Immune Responses Of Litopenaeus Vannamei Against White Spot Syndrome Virus After Oral Delivery Of VP28Using Nacillus Subtilis As Vehicles

White spot syndrome virus (WSSV) is currently the most serious viral pathogen of shrimp in China even worldwide, due to its high infectivity, wide host and high mortality. The shrimp culture industry has been severely hampered by outbreaks of WSSV. In previous work, we reported that the recombinant Bacillus subtilis strain with the ability of high-level secretion of VP28can evoke specific protection of crayfish and Fenneropenaeus chinensis against WSSV by oral delivery. However, the potential mechanism of this protective effect still remains to be studied for the further application. Therefore, this study examines the protective effect and immune responses of Iitopenaeus vannamei against WSSV after oral delivery of VP28using B. subtilis as vehicles (B.subtilis-VP28). Moreover, the variable regions of Dscam (Down syndrome cell adhesion molecule), a member of theimmunoglobulin superfamily (IgSF), from L. vannamei were cloned and analyzed after immunization, in order to reveal that Dscam-mediated phagocytosis may play an important role in the alternative adaptive immune system of shrimp.1. Influence of B. subtilis-VP28on humoral immune factors in L. vannameiShrimp were divided into three groups as B.subtilis-VP2S, B.subtilis and control. In our initial experiment, by oral delivery of B. sublilis-VP28to L. vannamei, the extremely high survival (Relative Percent Survival:83.3%) upon challenge with WSSV can be observed. After ’vaccination’ with B.subtilis-VP28, the activities of antibacteria, bacteriolysis, phenoloxidase, superoxide dismutase (SOD), acid phosphatase and alkaline phosphatase in haemolymph were significantly (p<0.05) enhanced. Moreover, immune-related genes (Toll receptor, Lysozyme, hemocyanin and Dscam) were significantly (p<0.05) upregulated in the group of B.subtilis-VP2%. This suggests that the humoral immune factors can be activated by B.subtilis-VP28leading to the high-level survival of shrimp towards WSSV challenge.2. Specific phagocytosis induced by oral delivery of B. subtilis-VP28in shrimpShrimp were vaccinated with B.subtilis-VP28, B.subtilis and algal polysaccharide (normal shrimp as negative control) for28days. On the4th,7th,14th,28th day of vaccination, phagocytic activities of haemocytes were significantly (p<0.05) increased in the group of B.subtilis-VP28compared to the other groups. In addition, the percentage of haemocytes phagocytosing WSSV in shrimp previously fed with B. subtilis-VP28was significantly higher (p<0.05) than that of phagocytosing vibrio. However, this phenomenon was not observed in both B.subtilis and algal polysaccharide groups. It was indicated that the heightened phagocytic activity, and thus the high-level survival of shrimp after B. subtilis-VP28’vaccination’are selective or specific towards WSSV.3. Identification of alternatively spliced exons locations and sequence analysis of the variable regions of Dscam from L. vannameiIn order to in identify the variable regions of Lv Dscam, we amplified two partial cDNA fragments (Igl-9; FNIII1-6) from shrimp hemocytes using specific primers designed by the full-length cDNA sequence of Dscam from Genbank. After PCR amplification, cloning and sequencing, multiple alignments among different sequences were demonstrated. Three alternatively spliced exons were found in the N-terminal regions of the Ig2(126-177aa), Ig3(228-268aa) domains and the entire Ig7(603-698aa) domain. A total of3,4and2expressed alternative isoforms were identified for N-terminal Ig2, N-terminal Ig3and the entire Ig7domain, respectively, implying that the LvDscam can present the molecular diversity. Furthermore, to investigate the evolutionary relatedness between LvDscam and other original Dscams, the conserved region was subjected to phylogenetic analysis. In the invertebrate Dscam clade, LvDscam is in a subclade on its own, while the other invertebrate Dscams form a second distinct subclade, suggesting that LvDscam is a unique, novel Dscam protein.4. Differential analysis of LvDscam specific isoforms induced by different immunogens activation in L. vannameiShrimp were vaccinated with B.subtilis-VP28, B.sublilis and algal polysaccharide (normal shrimp as negative control) for28days. The variable regions of LvDscam from each group were cloned, of which15clones in earch group were randomly selected and sequenced. Totally,13alternative amino acid sequences in the N-terminal of Ig2,15in the N-terminal of Ig3and11in the Ig7domain can be produced in groups of B.subtilis-VP28, B.sublilis and algal polysaccharide. This implies that LvDscam can potentially encode at least2145(13×15×11=2145) unique isoforms. Interestingly, two novel isoforms respective in the N-terminal of Ig2and Ig3were identified in the group of B.subtilis-VP28. It was supposed that the specific LvDscam isoforms can contribute to the mediation of specific phagocytosis, and thus lead to the selective protection of shrimp after B. sublilis-VP28’vaccination’ against WSSV.