A Peritrophin-44 gene in Litopenaeus vannamei is involved in disease resistance.

Peritrophic membrane (PM) is a pellicle structure present in the midgut of some invertebrates, such as insects and crustaceans. It could isolate harmful components and pathogens in food from intestinal epithelial cells; and it also plays a role in improving digestion and absorption efficiency. So PM is important for survival of its owner. In current study, 44 PM proteins were identified in Litopenaeus vannamei by PM proteome analysis. Among these PM proteins, the Peritrophin-44 homologous protein (LvPT44) was further studied. Chitin-binding assay indicated that LvPT44 could bind to colloidal chitin, and immunoeletron microscopy analysis shown that it was located to PM of L. vannamei. Furthermore, LvPT44 promoter was found to be activated by L. vannamei STAT and c-Jun. Besides, LvPT44 was induced by ER-stress as well as white spot syndrome virus infection. Knocked-down expression of LvPT44 by RNA inference increased the cumulative mortality of shrimp that caused by ER-stress or white spot syndrome virus. These results suggested that LvPT44 has an important role in disease resistance.

Study of the antivirus function mediated by STING in Micropterus salmoides.

Stimulator of interferon genes (STING) has been demonstrated as a critical mediator in the innate immune response to cytosolic DNA and RNA derived from different pathogens. While the role of Micropterus salmoides STING (MsSTING) in largemouth bass virus is still unknown. In this study, RT-qPCR assay and Western-blot assay showed that the expression levels of MsSTING and its downstream genes were up-regulated after LMBV infection. Pull down experiment proved that a small peptide called Fusion peptide (FP) that previously reported to target to marine and human STING as a selective inhibitor also interacted with MsSTING in vitro. Comparing with the RNA-seq of Largemouth bass infected with LMBV singly, 326 genes were significantly up-regulated and 379 genes were significantly down-regulated in the FP plus LMBV group in which Largemouth bass was treatment with FP before LMBV-challenged. KEGG analysis indicated that the differentially expressed genes (DEGs) were mainly related to signaling transduction, infectious disease viral, immune system and endocrine system. Besides, the survival rate of LMBV-infected largemouth bass was highly decreased following FP treatment. Taken together, our study showed that MsSTING played an important role in immune response against LMBV infection.

Preliminary study of the intranuclear function of Sma and Mad related protein 5 gene in Litopenaeus vannamei.

Litopenaeus vannamei Smad5 (LvSmad5) in cytoplasm has been proved to be involved in environmental stress response. As LvSmad5 could also locate in nucleus under specific stress, it was conjectured that LvSmad5 might participate in environmental stress response. While, the experimental evidence is still lacking. In this study, cytosolic LvSmad5 mutant or nuclear LvSmad5 mutant was expressed in Drosophila S2 cells, and then transcriptomic analysis of mentioned cells was performed using Illumina HiSeq based RNA-Seq, to reveal the function of LvSmad5 in nucleus. By comparing the two groups of cDNA libraries from S2 cells with cytosolic or nucleus LvSmad5 mutant, 86 differentially expressed genes as well as 765 differentially expressed transcripts were found. It was revealed that genes in the ER-stress response pathway, such as unfolded protein response and ER-associated degradation (ERAD) were enriched. Additionally, some kinds of metabolic reprogramming occurred in S2 cells with over-expressing nuclear LvSmad5, for significant changes in the expression of some metabolism-related genes. To test our infer that nuclear LvSmad5 was engaged in environmental stress response, homologous gene of Drosophila translocation in renal carcinoma on chromosome 8 in L.vannamei (LvTRC8) was chosen for further investigation. And studies about LvTRC8, a member of ERAD showed that it was induced by ER-stress or heat shock treatment. Suppressed the expression of LvTRC8 increased the cumulative mortality of shrimp upon stress. In some degree, these results support our speculation that nuclear LvSmad5 are involved in the environmental stress response of L. vannamei in fact.

Transcriptome analysis endoplasmic reticulum-stress response in Litopenaeus vannamei hemocytes.

Numerous studies have proved that endoplasmic reticulum (ER)-stress is an important cause of aquatic animal diseases. Therefore, for effectively preventing and controlling aquatic animal diseases, a systematic and in-depth understanding of the environmental stress response in aquatic animals is necessary. In present study, the influence of ER-stress in Litopenaeus vannamei was investigated using Illumina HiSeq based RNA-Seq. Comparing to the cDNA library of hemocytes treated with DMSO in L. vannamei, 286 unigenes were significantly upregulated and 473 unigenes were significantly down-regulated in the Thapsigargin treated group. KEGG analysis indicated that the differentially expressed genes (DEGs) are mainly related to ER-stress, immune as well as metabolism. Besides the classical ER-stress response pathways, the regulation of cell cycle and DNA replication are also important measures of ER-stress response. It has been suggested that the influence of ER-stress on immune genes might be an important factor in environmental stress inducing shrimp disease. Our investigation exhibited that immune-related DEG Prophenoloxidase activating enzyme 2 (LvPPAE2) roled in anti-pathogen immunity of shrimp. This study provides a solid foundation for uncovering the environmental adaptation response and especially its relationship with L. vannamei immune system.

A IFI27 gene contributes to ER-stress mediated apoptosis and benefits for white spot syndrome virus infection in Litopenaeus vannamei.

The interplay between virus and host has been one of the hot spot in virology, and it is also the important aspect of revealing the mechanism of virus infection. Increasing studies revealed that several key molecules took part in the process of virus-host interaction. White spot syndrome virus (WSSV) has been proved to affect several physiological processes of the host cells, especially apoptosis. While the relationship between them still remains unclear. In this study, a IFI27 gene (LvIFI27) of Litopenaeus vannamei was cloned. It is indicated that LvIFI27 was induced upon endoplasmic reticulum (ER)-stress and unfolded protein response activator Thapsigargin. Unlike human IFI27 locating to mitochondria, LvIFI27 lied to ER, and was involved in cell apoptosis process. Moreover, results of cumulative mortality analysis showed that LvIFI27 might contributed to WSSV proliferation by promoting apoptosis during the process of viral infection. Findings in this study enriched our understanding of the relationship between WSSV infection and ER-stress mediated apoptosis.

Transcriptome analysis of the influence of high plant protein based diet on Trachinotus ovatus liver.

Seeking out fish meal (FM) alternatives is an important requirement for aquaculture all over the world. And most practitioners believe that the plant protein is most potential for FM surrenal. While high plant protein feed caused some common problems in aquatic livestock: the absorption rate and growth rate are decreased, and even caused digestive tract inflammation. In present study, the inflence of high plant protien feed in Trachinotus ovatus was investigated using illumina HiSeqTM2000 based RNA-Seq. By comparing the two groups of cDNA libraries developed from high plant protien based diet or FM based diet fed T. ovatus livers, 836 unigenes were significantly upregulated, and 345 were significantly down regulated. KEGG analysis indicated that the differentially expressed genes (DEGs) are mainly metabolic-related genes. It was found that more than 28 DGEs beloned to the protein metabolism and absorption, lipid biosynthesis or other metabolic pathways. It indicated that high plant protein based diet had broad effects on metabolism on T. ovatus. There were also more DEGs belong to immune-related signaling pathways, include genes were involved in pathpathogen resistance and genes related to immunity system. These DEGs provided useful clues to explore the mechanisms that high plant protein based diet caused side effects on T. ovatus. These results improved our current understanding of the response of high plant protein based diet in T. ovatus, and outstanding the reasons of the side effect caused by high protein based diet.

Litopenaeus vannamei Sma and Mad related protein 5 gene is involved in stress response and white spot syndrome virus infection.

Cell survival is based on the stability of intracellular state. It was well known that biochemical reactions in cells require specific intracellular environments, such as pH and calcium concentration. While the mechanism of stabilizing the intracellular environment is complex and far from clear. In this study, a Sma and Mad related protein 5 gene (LvSmad5) of Litopenaeus vannamei was cloned. LvSmad5 was located to both cytoplasm and nucleus. And subcellular localization of LvSmad5 was responsed to the changing of cells internal and external environment. Besides, it was found that subcellular localization of LvSmad5 was also regulated by unfolded protein response. Moreover, it was proved that nucleic localization of LvSmad5 could significantly increase the white spot syndrome virus (WSSV) infection in shrimp, and knockdown expression of LvSmad5 decreased the cumulative mortality of WSSV infection shrimp. Further investigation revealed that cytoplasm LvSmad5 could interplay with shrimp hexokinase 1, and contribute to glycolysis. These results indicated that LvSmad5 played a role in L. vannamei environmental stress response, and was used by WSSV for its replication.

Identification and functional characterization of a systemic RNA interference defective 1 gene in Litopenaeus vannamei.

RNA interference (RNAi) is a conservative and important functional pathway in eukaryocyte. It regulates the expression of genes that are engaged in a variety of cellular physiological functions. Among the functions of RNAi, its antiviral function have attracted many attentions.The RNAi pathway molecules are able to recognize virus-related dsRNA and degrade it, therefore killing the virus. More importantly, RNAi could mediate systemic antiviral responses, transmit from cell to cell, and systemic RNA interference defective 1 (SID1) was thought to play an important role in this process. In the present study, a SID1 gene (LvSID1) of Litopenaeus vannamei was cloned. LvSID1 could locate to both plasma membrane and endoplasmic reticulum. Result of real-time RT-PCR assay showed that it was highly expressed in shrimp gills. Besides, it was shown that over-expressed LvSID1 in Sf9 cells could significant enchane RNAi efficiency. It was found that the expression of LvSID1was regulated by white spot syndrome virus (WSSV), and knockdown expression of LvSID1 increased the cumulative mortality of WSSV infection shrimp. These results suggested that LvSID1 likely to played a role in L. vannamei systemic RNAi, and was involved in WSSV resistence.

Identification and functional characterization of a C-type lectin gene from Litopenaeus vannamei that is associated with ER-stress response.

C-type lectins (CTLs), which bind carbohydrates in a Ca2+-dependent manner, are involved in many cellular activities, especially immunity. CTLs play important roles in both the antibacterial and the antiviral immune response and are also associated with autoimmunity. Several CTLs have been investigated in crustaceans, primarily with respect to their function in the immune response. In this study, we cloned a novel CTL gene (LvCTLU) from Litopenaeus vannamei. LvCTLU is involved in microbe agglutination and phagocytosis. Downregulating LvCTLU increased the cumulative mortality of L. vannamei after Vibrio parahemolyticus infection. Similar to other reported CTLs, LvCTLU also had antiviral properties. Downregulation of LvCTLU also increased the cumulative mortality of L. vannamei after infection with white spot syndrome virus. More importantly, LvCTLU expression was induced by the unfolded protein response (UPR), which is the key pathway in the endoplasmic reticulum (ER)-stress response of eukaryotic organism. Our results suggested that this protein might be involved in the shrimp ER-stress response. Reporter gene assay indicated that LvCTLU was regulated by X-box-binding protein 1, which is the key transcription factor in the UPR. Our study thus revealed that LvCTLU plays vital roles in both the anti-pathogen immune response and the ER-stress response.