Amazonian buriti and pracaxi as potential functional feed additives to improve shrimp immunity and resistance to WSSV.

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjβ-arrestin2. Further studies found that Mjβ-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus.

Most tripartite motif (TRIM) family proteins are critical components of the autophagy machinery and play important roles in host defense against viral pathogens in mammals. However, the roles of TRIM proteins in autophagy and viral infection have not been studied in lower invertebrates, especially crustaceans. In this study, we first identified a TRIM50-like gene from Penaeus monodon (designated PmTRIM50-like), which, after a white spot syndrome virus (WSSV) challenge, was significantly upregulated at the mRNA and protein levels in the intestine and hemocytes. Knockdown of PmTRIM50-like led to an increase in the WSSV quantity in shrimp, while its overexpression led to a decrease compared with the controls. Autophagy can be induced by WSSV or rapamycin challenge and has been shown to play a positive role in restricting WSSV replication in P. monodon. The mRNA and protein expression levels of PmTRIM50-like significantly increased with the enhancement of rapamycin-induced autophagy. The autophagy activity induced by WSSV or rapamycin challenge could be inhibited by silencing PmTRIM50-like in shrimp. Further studies showed that rapamycin failed to induce autophagy or inhibit WSSV replication after knockdown of PmTRIM50-like. Moreover, pull-down and in vitro ubiquitination assays demonstrated that PmTRIM50-like could interact with WSSV envelope proteins and target them for ubiquitination in vitro. Collectively, this study demonstrated that PmTRIM50-like is required for autophagy and is involved in restricting the proliferation of WSSV through its ubiquitination. This is the first study to report the role of a TRIM family protein in virus infection and host autophagy in crustaceans.

β-Thymosins participate in antiviral immunity of red swamp crayfish (Procambarus clarkii)

Identification of differentially transcribed genes in shrimp Litopenaeus vannamei exposed to osmotic stress and challenged with WSSV virus

Dietary Hizikia fusiforme enhance survival of white spot syndrome virus infected crayfish Procambarus clarkii

Simple SummaryDisease is a frequently encountered problem in aquaculture, which always causes global economic losses. White spot syndrome virus (WSSV) and Vibrio parahaemolyticus are two of the most destructive pathogens causing severe loss of shrimp aquaculture. Understanding the host immune responses against different pathogens is vital for developing effective disease control technologies. The lymphoid organ is a vital part of the shrimp immune system and exhibits important immune functions including cellular and humoral immunity. However, the immune function of the lymphoid organ and its responses against different pathogens are still largely unclear. In the present study, transcriptomic analysis was applied to compare the differentially expressed genes (DEGs) in the lymphoid organ of shrimp after Vibrio or WSSV challenge. Data showed that Vibrio challenge induced broad immune responses in the lymphoid organ including activation of several pattern recognition receptors, the proPO activating system, phagocytosis related genes, and immune effectors. In contrast, the immune responses seemed to be inhibited after WSSV infection. The present study suggests that the shrimp lymphoid organ plays different functions in response to the infection of distinct pathogens at early stage, which provides new insights into the immune functions of lymphoid organ in shrimp.The lymphoid organ is an essential part of the immune system involved in cellular and humoral immune responses in shrimp. However, its roles in the immune responses against different pathogens are still largely unclear. In the present study, transcriptomic analysis was applied to compare the differentially expressed genes (DEGs) in the lymphoid organ of shrimp after Vibrio or WSSV challenge. In total, 2127 DEGs were screened in the lymphoid organ of shrimp at 6 h post Vibrio parahaemolyticus injection, and 1569 DEGs were obtained at the same time after WSSV challenge. KEGG pathway enrichment analysis of these DEGs revealed that two significantly enriched pathways including “neuroactive ligand–receptor interaction” and “protein digestion and absorption” were responsive to both pathogens. In contrast, “lysosome” was the significantly enriched pathway only in Vibrio challenge whereas carbohydrate metabolism related pathways were the significantly enriched pathways only in WSSV challenge. Further analysis on immune-related DEGs showed that Vibrio challenge induced broad immune responses in the lymphoid organ including activation of several pattern recognition receptors, the proPO activating system, phagocytosis related genes, and immune effectors. In contrast, the immune responses seemed to be inhibited after WSSV infection. The data suggest that the shrimp lymphoid organ plays different functions in response to the infection of distinct pathogens at the early stage, which provides new insights into the immune functions of lymphoid organ in shrimp.

Fusion of flagellin 2 with bivalent white spot syndrome virus vaccine increases survival in freshwater shrimp

Exposure to probiotics and β-1,3/1,6-glucans in larviculture modifies the immune response of Penaeus vannamei juveniles and both the survival to White Spot Syndrome Virus challenge and pond culture

Adjuvant effects of poly I:C and imiquimod on the immunization of kuruma shrimp (Marsupenaeus japonicus) with a recombinant protein, VP28 against white spot syndrome virus

Molecular characterization of a novel white spot syndrome virus response protein (dubbed LvWRP) from Litopenaeus vannamei

An outbreak of white spot syndrome virus (WSSV) can hit shrimp culture with a devastating blow, and there are no suitable measures to prevent infection with the virus. In this study, the activity of active molecules from Chinese herbs against WSSV was evaluated and screened. Taxifolin had the highest rate (84%) of inhibition of the WSSV infection. The viral infectivity and genome copy number were reduced by 41% when WSSV virion was pretreated with taxifolin prior to shrimp infection. A continuous exchange of taxifolin significantly reduced the mortality of shrimp infected with WSSV. Due to the WSSV virion infectivity being affected by taxifolin, the horizontal transmission of the virus was blocked with an inhibition rate of up to 30%, which would further reduce the cost of a viral outbreak. Additionally, the viral genome copy number was also reduced by up to 63% in shrimp preincubated in taxifolin for 8 h. There may be a connection to the enhancement of innate immunity in shrimp that resulted in a 15% reduction in mortality for taxifolin-fed shrimp after the WSSV challenge. After dietary supplementation with taxifolin, the resistance of larvae to WSSV was improved, indicating that taxifolin may be a potential immunostimulant for shrimp to prevent WSD. Therefore, the results indicate that taxifolin has application potential for blocking a WSSV outbreak and reducing the loss of shrimp culture.

Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up-regulated in WSSV-injected Macrobrachium rosenbergii and is responsible for protecting M.rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA-proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331bp were used to synthesize dsRNA using LITMUS38i vector and E.coli. The bacterially synthesized dsRNA-proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene-silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene-silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real-time PCR at different time intervals. Real-time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV-challenged proPO-silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO-silenced prawn when compared to prawn injected with PBS.

Double-dose β-glucan treatment in WSSV-challenged shrimp reduces viral replication but causes mortality possibly due to excessive ROS production

Dietary Bacillus amyloliquefaciens enhance survival of white spot syndrome virus infected crayfish

Involvement of WSSV–shrimp homologs in WSSV infectivity in kuruma shrimp: Marsupenaeus japonicus