes were observed in the histological structure of some organs in crabs exposed to high concentrations of ammonia-N. The chitin layer of the gills became thin and was partly ruptured, the epithelial lamina became disorganized, and the chromatin condensed. The quantity of hemolymph in the gill cavity was reduced and its density decreased. Hemocytes were condensed with ruptured cytomembranes and abundant vacuoles in cytoplasm. The glandular epithelium exhibited an irregular morphology, the number of secretory cells (B-cells) decreased and numerous vacuoles appeared. There were fewer organelles and the chromatin was condensed. The chitin layer of the stomach ruptured and the gastric epithelium became disordered, with abundant residual bodies formed in the cytoplasm.
We conclude that high concentrations of ammonia-N have significant effects on the activities of immune-related enzymes and on organ morphology in Charybdis japonica. Changes in the activities of SOD and in the content of MDA, may be used as indices for evaluating the immune state of C. japonica under ammonia-N stress.