氨氮胁迫对日本蟳免疫生理指标及器官结构的影响

Effects of ammonia-N stress on immunity-related indicators and histological structure of some organs of the marine crab,Charybdis japonica(A.Milne-Edwards)

采用生物酶测定及组织学方法,研究了水体中不同浓度氨氮胁迫下日本蟳免疫相关指标的变化,以及对鳃、肝胰腺和胃等器官结构的影响。结果表明:在氨氮胁迫下,各实验组的血细胞密度(DHC)、血蓝蛋白含量、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活力,以及低浓度胁迫组的酚氧化酶(PO)和溶菌酶(LSZ)活力呈"先升后降"的变化趋势;高浓度组LSZ活力持续下降。在低浓度氨氮胁迫下,过氧化代谢产物丙二醛(MDA)含量呈"先升后降再升",而高浓度氨氮胁迫导致MDA含量持续上升。胁迫第15天,除低浓度组的DHC、血蓝蛋白含量和PO活力略高于对照组外,其他实验组所测指标均低于对照组;PO、LSZ、SOD和CAT等酶活力以及DHC均与氨氮胁迫浓度呈显著负相关,MDA含量则与胁迫浓度呈显著正相关(P<0.01)。高浓度氨氮胁迫会导致鳃几丁质膜变薄、断裂,鳃上皮排列紊乱、染色质异固缩;鳃腔中血淋巴减少,密度降低,血细胞凝集、质膜破裂,胞质严重空泡化;肝胰腺上皮形态不规则,B细胞减少,腺细胞出现大量空泡,染色质凝聚;胃几丁质膜断裂,胃上皮排列不规则,胞质中出现大量残余体。研究表明高浓度氨氮胁迫对日本蟳免疫相关指标和器官结构产生显著影响,SOD活力和MDA含量长期变化情况可作为衡量日本蟳在氨氮胁迫下免疫状态的指标。

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.