| The Chinese shrimp(Fenneropenaeus chinensis)“Huanghai No. 2†were selected as the experimental subject. The WSSV, which has been inactivated by dry-heating, 60 Co γ-ray and BEI, is used to induce the immune priming of Chinese shrimp. This present study was aimed to analyze the influence of different inactivated ways on the death rate, mean survival time and the relative percent survival of F. chinensis which were infected again with WSSV. We analyzed the relative expression level of Dscam, which was supposed to have close connection with the immune priming, at spatial-temporal and organization level through real-time PCR. We hoped that we could explore the mechanism of immune priming response in F. chinensis and find new ways for the prevention of shrimp disease. The concrete research content is as follows:This study was designed to explore whether the heat-inactivated WSSV could induce immune priming response or not in F. chinensis. During the initial 6 days, four experiment groups(E15℃, E23℃, E28℃, E32℃) were fed with typical symptom of WSSV prawn muscle, which was dealt with at 60℃ for 1 h by the equivalent and quantitative test method. At the same time, we set a positive control group C23℃(fed with untreated WSSV muscle) and a control group CF23℃(fed with commercial compound feed only). At the 13 th day of the experiment, all shrimps were infected with WSSV again. The result showed that 60℃ could decrease the activity of WSSV absolutely. And there were no dead shrimp in the initial 12 d, but the mortality of positive control group C23℃ was 100%. The survival rates of different experiment(E15℃, E23℃, E28℃, E32℃) and control groups were 80.41%, 33.29%, 8.47%, 16.43% and 8.89%, respectively, the survival rate of E15 ℃was significantly different from other experiments(P<0.01) after 19 days. In addition, there was a significant difference between E23 ℃ experiment group and other experiment groups(P < 0.05). Nevertheless, there was no difference between E28 ℃ and E32 ℃ groups(P>0.05). Compared to the results of absolute quantitation, it was found that the multiplication rate of WSSV was the highest under 28℃. High(32℃) and low(15℃) temperature would inhibit the multiplication of WSSV in the shrimps which accepted the subsequent re-challenge of WSSV after the heat-inactivated WSSV induced. Experimental results show that heat-inactivated WSSV could induce immune priming response and provide some protection to the infected shrimp. The multiplication rate of WSSV is closely related to the temperature.We used the rapid amplification of c DNA ends(RACE) to clone the cDNA sequence of F. chinensis Dscam gene, then analyzed the features of gene by bioinformatics software. The full length of Dscam cDNA was 6624 bp. It contained a 5’-untranslate region of 171 bp, a 3’-untranslate region of 459 bp, and an opening reading frame of 5994 bp which encoded 1997 amino acids. The gene was named as FcDscam. The protein encoded by FcDscam contains one signal peptide, one cytoplasmic tail, one transmembrane domain, six of fibronectin type Ⅲ(FNⅢ) and ten of immunoglobulin(Ig). The analysis of the homology search and neighbor-joining phylogenetic tree showed that the highest similarity occurs between Litopenaeus vannamei and F. chinensis, reaching 92.4%, and these two species were belong to the same branch of a bigger trunk which contains other crustacean species such as Cherax quadricarinatus, Pacifastacus leniusculus, Carcinus maenas and Eriocheir sinensis. The shrimps were feed heat inactivated WSSV bait for six days to induce the immune response and infected again 12 days later. The shrimps were sampled at 0 d, 6 d, 12 d of first-time infection and 12 h, 24 h, 48 h, 72 h, 168 h of second-time infection. The mRNA expression level of FcDscam were analyzed by Realtime PCR. On the 12 th day, the expression level began to increase and showed significant difference with the control group(P<0.05). After 24 h of second-time infection, the expression level reached a maximum value and showed extremely significant with the control group(P<0.01). The expression level started to decrease at the 48 h of secondtime infection. It was showed that the Dscam gene exists in Chinese shrimp, and played an important role in the process of immune sensitization.In order to explore how to induce the immune priming response effectively in shrimp and other invertebrates, the muscle were inactivated which is obtained from the shrimp with typical WSSV symptom by γ-radiation, then the shrimps were fed on muscle for 10 days. The WSSV suspension was inactivated by adding BEI solution, the shrimps were given twice injections at the 1st and 6th day of the experiment with the inactivated suspension solution. After 10 d, shrimps induced by different kinds of inactivated methods of feeding and injection, respectively, and the positive control and negative control were set respectively. The results showed that the two ways of WSSV inactivation can induce the immune priming. At 7th day after subsequent re-challenge of WSSV, the relative protection rate was 20.94%(feeding), 13.64%(Injection) ofγ-radiation group, and the relative protection rate were 45.23%(feeding), 11.11%(Injection). We set sampling points at 0 d, 3 d, 10 d at the inducing stage and 0 h(10 d), 6 h, 12 h, 24 h, 48 h at the infection stage in the group of BEI inactivated. Then the spatial-temporal and organization expressional characteristics through real-time PCR were analyzed. The results showed that the expression of Dscam in hemolymph rose up at 3 d of first inducing, and reached the maximum value after 10 d of inducing stage. There was a significant difference between the BEI inducing group and the control groups(P < 0.05). The expression of Dscam kept at high level after the second feeding and injection. The expression of Dscam in the survival shrimps reached the maximum value. In conclusion, these two methods could inactivate the WSSV effectively and induce the immune priming of F. chinensis, and the immune priming could provide protection to feeding and injection infection. Dscam could be induced to be raised by inactivated WSSV, which suggested that Dscam may be closely related to immune priming. |
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