Glycogen synthase kinase-3 (GSK3), a cytoplasmic serine/threonine-protein kinase involved in a large number of key cellular processes, is a little-known signaling molecule in virus study. In this study, a GSK3 protein which was highly similar to GSK3β homologs from other species in Litopenaeus vannamei (designated as LvGSK3β) was obtained. LvGSK3β was expressed constitutively in the healthy L. vannamei, at the highest level in the intestine and the lowest level in the eyestalk. White spot syndrome virus (WSSV) reduced LvGSK3β expression was in immune tissues including the hemocyte, intestine, gill and hepatopancreas. The inhibition of LvGSK3β resulted in significantly higher survival rates of L. vannamei during WSSV infection than the control group, and significantly lower WSSV viral loads in LvGSK3β-inhibited L. vannamei were observed. Knockdown of LvGSK3β by RNAi resulted in increases in the expression of LvDorsal and several NF-κB driven antimicrobial peptide (AMP) genes (including ALF, PEN and crustin), but a decrease in LvCactus expression. Accordingly, overexpression of LvGSK3β could reduce the promoter activity of LvDorsal and several AMPs, while the promoter activity of LvCactus was increased. Electrophoretic mobility shift assays (EMSA) showed that LvDorsal could bind to the promoter of LvGSK3β. The interaction between LvGSK3β and LvDorsal or LvCactus was confirmed using co-immunoprecipitation (Co-IP) assays. In addition, the expression of LvGSK3β was dramatically reduced by knockdown of LvDorsal. In summary, the results presented in this study indicated that LvGSK3β had a negative effect on L. vannamei by mediating a feedback regulation of the NF-κB pathway when it is infected by WSSV.

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Wnt/β-catenin signalling plays an essential role in the immunity of Penaeus vannamei. In this study, the effects of dietary Wnt/β-catenin pathway activator TWS119 on the growth, immunity, and transcriptome response in P. vannamei were investigated. Penaeus vannamei were fed diets with added TWS119 at doses of 0 (T0), 0.25 (T0.25), 1 (T1), 4 (T4), 16 (T16), or 64 mg·kg−1 (T64), respectively. LvGSK3β activity was effectively inhibited in P. vannamei given TWS119. The growth of P. vannamei in the T16 group was significantly improved when compared with the control group. After Vibrio parahaemolyticus infection, the survival rates (SRs) of P. vannamei in all experimental groups except the T64 group were significantly higher than in the T0 group. Compared with the control group, the immune enzymes’ activities in the serum of P. vannamei increased in all the experimental groups, while the malondialdehyde (MDA) contents decreased. Transcriptome analysis identified 5,073 differentially expressed genes (DEGs) for P. vannamei in the T0 and T16 groups. Most of the DEGs are involved in the ribosome pathway, endocytosis, glycerophospholipid metabolism, Wnt signalling, and FoxO =signalling pathways. The majority of the DEGs were from the ribosome pathway, which is also the most significantly enriched pathway. The study confirmed that the growth and immunity status of P. vannamei could improve by increasing dietary TWS119, which probably regulates the activity of the Wnt/β-catenin pathway and may be closely related to ribosome function and energy metabolism.

The effects of dietary zymosan A on the growth performance and histological structure, digestive capacity, and microbiota were investigated in Litopenaeus vannamei, an important shrimp species used in aquaculture. L. vannamei (initial body weight = 0.41 ± 0.05 g) were fed diets supplemented with zymosan-A at doses of 0 mg/kg (Z0, control), 1 mg/kg (Z1), 5 mg/kg (Z5), 25 mg/kg (Z25), 125 mg/kg (Z125), or 625 mg/kg (Z625) for 8 weeks. The results showed that doses of 25 and 125 mg/kg significantly increased the final body weight, weight gain rate (WGR), and specific growth rate (SGR) and significantly decreased the feed conversion rate compared with the controls (p < 0.05). Analyses of the WGR and SGR revealed that the optimum dietary zymosan-A levels were 308.56 and 300.00 mg/kg, respectively. Compared with the controls, the intestinal villus height was significantly increased by the addition of zymosan-A at doses ≥25 mg/kg (p < 0.05). An obvious upregulation of the activities of trypsin and amylase was observed in all groups that received zymosan-A supplementation, while the activity of lipase was significantly increased in groups Z125 and Z625 (p < 0.05). Consistently, the gene expressions of trypsin and lipase were significantly higher in group Z125, while the gene expression of amylase was significantly increased in all zymosan-A-supplemented groups (p < 0.05). Analysis of the intestinal microbiota showed significant differences in the alpha diversity between group Z125 and controls. The supplemented groups showed altered intestinal bacterial community structures and compositions at the phylum, family, and genus levels, with statistical differences observed in the relative abundances of some dominant species. Tax4Fun predictions of the microbiota indicated that multiple intestinal functions were significantly altered in all zymosan-A-supplemented groups, except Z1. Among these groups, the functions related to transport and catabolism, substance dependence, cardiovascular disease, and signaling molecules and interactions were obviously increased by the addition of zymosan-A at different levels. In conclusion, dietary supplementation of the optimum amount of zymosan-A can improve growth and intestinal function in L. vannamei, which will be valuable in aquaculture.

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