Studies on immunological characteristics and transcriptomic analysis of Litopenaeus vannamei low salt-tolerance family.

Litopenaeus vannamei is a widely distributed euryhaline aquatic animal, affected by low salinity, which can impact its disease resistance and immunity. However, there is a limited understanding of the adaptation mechanisms of L. vannamei with different genetic backgrounds to low salinity. Therefore, the present study aimed to compare the immunity characteristics and transcriptomics of L. vannamei low salt-tolerant (FG I/J) and low salt-sensitive (control) families. Also, the disease resistance and immune parameters (including [THC], hemolymph cell viability, lysozyme activity [LZM], phenoloxidase content [PO], interleukin-6 [IL-6], and tumor necrosis factor-alpha [TNF-α]) of the FG I/J and control families of L. vannamei under low salinity (5‰) and ambient salinity (24‰) were examined. Additionally, hepatopancreas transcriptomics of the FG I/J and control families were analyzed at a salinity of 5‰. The results showed that the FG I/J family had higher disease resistance to Vibrio parahaemolyticus and stronger immunological capacity than the control family. Transcriptomic analysis showed significantly enriched energy metabolism and immune regulation pathways. Therefore, we speculated that energy metabolism provides sufficient energy for immunological modulation in the FG I/J family to deal with long-term low-salt stress and achieve high growth and survival rates.

Effects of dietary supplementation with medicinal plant mixtures and immunostimulants on the immune response, antioxidant capacity, and hepatopancreatic health of Chinese mitten crab (Eriocheir sinensis).

Introduction: This study aimed to evaluate the efficiency of tea polyphenols (TP) and medicinal plant mixtures (Astragalus membranaceus + Lonicera japonica, Rheum officinale Bail + Scutellaria baicalensis + Platycladus orientalis) combined with astaxanthin (AST), benzoic acid (BA), and yeast complex on the health status of Eriocheir sinensis. Method: A total of 630 crabs (male crabs: 41.51 ± 1.63 g; female crabs: 47.27 ± 0.79 g) were randomly distributed into seven groups with three replicates (male: female, 1:1). These crabs were fed as follows for 8 weeks: basal diet (M1), M2 (M1 + 100 mg/kg TP), M3 (M1 + 2.0 g/kg A. membranaceus + 20 g/kg L. japonica), M4 (M1 + 2.5 g/kg R. officinale Bail + 1.5 g/kg S. baicalensis + 1.0 g/kg P. orientalis), and M5, M6, M7 (M2, M3 and M4 with 600 mg/kg AST +1.0 g/kg BA + 20 mg/kg yeast complex added, respectively). Results and discussion: The results showed that the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), and lysosome (LZM) in the hemolymph were significantly increased in M5, M6, and M7 (P < 0.05), and the highest phagocytosis index (PI) and LZM activity were observed in M7 of female crabs. Moreover, the antioxidant indicators superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and catalase (CAT) of hepatopancreas were also significantly improved in M5, M6, and M7 (P < 0.05), while the malondialdehyde (MDA) contents showed an opposite trend. Furthermore, a morphological examination also showed the improved histological structure of hepatopancreas in M7, especially as seen in the clear lumens, no vacuolation, and integrity of the basal membrane of the hepatopancreatic tubule. Taken together, these results suggested that 2.5 g/kg R. officinale Bail, 1.5 g/kg S. baicalensis, and 1.0 g/kg P. orientalis in combination with 600 mg/kg AST, 1.0 g/kg BA, and 20 mg/kg yeast complex could improve the non-specific immunity, antioxidant capacity, and hepatopancreatic health of E. sinensis.

Effects of a phytobiotic-based additive on the growth, hepatopancreas health, intestinal microbiota, and Vibrio parahaemolyticus resistance of Pacific white shrimp, Litopenaeus vannamei.

Vibrio genus is a common pathogen in aquaculture and causes acute hepatopancreatic necrosis disease (AHPND) and massive mortality of shrimp. Many studies have suggested that a single functional ingredient such as plant extract or organic acid can reduce the dependence on antibiotics and promote the growth and immunity of aquatic animals. In this study, we evaluated the effects of a phytobiotic-based compound additive (Sanacore® GM, SNGM), which had a successful trajectory of commercial application in fish farming. However, its effects on the hepatopancreas health and intestinal microbiota of shrimp after Vibrio challenge have not been well evaluated. In the present study, Pacific white shrimp were fed diets with or without supplementation of SNGM, and the SNGM grades were 0-g/kg (CON), 3-g/kg (SNGM3), and 5-g/kg (SNGM5) diets. The feed trial lasted 60 days, after which a Vibrio parahaemolyticus challenge was performed. The results showed that compared to the CON group, both the SNGM3 and SNGM5 groups had a significantly higher weight gain and a lower feed conversion ratio as well as higher survival after Vibrio parahaemolyticus challenge. In the growth trial, the SNGM3 group had a significantly increased total protein, albumin concentration, and acid phosphatase activity in hemolymph compared to the CON group. In the challenge experiment, the SNGM3 and SNGM5 groups had increased albumin and glucose contents as well as the activities of phenoloxidase, lysozyme, alkaline phosphatase, and superoxide dismutase in hemolymph. Both the SNGM3 and SNGM5 groups had improved morphology of the hepatopancreas and intestine. The SNGM5 group had alleviated gut microbiota dysbiosis induced by Vibrio infection by increasing the potential probiotic bacterium abundance (Shewanella) and decreasing the potential pathogenic bacteria abundance (Vibrio, Photobacteriuma, Pseudoalteromonas, and Candidatus_Bacilloplasma). In conclusion, the dietary phytobiotic-based additive at 3-g/kg level increased the growth and Vibrio parahaemolyticus resistance of Pacific white shrimp by promoting immune-related enzyme activities and improving the morphological structure of the hepatopancreas and intestine and the intestinal microbiota composition.

Comparative transcriptomic analysis of hepatopancreas reveals that more genes are involved in the exposure response of Vibrio parahaemolyticus PirAvp compared to PirBvp.

Vibrio parahaemolyticus (VP-AHPND) is regarded as one of the main pathogens that caused acute hepatopancreatic necrosis disease (AHPND) in the Pacific white shrimp Litopenaeus vannamei. PirAvp and PirBvp toxin proteins are the main pathogenic proteins of AHPND in shrimp. Knowledge about the mechanism of shrimp response to PirAvp or PirBvp toxin is very helpful for developing new prevention and control strategy of AHPND in shrimp. In this study, the pathological sections showed that after 4 h treatment, significant pathological changes were observed in the PirBvp treated group, and no obvious pathological changes was found in PirAvp treated group. In order to learn the mechanism of shrimp response to PirAvp and PirBvp, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp after treatment with PirAvp or PirBvp. A total of 9978 differentially expressed genes (DEGs) were identified between PirAvp or PirBvp-treated and PBS control shrimp, including 6616 DEGs in the PirAvp treated group and 3362 DEGs in the PirBvp treated group. There were 2263 DEGs that were commonly expressed, 4353 DEGs were only expressed in PirAvp VS PBS group and 1099 DEGs were uniquely expressed in PirBvp VS PBS group. Among these DEGs, the anti-apoptosis related pathways and immune response related genes significantly expressed in the commonly expressed DEGs of PirAvp VS PBS group and PirBvp VS PBS group, and small GTPase-mediated signaling and DNA metabolic process might relate to the host special reaction towards PirAvp and PirBvp exposure. The data suggested that the differential expression of these immune and metabolic-related genes in hepatopancreas might contribute to the pathogenicity variations of shrimp to VP-AHPND. The identified genes in this study will be useful for clarifying the response mechanism of shrimp toward different toxins of VP-AHPND and will further provide molecular basis for understanding the pathogenic mechanism of VP-AHPND.

Effects of Vibrio harveyi infection on the biochemistry, histology and transcriptome in the hepatopancreas of ivory shell (Babylonia areolata).

The ivory shell (Babylonia areolata) is one of the most promising high quality marine products. However, ivory shell is susceptible to Vibrio harveyi infection during the culture period. In this study, we investigated the biochemical indicators, histological changes and transcriptomic response in the hepatopancreas of ivory shells from the PBS control group (PC) and infection group (A3) with 1 × 109 CFU/mL V. harveyi after 24 h. Results showed that compared to the PC group, biochemical indicators, including malondialdehyde (MDA), reactive oxygen species (ROS), acid phosphatase (ACP), and Caspase 3 (Casp-3) were significantly increased (p < 0.05) in A3 group after V. harveyi infection for 24 h. Compared with the PC group, the hepatopancreas of A3 group were seriously damaged, the columnar epithelial cells of the tissue were enlarged, the space of digestive cells was increased, and vacuolar cavities appeared. A total of 95,581 unigenes were obtained and 2949 (1787 up-regulated and 1162 down-regulated) differential expressed genes (DEGs) were identified in the A3 group. GO and KEGG enrichment analysis showed that DEGs were mainly enriched in immune system process (GO:0002376), antioxidant activity (GO:0016209), lysosome (ko04142), toll and IMD signaling pathway (ko04624), and etc. These biological functions and pathways are associated with immune and inflammatory responses and apoptosis. 12 DEGs were randomly selected for real-time quantitative PCR (RT-qPCR) validation, and the expression profiles of these DEGs were consistent with the transcriptome data, confirming the accuracy and reliability of the transcriptome results. In summary, V. harveyi infection of ivory shells inducing oxidative stress, leading to severe hepatopancreatic damage, stimulating glutathione production to neutralize excessive ROS, and stimulating antimicrobial peptides production to counteract the deleterious effects of bacterial infection, which in turn modifying the immune and inflammatory response, ultimately resulting in apoptosis. This study provided valuable information to explore the immune regulation mechanism after V. harveyi infection and established molecular basis to support the prevention of V. harveyi infection.

Effects of monoglycerides of short and medium chain fatty acids and cinnamaldehyde blend on the growth, survival, immune responses, and tolerance to hypoxic stress of Pacific white shrimp (Litopenaeus vannamei).

Free fatty acids have long been used as dietary supplements in aquaculture, but the application of monoglycerides has increased interest in more recent times. The study aimed to investigate the effects of dietary short- and medium-chain fatty acid monoglyceride and cinnamaldehyde (SMMG) on the growth performance, survival, immune responses, and tolerance to hypoxic stress of Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, shrimp post-larvae were divided into 4 groups with 6 replicates and fed with diets supplemented with 0 (control), 0.3, 0.4, and 0.5% diet for 30 days. The final body weight and survival rate were determined. In Experiment 2, the juvenile shrimp from Experiment 1 were subjected to hypoxic stress conditions (dissolved oxygen level 2-2.5 mg/L) for 14 days, then the specific growth rate (SGR), survival rate, intestinal Vibrio spp. count, immune responses, and histopathological change of the hepatopancreas were analyzed. Following the 30-day feeding trial, the results revealed that the final body weight and survival of the 0.3-0.5% SMMG groups (2.81-3.06 g and 74.00-84.33%, respectively) were significantly higher than the control shrimp (1.96 g and 68.33%, respectively). In the hypoxic stress experiment, the survival rates of shrimp fed 0.4-0.5% SMMG (71.67-80.00%) were significantly higher than the control (51.67%). Although the SGR were not affected by SMMG supplementation, all immune parameters evaluated were significantly enhanced, and the intestinal Vibrio spp. counts were significantly decreased in the 0.4-0.5% SMMG-fed shrimp; the histopathological structure of the hepatopancreas was also improved in these shrimp compared to the control. Our findings indicated that SMMG as a feed additive has beneficial effects in improving shrimp health and increasing tolerance to hypoxic conditions.

In silico identification and functional study of long non-coding RNA involved in acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus infection in white shrimp, Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) caused by toxin-producing Vibrio parahaemolyticus (VpAHPND) has severely affected shrimp production. Long non-coding RNA (lncRNA), a regulatory non-coding RNA, which can play important function in shrimp disease responses. This study aimed to identify and investigate the role of lncRNA involved in VpAHPND infection in Pacific white shrimp, Litopenaeus vannamei. From a total of 368,736 de novo assembled transcripts, 67,559 were identified as putative lncRNAs, and only 72 putative lncRNAs showed differential expression between VpAHPND-infected and normal shrimp. The six candidate lncRNAs were validated for their expression profiles during VpAHPND infection and tissue distribution using RT-qPCR. The role of lnc2088 in response to VpAHPND infection was investigated through RNA interference. The result indicated that the suppression of lnc2088 expression led to an increase in shrimp mortality after VpAHPND infection. To explore the set of genes involved in lnc2088 knockdown, RNA sequencing was performed. A total of 275 differentially expressed transcripts were identified in the hepatopancreas of lnc2088 knockdown shrimp. The expression profiles of five candidate metabolic and immune-related genes were validated in lnc2088 knockdown and VpAHPND-infected shrimp. The result showed that the expression of ChiNAG was significantly increased, while that of NCBP1, WIPF2, and NFKB1 was significantly downregulated in ds2088-injected shrimp. Additionally, the expression of NFKB1, NCBP1 and WIPF2 was significantly increased, whereas that of ChiNAG and CUL5 were significantly decreased after infection with VpAHPND. Our work identified putative lncRNA profiles in L. vannamei in response to VpAHPND infection and investigated the role of lncRNA in shrimp immunity.

Transcriptome-based network analysis reveals hub immune genes and pathways of hepatopancreas against LPS in Amphioctopus fangsiao.

The hepatopancreas is the biggest digestive organ in Amphioctopus fangsiao (A. fangsiao), but also undertakes critical functions like detoxification and immune defense. Generally, pathogenic bacteria or endotoxin from the gut microbiota would be arrested and detoxified in the hepatopancreas, which could be accompanied by the inevitable immune responses. In recent years, studies related to cephalopods immune have been increasing, but the molecular mechanisms associated with the hepatopancreatic immunity are still unclear. In this study, lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, was used for imitating bacteria infection to stimulate the hepatopancreas of A. fangsiao. To investigate the immune process happened in A. fangsiao hepatopancreas, we performed transcriptome analysis of hepatopancreas tissue after LPS injection, and identified 2615 and 1943 differentially expressed genes (DEGs) at 6 and 24 h post-injection, respectively. GO and KEGG enrichment analysis showed that these DEGs were mainly involved in immune-related biological processes and signaling pathways, including ECM-receptor interaction signaling pathway, Phagosome signaling pathway, Lysosome signaling pathway, and JAK-STAT signaling pathways. The function relationships between these DEGs were further analyzed through protein-protein interaction (PPI) networks. It was found that Mtor, Mapk14 and Atm were the three top interacting DEGs under LPS stimulation. Finally, 15 hub genes involving multiple KEGG signaling pathways and PPI relationships were selected for qRT-PCR validation. In this study, for the first time we explored the molecular mechanisms associated with hepatopancreatic immunity in A. fangsiao using a PPI networks approach, and provided new insights for understanding hepatopancreatic immunity in A. fangsiao.

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.

Protective effects of butyric acid during heat stress on the survival, immune response, histopathology, and gene expression in the hepatopancreas of juvenile pacific shrimp (L. Vannamei).

This study looked at the effects of adding butyric acid (BA) to the diets of juvenile Pacific shrimp and how it affected their response to survival, immunity, histopathological, and gene expression profiles under heat stress. The shrimp were divided into groups: a control group with no BA supplementation and groups with BA inclusion levels of 0.5 %, 1 %, 1.5 %, 2 %, and 2.5 %. Following the 8-week feeding trial period, the shrimp endured a heat stress test lasting 1 h at a temperature of 38 °C. The results showed that the control group had a lower survival rate than those given BA. Interestingly, no mortality was observed in the group receiving 1.5 % BA supplementation. Heat stress had a negative impact on the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the control group. Still, these activities were increased in shrimp fed the BA diet. Similar variations were observed in AST and ALT fluctuations among the different groups. The levels of triglycerides (TG) and cholesterol (CHO) increased with high temperatures but were reduced in shrimp-supplemented BA. The activity of an antioxidant enzyme superoxide dismutase (SOD) increased with higher BA levels (P < 0.05). Moreover, the groups supplemented with 1.5 % BA exhibited a significant reduction in malondialdehyde (MDA) content (P < 0.05), suggesting the potential antioxidant properties of BA. The histology of the shrimp's hepatopancreas showed improvements in the groups given BA. Conversely, the BA significantly down-regulated the HSPs and up-regulated MnSOD transcript level in response to heat stress. The measured parameters determine the essential dietary requirement of BA for shrimp. Based on the results, the optimal level of BA for survival, antioxidant function, and immunity for shrimp under heat stress is 1.5 %.

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.

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.

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.

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.

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.

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.

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.