白斑综合征病毒对红螯光壳螯虾幼虾肝胰腺免疫酶活性及其超微结构的影响

利用生物化学方法和电镜技术,研究人工注射免疫多糖及WSSV对红螯光壳螯虾幼虾肝胰腺POD、LSZ、SOD、PO的活性变化及肝胰腺超微结构的影响。试验分对照组、实验组Ⅰ(注射WSSV)、实验组Ⅱ(注射免疫多糖)、实验组Ⅲ(注射免疫多糖48 h后注射WSSV)4组,结果显示,随着处理时间的增加,实验组Ⅰ与对照组相比POD、PO、LSZ活性均呈现明显降低趋势(P<0.01),而SOD活性呈现先升高后降低的趋势(P<0.01);实验组Ⅱ的螯虾4种酶活性呈现先升后降,与对照组相比酶活性增加(P<0.05);实验组Ⅲ4种酶活性均高于实验组Ⅰ(P<0.05),但与对照组相比SOD、POD、LSZ活性降低(P<0.01)。红螯光壳螯虾幼虾肝胰腺组织由肝小管组成,肝小管由基膜和上皮细胞组成。超微结构显示,对照组螯虾幼虾肝胰腺单层柱状上皮细胞的表面微绒毛排列整齐,各细胞器结构完整;实验组Ⅰ上皮细胞微绒毛受损、断裂,核膜解体,细胞核破裂,粗面内质网断裂;实验组Ⅱ上皮细胞粗面内质网核糖体增多;实验组Ⅲ与实验组Ⅰ相比,细胞核结构完整,粗面内质网肿胀,线粒体部分畸变。结果说明感染WSSV的幼虾肝胰腺形态结构受损,并进一步影响其生物学功能。人工注射免疫多糖能提高幼虾免疫相关酶活性,在一定程度上能抵抗WSSV的侵染。

WSSV stress on the immunoe-related enzyme and ultrastructure of hepatopancreas in juvenile Cherax quadricarinatus

In present study,the enzymatic activities of the peroxidase(POD),lysozyme(LSZ),superoxide dismutase(SOD)and phenoloxidase(PO)have been monitored by use of biochemical techniques.Meanwhile,the transmission electron microscopy(TEM)method has been applied to compare the morphological changes in hepatopancreas of juvenile redclaw crayfish,Cherax quadricarinatus,under two experimental conditions:one manual injection of immune polysaccharide;two White Spot Syndrome Virus(WSSV)stress exposure.Four experimental groups have been set up in this study:control group[with PBS(pH 7.4)injection];experimental group Ⅰ(with WSSV injection);experimental group Ⅱ(with immune polysaccharides injection);experimental group Ⅲ(with immune polysaccharides injection first and with WSSV injection after 48 hours).The results of biochemical studies showed that:in comparison with the control group,the activities of POD,PO,LSZ in experimental group Ⅰ were decreasing significantly(P<0.01)in 72 hours.In contrast,the activity of SOD in experimental group Ⅰ was increasing first and then decreasing,the enzyme activity was significantly lower(P<0.01)in 72 hours;in the experimental group Ⅱ,the enzymatic activities of POD,PO,LSZ,SOD were all increasing first and then decreasing.Their enzymatic activities were all higher than the control group(P<0.05).In detail,the SOD and LSZ activity reached the threshold after 24 hours immune polysaccharide injection,the POD had the highest activity in 48 hours,the maximum activity of PO reached after 12 hours injection;experimental group Ⅲ,the enzymatic activities of POD,PO,LSZ,SOD were all higher than experimental group Ⅰ(P<0.05)after 72 hours exposure.However,in comparison with the control group,the SOD,POD,LSZ activities were decreasing,with only PO enzymatic activity increasing first and then decreasing,four enzymes,activities were all decreasing significantly(P<0.01)in 72 hours in contrast with the control group.Four enzymatic activities in experimental group Ⅱ is greater than control group and enzyme activity in experimental group Ⅲ is greater than experimental group Ⅰ and this proves a direct evidence that the immune polysaccaride injection can improve the resistance of juvenile prawn to WSSV and be able to actively suppress the infection with WSSV.Moreover,the morphological study in hepatopancreas of C.quadricarinatus showed that:in control group,the hepatopancreas is composed of liver-tubules,the liver-tubules is made up of simple columnar epithelium cells and basement layers,with the simple columnar epithelial cells the neat microvilli on the surface and the liver-tubules epithelial cells structurally-complete.In experimental group Ⅰ,the mitochondria were seriously harmed and had abnormality;the karyotheca disassembly,substance external flow in nucleolus and cell nucleus become torn,at the same time the cell nucleus had the chromosome heterochromatin phenomenon.In addition,more serious of the rough endoplasmic reticulum becoming split liminated structure and ribosome is to reduce in experimental group Ⅰ;In experimental group Ⅱ,the cell nucleus chromatin uniform distribution on the rough endoplasmic reticulum(RER)ribosome showed an increase;In experimental group Ⅲ,RER become tumescence and few split.Meanwhile,the number of vesica increased,and the vacuole were also found increased.Certain portion of mitochondria turned into abnormality,regular ellipse or length structure mitochondria develop into irregular.Our results indicate that WSSV stress can damage the ultrastructure of hepatopancreas in juvenile C.quadricarinatus,and further influence the normal physiological function of C.quadricarinatus.Present study by biochemical techniques and TEM method further shows that the immune polysaccharides can promote the resistance of juvenile prawn to the WSSV infection.