Dietary Bacillus velezensis T23 fermented products supplementation improves growth, hepatopancreas and intestine health of Litopenaeus vannamei.

This study aimed to elucidate the effects of dietary fermented products of Bacillus velezensis T23 on the growth, immune response and gut microbiota in Pacific white shrimp (Litopenaeus vannamei). Shrimp were fed with diets containing fermentation products of B. velezensis T23 at levels of (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 g/kg) for 4 weeks, to assess the influence on shrimp growth. The results showed that 0.3 and 0.4 g/kg T23 supplementation improved shrimp growth and feed utilization. Based on these results we selected these three diets (Control, 0.3T23 and 0.4T23) to assess the effect on immune response and gut microbiota of shrimp. Compared with the control, the 0.3T23 and 0.4T23 groups enhanced lipase and α-amylase activities in the gut significantly. Moreover, the 0.4T23 group decreased TAG and MDA levels in hepatopancreas, ALT and AST levels of serum significantly (P < 0.05). In hepatopancreas, CAT and SOD activities were improved observably and the MDA content was reduced markedly in both T23 groups. The expressions of antimicrobial related genes, Cru and peroxinectin in the 0.3T23 group, and proPO and peroxinectin in the 0.4T23 group were up-regulated remarkably (P < 0.05). Moreover, hepatopancreas of shrimp fed with a diet amended with T23 showed a significant down-regulated expression of nf-kb and tnf-α genes, while expressions of tgf-ß was considerably up-regulated. Furthermore, serum LPS and LBP contents were reduced markedly in T23 groups. Intestinal SOD and CAT were noteworthy higher in T23 groups (P < 0.05). In the intestine of shrimp fed on the diet enriched with T23 the expression of nf-κb and tnf-α exhibited markedly down-regulated, whereas hif1α was up-regulated (P < 0.05). Besides, in the intestine of shrimp grouped under T23, Cru and peroxinectin genes were markedly up-regulated (P < 0.05). Dietary 0.3 g/kg T23 also upregulated the ratio of Rhodobacteraceae to Vibrionaceae in the gut of the shrimp. Taken together, the inclusion of B. velezensis T23 in the diet of shrimp enhanced the growth and feed utilization, enhanced hepatopancreas and intestine health.

Effects of replacing fishmeal with soybean meal on the immune and antioxidant capacity, and intestinal metabolic functions of red swamp crayfish Procambarus clarkii.

Excess utilization of plant protein sources in animal feed has been found to adversely affect the antioxidant properties and immunity of animals. While the role of gut microbes in plant protein-induced inflammation has been identified in various models, the specific mechanisms regulating gut microbes in crustaceans remain unclear. Accordingly, this study was designed to investigate the effects of replacing fishmeal with soybean meal (SM) on the hepatopancreas antioxidant and immune capacities, and gut microbial functions of crayfish, as well as the potential microbial regulatory mechanisms. 750 crayfish (4.00 g) were randomly divided into five groups: SS0, SS25, SS50, SS75, and SS100, and fed diets with different levels of soybean meal substituted for fishmeal for six weeks. High SM supplementation proved detrimental to maintaining hepatopancreas health, as indicated by an increase in hemolymph MDA content, GPT, and GOT activities, the observed rupture of hepatopancreas cell basement membranes, along with the decreased number of hepatopancreatic F cells. Moreover, crayfish subjected to high SM diets experienced obvious inflammation in hepatopancreas, together with up-regulated mRNA expression levels of nfkb, alf, and tlr (p<0.05), whereas the lzm mRNA expression level exhibited the highest value in the SS25 group. Furthermore, hepatopancreas antioxidant properties highly attenuated by the level of dietary SM substitution levels, as evidenced by the observed increase in MDA content (p<0.05), decrease in GSH content (p<0.05), and inhabitation of SOD, CAT, GPx, and GST activities (p<0.05), along with down-regulated hepatopancreas cat, gpx, gst, and mmnsod mRNA expression levels via inhibiting nrf2/keap1 pathway. Functional genes contributing to metabolism identified that high SM diets feeding significantly activated lipopolysaccharide biosynthesis, revealing gut dysfunction acted as the cause of inflammation. The global microbial co-occurrence network further indicated that the microbes contributing more to serum indicators and immunity were in module eigengene 17 (ME17). A structural equation model revealed that the genes related to alf directly drove the serum enzyme activities through microbes in ME17, with OTU399 and OTU533 identified as major biomarkers and classified into Proteobacteria that secrete endotoxins. To conclude, SM could replace 25 % of fishmeal in crayfish diets without negatively affecting immunity, and antioxidant capacity. Excessive SM levels contributed to gut dysfunction and weakened the innate immune system of crayfish.

Glycosaminoglycan lyase: A new competition between bacteria and the pacific white shrimp Litopenaeus vannamei.

Horizontal gene transfer (HGT) is an important evolutionary force in the formation of prokaryotic and eukaryotic genomes. In recent years, many HGT genes horizontally transferred from prokaryotes to eukaryotes have been reported, and most of them are present in arthropods. The Pacific white shrimp Litopenaeus vannamei, an important economic species of arthropod, has close relationships with bacteria, providing a platform for horizontal gene transfer (HGT). In this study, we analyzed bacteria-derived HGT based on a high-quality genome of L. vannamei via a homology search and phylogenetic analysis, and six HGT genes were identified. Among these six horizontally transferred genes, we found one gene (LOC113799989) that contains a bacterial chondroitinase AC structural domain and encodes an unknown glycosaminoglycan (GAG) lyase in L. vannamei. The real-time quantitative PCR results showed that the mRNA expression level of LOC113799989 was highest in the hepatopancreas and heart, and after stimulation by Vibrio parahaemolyticus, its mRNA expression level was rapidly up-regulated within 12 h. Furthermore, after injecting si-RNA and stimulation by V. parahaemolyticus, we found that the experimental group had a higher cumulative mortality rate in 48 h than the control group, indicating that the bacteria-derived GAG lyase can reduce the mortality of shrimp with respect to infection by V. parahaemolyticus and might be related to the resistance of shrimp to bacterial diseases. Our findings contribute to the study of the function of GAGs and provide new insights into GAG-related microbial pathogenesis and host defense mechanisms in arthropods.

Integrated analysis of intestinal microbiota and transcriptome reveals that a coordinated interaction of the endocrine, immune system and gut microbiota response to heat stress in Litopenaeus vannamei.

Due to the ongoing global warming, the risk of heatwaves in the oceans is continuously increasing while our understanding of the physiological response of Litopenaeus vannamei under extreme temperature conditions remains limited. Therefore, this study aimed to evaluate the physiological responses of L. vannamei under heat stress. Our results indicated that as temperature rose, the structure of intestinal and hepatopancreatic tissues was damaged sequentially. Activity of immune-related enzymes (acid phosphatase/alkaline phosphatase) initially increased before decreased, while antioxidant enzymes (superoxide dismutase and glutathione-S transferase) activity and malondialdehyde content increased with rising temperature. In addition, the total antioxidant capacity decreased with rising temperature. With the rising temperature, there was a significant increase in the expression of caspase-3, heat shock protein 70, lipopolysaccharide-induced tumor necrosis factor-α, transcriptional enhanced associate domain and yorkie in intestinal and hepatopancreatic tissues. Following heat stress, the number of potentially beneficial bacteria (Rhodobacteraceae and Gemmonbacter) increased which maintain balance and promote vitamin synthesis. Intestinal transcriptome analysis revealed 852 differentially expressed genes in the heat stress group compared with the control group. KEGG functional annotation results showed that the endocrine system was the most abundant in Organismal systems followed by the immune system. These results indicated that heat stress leads to tissue damage in shrimp, however the shrimp may respond to stress through a coordinated interaction strategy of the endocrine system, immune system and gut microbiota. This study revealed the response mechanism of L. vannamei to acute heat stress and potentially provided a theoretical foundation for future research on shrimp environmental adaptations.

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

Two NF-κB subunits are associated with antimicrobial immunity in Hyriopsis cumingii.

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.

Expression and functional characterization of peptidoglycan recognition protein-S6 involved in antibacterial responses in the razor clam Sinonovacula constricta.

It has been recognized that peptidoglycan recognition proteins (PGRPs), structurally conserved molecules, play crucial roles in the innate immunity of invertebrate. However, few studies have been taken to explore their potential functions. In this study, a novel PGRP from the razor clam Sinonovacula constrict designated as ScPGRP-S6 was identified and characterized. The open reading frame (ORF) of ScPGRP-S6 was 666 bp in length, encoding a protein of 221 amino acid with a signal peptide (1-30) and a typical PGRP domain (39-187). The sequence alignment combined with phylogenetic analysis collectively confirmed that ScPGRP-S6 was a novel member belonging to PGRP-S family. The mRNA transcript of ScPGRP-S6 in the hepatopancreases was significantly up-regulated after peptidoglycan (PGN) stimulation, while it was moderately up-regulated after lipopolysaccharide (LPS) stimulation. The result of immunofluorescence detection demonstrated that the positive signal enhanced obviously after Vibrio parahaemolyticus challenge. Notably, the recombinant protein of ScPGRP-S6 (designed as rScPGRP-S6) exhibited high agglutination activity towards V. parahaemolyticus but weak to Staphylococcus aureus. Furthermore, rScPGRP-S6 showed strong amidase and antibacterial activity in the presence of Zn2+. Collectively, our results manifested that ScPGRP-S6 could act as a scavenger in the innate immune response of S. constricta.

The Microbial Composition of Penaeid Shrimps' Hepatopancreas Is Modulated by Hemocyanin.

Aquatic organisms have to produce proteins or factors that help maintain a stable relationship with microbiota and prevent colonization by pathogenic microorganisms. In crustaceans and other aquatic invertebrates, relatively few of these host factors have been characterized. In this study, we show that the respiratory glycoprotein hemocyanin is a crucial host factor that modulates microbial composition and diversity in the hepatopancreas of penaeid shrimp. Diseased penaeid shrimp (Penaeus vannamei), had an empty gastrointestinal tract with atrophied hepatopancreas, expressed low hemocyanin, and high total bacterial abundance, with Vibrio as the dominant bacteria. Similarly, shrimp depleted of hemocyanin had mitochondrial depolarization, increased reactive oxygen species (ROS) levels, and dysregulation of several energy metabolism-related genes. Hemocyanin silencing together with ROS scavenger (N-acetylcysteine) treatment improved microbial diversity and decreased Vibrio dominance in the hepatopancreas. However, fecal microbiota transplantation after hemocyanin knockdown could not restore the microbial composition in the hepatopancreas. Collectively, our data provide, to our knowledge, new insight into the pivotal role of hemocyanin in modulating microbial composition in penaeid shrimp hepatopancreas via its effect on mitochondrial integrity, energy metabolism, and ROS production.

Combined stress of acute cold exposure and waterless duration at low temperature induces mortality of shrimp Litopenaeus vannamei through injuring antioxidative and immunological response in hepatopancreas tissue.

High mortality is a frequent occurrence during live transport of shrimp species and the biochemical mechanism remains unknown. This study aimed to explore the influence of combined stress of acute cold exposure (AC) and waterless duration (WD) on survivability and biochemical response of shrimp L. vannamei during live transport. The shrimps in NC and AC groups remained the total survivability throughout the experiment while the shrimps exposed to AC + WD stress exhibited significantly higher mortality since 6h afterwards (P < 0.05) and the median survival time was calculated at 10.46 h. Moreover, the typical combined stress points at AC + WD3h, AC + WD6h and AC + WD9h were assigned for exploring the immunological and antioxidative responses. For immunity response, the total hemocyte counts (THC) decreased with the prolongation of duration time and the activities of non-specific immunity enzymes such as phenol oxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), aspartate aminotransferase (AST) and alanine transaminase (ALT) were significantly elevated in AC + WD9h groups (P < 0.05). Moreover, compared with that in NC group, the significant accumulation of reactive oxygen species (ROS) was observed in AC group and then reduced in combined stress groups (P < 0.05), with the highest level of malonaldehyde (MDA) in AC and AC + WD3h groups. Overall, the significant elevation of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) was detected in AC + WD9h group (P < 0.05). Furthermore, the accumulative pathological impairment on hepatopancreas tissue revealed the cytoskeleton degradation. In addition, correlation analyses visualized the correlation between oxidative stress and biochemical response. This study not only deepens our understanding on the biochemical mechanism of shrimp mortality induced by combined stress, but also provides a potential strategy for improving the management of L. vannamei during live transport.

The effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

A proteomics investigation of 'immune priming' in Penaeus vannamei as shown by isobaric tags for relative and absolute quantification.

Invertebrates are considered completely dependent on their innate immunity to defend themselves against pathogens as they lack an adaptive immunity. However, a growing body of evidence has indicated a specific acquired immunity called 'immune priming' may exist. The Pacific white shrimp, Penaeus vannamei is one of the most economically important shrimp species in the world. In the previous research, we investigated the hepatopancreas immune response of shrimp immunized with trans -vp28 gene Synechocystis sp. PCC6803 at the protein level. In this study, on the basis of the previous research, the shrimp were then challenged with WSSV, and hepatopancreas analyzed using isobaric tags for relative and absolute quantification (i TRAQ) labeling. In total, 308 differentially expressed proteins (DEPs) were identified including 84 upregulated and 224 downregulated. Upregulated proteins such as calmodulin B and calreticulin, and downregulated proteins such as calnexin, and signaling pathways like Ras, mTOR were differentially expressed in both studies. Data from this study are more significant than previous work and indicate increased sensitivity to WSSV after immunization with trans-vp28 gene Synechocystis sp. PCC6803. In addition, selected DEPs (upregulated: A0A3R7QHH6 and downregulated: A0A3R7PEF6, A0A3R7MGX8, A0A423TPJ4, and A0A3R7QCC2) were randomly analyzed using parallel reaction monitoring (PRM). These data preliminarily confirm immune priming in P. vannamei, and show that the initial stimulation with trans -vp28 gene Synechocystis sp. PCC6803 regulate P. vannamei immune responses and they provide shrimp with enhanced immune protection against secondary stimulation.

Comparative transcriptome analysis of Chinese grass shrimp (Palaemonetes sinensis) hepatopancreas under ectoparasitic isopod (Tachaea chinensis) infection.

Tachaea chinensis, a parasitic isopod, negatively affects the production of several commercially important shrimp species. To better understand the interaction between shrimp immunity and isopod infection, we performed a transcriptome analysis of the hepatopancreas of Palaemonetes sinensis challenged with T. chinensis. After assembly and annotation, 75,980 high-quality unigenes were obtained using RNA-seq data. Differential gene expression analysis revealed 896 significantly differently expressed genes (DEGs) after infection, with 452 and 444 upregulated and downregulated genes, respectively. Specifically, expression levels of genes involved in detoxification, such as the interferon regulatory factor, venom carboxylesterase-6, serine proteinase inhibitor, and cytochrome P450, were upregulated. Furthermore, expression levels of genes corresponding to retinol dehydrogenase, triosephosphate isomerase, variant ionotropic glutamate receptor, and phosphoenolpyruvate carboxykinase were significantly upregulated after isopod parasitization, indicating that the shrimp's visual system was influenced by isopod parasitization. Moreover, quantitative real-time PCR of 10 DEGs helped validate the RNA-seq findings. These results provide a valuable basis for future studies on the elucidation of immune responses of P. sinensis to T. chinensis infection.

Histopathology, antioxidant responses, transcriptome and gene expression analysis in triangle sail mussel Hyriopsis cumingii after bacterial infection.

Bacterial disease outbreaks in filter feeder bivalve Hyriopsis cumingii as water contamination become more frequent in the water ecosystem, especially in intensive aquaculture habitats. To characterize host-pathogen interactions between H. cumingii and bacterial infection, we investigated the effects of Stenotrophomonas maltophilia HOP3 and Aeromonas veronii GL1 on the antioxidant response, tissue invasion and transcriptome expression of H. cumingii by infectivity trials. We showed that bacterial infections resulted in tubular necrosis of the hepatopancreas and induced the acute immune response in H. cumingii. The transcriptomic study identified a total of 5957 differentially expressed genes (DEGs) after A. veronii challenge. These DEGs were implicated in 302 KEGG pathways, notably in Apoptosis, Phagosome and Lysosome. The results showed that the relative expressions of all six immune-related DEGs were effectively stimulated with A. veronii, accompanied by tissue differences. Overall, these findings will contribute to an analysis of the immune response of H. cumingii to bacterial infection at the transcriptomic level and its genomic resource for research.

Differentially Expressed Genes in Hepatopancreas of Acute Hepatopancreatic Necrosis Disease Tolerant and Susceptible Shrimp (Penaeus vannamei).

Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pirA and pirB in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei. This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei.

An invertebrate gene encoding a Mab21-containing protein involves in antiviral response through regulating the STING pathway.

The cGAS-STING pathway plays essential roles in detecting cytosolic dsDNA and initiating antiviral and antibacterial responses in vertebrates. However, knowledge about its function in antiviral response of invertebrates is very limited. In the present study, a gene encoding a Mab21-containing protein, a cGAS homologue, was identified from a decapod crustacean Litopenaeus vannamei and designated as LvMab21cp. LvMab21cp was mainly distributed in intestine and hepatopancreas, showing similar expression profile with other genes in the cGAS-STING pathway, such as LvSTING and LvIRF. The expression levels of LvMab21cp, LvSTING and LvIRF were up-regulated in intestine and hepatopancreas of shrimp after white spot syndrome virus (WSSV) infection. Knockdown of LvMab21cp by dsRNA-mediated RNA interference could decrease the expression levels of its putative downstream genes, including LvSTING, LvIRF, LvVago4 and LvVago5, and enhance the in vivo propagation of WSSV in shrimp. Overexpression of LvMab21cp and LvSTING in HEK 293T cells activated the expression of mammalian IFNs upon simulation with interferon stimulatory DNA (ISD). These data suggest that LvMab21cp was a cGAS homologue, a member of the shrimp cGAS-STING pathway, and play an important role during WSSV infection. To our knowledge, this is the first report to show the role of the cGAS-STING pathway in the antiviral response of invertebrates, which will provide new insights into the innate immunity of invertebrates.

The T cell factor, pangolin, from Litopenaeus vannamei play a positive role in the immune responses against white spot syndrome virus infection.

As a downstream interactor of ß-catenin, Pangolin which is the homologous protein of the T cell factor/lymphoid enhancer factor (TCF/LEF) in vertebrates is less understood in the research field of immunity. In this study, two isoforms of Litopenaeus vannamei Pangolin (LvPangolin1 and LvPangolin2) were identified. Phylogenetic tree analysis revealed that all of the Pangolin proteins from invertebrates were represented the same lineage. The mRNA expression profiles of the LvPangolin1 and LvPangolin2 genes differed across different tissues. The expression of LvPangolin1 and the amount of LvPangolin1and LvPangolin2 combined (LvPangolinComb) were significantly increased in the haemocyte, intestine and gill but reduced in the hepatopancreas after white spot syndrome virus (WSSV) challenge. The inhibition of LvPangolin1 but not LvPangolinComb significantly reduced the survival rates of L. vannamei after WSSV infection, while significantly higher WSSV viral loads in both LvPangolin1-inhibited and LvPangolinComb-inhibited L. vannamei were observed. Knockdown of LvPangolin by RNAi could distinctly decrease the expression of antimicrobial peptide (AMP) genes and their related transcription factors. All of these results indicate that LvPangolin plays a positive role in the response to WSSV infection and that this may be mediated through regulating the immune signalling pathways which control the expression of AMPs with antiviral abilities.

In Litopenaeus vannamei, the cuticular chitin-binding proteins LvDD9A and LvDD9B retard AHPND pathogenesis but facilitate WSSV infection.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication.

Transcriptome analysis of the Macrobrachium nipponense hepatopancreas provides insights into immunoregulation under Aeromonas veronii infection.

The oriental river prawn Macrobrachium nipponense is a commercially important freshwater shrimp that is widely farmed in China. Aeromonas veronii is a conditional pathogen of farmed shrimp, which has caused huge economic losses to the industry. Therefore, there is urgency to study the host-pathogen interactions between M. nipponense and A. veronii to screen individuals with antimicrobial resistance. In this study, we examined the hepatopancreas of moribund M. nipponense infected with A. veronii and healthy individuals at both the histopathological and transcriptomic levels. We showed that A. veronii infection resulted in tubular necrosis of the M. nipponense hepatopancreas. Such changes likely affect assimilation, storage, and excretion by the hepatopancreas, which could ultimately affect the survival and growth of infected individuals. Among the 61,345 unigenes obtained through RNA sequencing and de novo transcriptome assembly, 232 were differentially expressed between the two groups. KEGG and GO analyses revealed that these differentially expressed genes were implicated in pathways, including PPAR, PI3K/AKT, and AMPK signaling. The results of this study will contribute to an analysis of the immune response of M. nipponense to A. veronii infection at the transcriptomic level. Furthermore, the RNA-seq data generated here provide an important genomic resource for research on M. nipponense in the absence of a reference genome.

Transcriptomic analysis of Procambarus clarkii affected by "Black May" disease.

Each year from April to May, high mortality rates are reported in red swamp crayfish (Procambarus clarkii) cultured in Jiangsu and other regions, in China, and this phenomenon has come to be known as "Black May" disease (BMD). Therefore, in order to investigate the possible causes of this disease, this study gathered BMD-affected P. clarkii samples and performed transcriptome analysis on hepatopancreas, gill, and muscle tissues. A total of 19,995,164, 149,212,804, and 222,053,848 clean reads were respectively obtained from the gills, muscle, and hepatopancreas of BMD-affected P. clarkii, and 114,024 unigenes were identified. The number of differentially expressed genes (DEGs) in gill, muscle, and hepatopancreas was 1703, 964, and 476, respectively. GO and KEGG enrichment analyses of the DEGs were then conducted. Based on KEGG pathway enrichment analysis, the most significantly differentially expressed pathways were mainly those involved with metabolism, human disease, and cellular processes. Further analysis of the significantly DEGs revealed that they were mainly related to the mitochondrial-mediated apoptosis pathway and that the expression of these DEGs was mostly down-regulated. Moreover, the expression of genes related to immune and metabolism-related pathways was also significantly down-regulated, and these significantly-inhibited pathways were the likely causes of P. clarkii death. Therefore, our results provide a basis for the identification of BMD causes.

Enhanced Immune Protection of Mud Crab Scylla paramamosain in Response to the Secondary Challenge by Vibrio parahaemolyticus.

"Immune priming" plays a vital part in the immune system of invertebrates, protecting against recurrent infections by pathogens, and can provide some ideas for the prevention and treatment of invertebrate diseases. Many invertebrates have been demonstrated recently to have immune priming, but the relevant mechanisms are not known. Expression of immune system-related genes in the hemocytes and hepatopancreas of the mud crab (Scylla paramamosain) before and after repeated stimulation with Vibrio parahaemolyticus were analyzed by real-time fluorescence quantitative polymerase chain reaction. Some molecules that may participate in the immune priming of S. paramamosain were screened out, and their possible roles in immune priming were interpreted. Crabs injected first with heat-killed V. parahaemolyticus (HkVp group) or physiologic (0.9%) saline (PS group) were rechallenged at 168 h with live V. parahaemolyticus (HkVp+Vp group and PS+Vp group, respectively). The log-rank test shows a significant difference in survival rate between the HkVp+Vp group and the other groups after the ICH (p < 0.05). Expression of genes involved in the toll-like receptor (TLR) signaling pathway and some antimicrobial peptide genes were detected. By, respectively, comparing gene quantification at different time points in hemocytes and the hepatopancreas, the molecules that may play a part in the early stage of the immune priming of S. paramamosain in the hemocytes are found to be down syndrome cell adhesion molecule (Dscam), Hyastatin, Cactus, Arasin, antilipopolysaccharide factor 3 (ALF3), ALF4, ALF5, and ALF6 as well as later acting molecules, such as Crustin, Dorsal, Pelle, and myeloid differentiation factor 88 (MyD88). The molecules that functioned throughout the entire period are TLR and Spaetzle. In the hepatopancreas, the molecules that may play a part in the early stages of immune priming are Dscam, Hyastatin, Arasin, ALF6, Pelle, Spaetzle, Dorsal and, in the later stage, ALF4. The molecules that functioned throughout the entire period are TLR, Crustin, Cactus, MyD88, ALF3, and ALF5. In summary, the immune function of S. paramamosain is enhanced after it receives the same repetitive stimulation by V. parahaemolyticus, indicating immune priming in S. paramamosain. Our study enriches research on immune priming in invertebrates and lays the foundation for further studies revealing the molecular mechanism of immune priming in crabs.