Activities Of Immune-related Enzymes Research Articles

Biomimetic nanovaccine based on erythrocyte membrane enhances immune response and protection against tilapia lake virus

An infectious disease emerged in recent years, Tilapia Lake Virus Disease (TiLVD), has severely restricted the development of global tilapia industry. Vaccination has proved potential strategy to prevent its causative agent Tilapia Lake Virus (TiLV) infectious. However, the response intensity of subunit vaccine is limited by its low immunogenicity, thus inclusion of adjuvants is required. Thus, we prepared a biomimetic nano-system (Cs-S2@M-M) with a particle size of ∼100 nm and an encapsulation efficiency of about 79.15% based on erythrocyte membrane. The immune response was detected after intramuscular injection to assess the effectiveness of the vaccine. The biomimetic system significantly up-regulates the expression of immune genes, enhances the activity of non-specific immune-related enzymes (P < 0.05) and improved relative percentage survival by 17.4%–26.1% in TiLV challenge. The biomimetic nano-system based on erythrocyte membrane induced significant immune response in tilapia and enhanced protection against TiLV, promising as a model for fish vaccines.

Pathology, Enzyme Activity and Immune Responses after Cryptocaryon irritans Infection of Golden Pompano Trachinotus ovatus (Linnaeus 1758)

Golden pompano (Trachinotus ovatus) has become an economically important fish in China in the past decade. However, Cryptocaryon irritans, a parasitic ciliate, causes considerable economic losses in the mariculture of T. ovatus. To characterize the pathogenesis of C. irritans in T. ovatus, the pathological properties, immune-related enzyme activity and expression of the NEMO gene was analysed. The results from the histological sections showed that there was considerable metamorphosis and hyperplasia in the parasitized sites (skin) with leukocyte aggregation and mucous cell increases after C. irritans infection. Moreover, the activities of four enzymes, that is, alkaline phosphatase (AKP), acid phosphatase (ACP), superoxide dismutase (SOD) and lysozyme (LZM), were significantly increased in different tissues after C. irritans infection. Furthermore, the ORF of T. ovatus NF-kappa-B essential modulator (ToNEMO) measures 1650 bp, encoding 548 amino acids. The ToNEMO transcripts were universally expressed in all examined tissues, with higher levels being observed in the immune-relevant and central nervous tissues. The mRNA levels of ToNEMO after C. irritans infection were significantly increased in the gill, skin, liver, spleen and head kidney. These results suggested that ToNEMO might be involved in immune responses and helped to elucidate the physiological response after the C. irritans infection of fish.

Transcriptome reveals the immune and antioxidant effects of residual chlorine stress on Cyclina sinensis

Residual chlorine is a common by-product of warm drainage in coastal nuclear power plants. when accumulating to some limit, it may threaten marine ecosystem especially for benthic clam. However, there are few studies on the molecular mechanisms related to immunity and antioxidant of residual chlorine stress on clams. In this study, the clam (Cyclina sinensis) was exposed for 96 h at different concentrations (0, 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 mg/L) of residual chlorine to observe its mortality, measure the activity of antioxidant and immune-related enzymes, and analyses the gene expression level in the hepatopancreas by using the transcriptome sequencing. The results showed that the mortality rate increased with the increase of stress time and concentration, and the mortality rate in the 400, 450 and 500 mg/L groups reached 100% at 96 h. The tolerance to residual chlorine of C. sinensis decreased with the increase of chlorine dioxide concentration, and the LC50 of 96 h was 217.6 mg/L by linear regression method. After residual chlorine stress, the activity of antioxidant-related enzymes (T-AOC and SOD) in the hepatopancreas showed a trend of first increase and then decrease with the extension of stress time. The immune-related enzyme activities of AKP and LZM showed a downward trend between 0 and 96 h, while the ACP enzyme activity showed a trend of first rising and then decreasing. Transcriptome analysis showed that residual chlorine stress significantly changed the expression levels of immune-related molecules associated with signal transduction, prophenoloxidase cascade, cell apoptosis and pattern recognition protein/receptor. Moreover, glutathione S-transferase (GST), heat shock protein (HSP) and other antioxidant-related genes were significantly affected under residual chlorine stress. This study provided valuable information for understanding the effects of residual chlorine stress on survival, physiological metabolism and molecular mechanisms of immune and antioxidant functions of C. sinensis.

The effects of dietary supplementation of ginseng stem and leaf saponins on the antioxidant capacity, immune response, and disease resistance of crucian carp, Carassius auratus.

This is the first study to explore the positive effects of ginseng stem and leaf saponins (GSLS) on antioxidant capability, immunity, and disease resistance of crucian carp. Seven hundred fifty crucian carps (initial body weight: 25 ± 0.15g (mean ± SE)) were randomly allocated into five groups with three replicates each; five diets supplemented with the final concentration of 0, 1, 2, 4, and 8g/kg GSLS were fed to crucian carp for 5weeks. The results demonstrated that, at a concentration of 8g/kg, the contents of IgM, C4, SOD, GSH-Px, and the activity of AKP in serum of crucian carp gradually increased at 7, 14, 21, 28, and 35days, and the expression of immune-relative cytokine genes (TNF-α, IL-10, IFN-γ) in the liver, spleen, and the intestinal tract also had a significant up-regulation (P < 0.05), and which were significant difference compared with control (P < 0.05). The above results demonstrated that dietary GSLS showed enhancement effects on the antioxidant and anti-inflammatory capability, and innate immune response of crucian carp. The feed of 8g/kg GSLS for 1week could improve the survival rate 44% more than the control group when crucian carp infected Aeromonas hydrophila (A. hydrophila). In conclusion, the addition of GSLS at a concentration of 8g/kg in the diet improve immune-related enzyme activity better, immune-relative cytokine expression, and disease resistance.

Defensive thrashing behavior of caterpillars incurs a fatal retaliation from predatory stinkbugs

Thrashing behavior is generally assumed to be an effective means of defense of caterpillars to escape arthropod predators. Yet, scant attention has been paid to subsequent performances of escaped caterpillars. To address this knowledge gap, we conducted experiments with predatory stinkbug Arma chinensis nymphs or adults. As prey, we used armyworm Mythimna separata caterpillar and examined developmental consequences of the prey escaping the predator by thrashing. Surprisingly, we found that caterpillars which escaped from the stylet insertion of the stinkbug by thrashing underwent poor development and were less likely to survive to pupation. This effect did not vary with the life stage of the stinkbug or the duration of its stylet insertion. On the other hand, sham injuries caused by needle puncturing did not significantly affect the caterpillar’s survival. We also examined the overall activities of antioxidant enzymes, immune-related enzymes, and neuroregulatory bioamines as a known insect response to envenomation. We found that these biochemicals were more abundant in caterpillars exercising thrashing behavior. Based on these findings, we speculate that the increased defensive behavior by thrashing results in an increased venom injection by the predator, which is responsible for the observed increased mortality in those caterpillars that practice thrashing.

Immune and intestinal microbiota responses to heat stress in Chinese mitten crab (Eriocheir sinensis)

Temperature is an important environmental stressor that affects the metabolism and immunity of Chinese mitten crab. In this study, we investigated immune-related enzyme activity, metabolism-related substances in haemolymph and intestinal microbiota responses of Eriocheir sinensis assigned to the control (24 °C) and heat stress (32 °C) groups. Based on the data obtained, the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase in the heat stress group exhibited a trend of increasing first and then decreasing, and all peaked at 12 h or 24 h. The metabolism-related substances in the haemolymph, including glucose, total cholesterol, and total protein, all showed significant differences between the control and heat stress groups from the mid to late period (P < 0.05). Under heat stress, the levels of alkaline phosphatase and acid phosphatase were both significantly higher than those of the control group from 12 h to 24 h and significantly lower at 72 h and 96 h (P < 0.05). The T-AOC of the heat stress group was significantly lower than that of the control group from 48 h to 96 h (P < 0.05). High-throughput 16S ribosomal DNA (rDNA) sequencing revealed that heat stress altered the composition of intestinal microbes of Eriocheir sinensis. Specifically, the abundance of the beneficial bacteria Candidatus, Hepatoplasma and Marinifilum decreased significantly, while the abundance of the pathogenic bacteria Rhodococcus and Morganella increased significantly (P < 0.05). The results revealed that acute heat stress exposure affected immune function and intestinal microbial community in E. sinensis. This work can help to uncover the mechanisms of heat stress responses in this species.

Oxidative stress and immune response of hepatopancreas in Chinese mitten crab Eriocheir sinensis under lipopolysaccharide challenge

Chinese mitten crab (Eriocheir sinensis; H. Milne Edwards, 1853) is one of the important farmed crustaceans in China. Lipopolysaccharide (LPS), as a harmful factor, is prone to occur during the farming process of crabs. Aiming to test the hypothesis that damage degrees of the hepatopancreas in E. sinensis is correlated to LPS concentrations, in this study, E. sinensis were injected with LPS (50 μg/kg, and 500 μg/kg) and analyzed for the activity of antioxidant and immune-related enzymes, immune-related gene expression, and histopathological of hepatopancreas. As result, the hepatopancreas of E. sinensis immune-related genes, i.e., Dorsal, HSP90, Toll2, TLRs, Tube, and proPO, were significantly affected by LPS challenge. Among immune-related genes, Dorsal and proPO might play key roles in combating the LPS challenge. The activity of CAT gradually decreased with the increase of time, and the total antioxidant capacity was decreased after LPS challenge, indicating the inhibition of LPS on the antioxidant system. Interestingly, the decreasing trend of AKP and ACP activity suggested the immune system of crabs was affected by LPS challenge. The hepatopancreas section showed that the damage degree of hepatopancreas was different under the challenge of LPS with different concentrations, and the damage degree was proportional to the concentration. Our findings provide useful information for understanding the mechanism of hepatopancreas injury of E. sinensis induced by LPS infection.

Toxicity of the microcystin-producing cyanobacteria Microcystis aeruginosa to shrimp Litopenaeus vannamei

Microcystis aeruginosa is reported to cause cyanobacterial blooms in shrimp breeding ponds, which can result in significant shrimp mortality. However, the toxic effects of M. aeruginosa on Litopenaeus vannamei are still not completely understood. In this paper, the toxicity of M. aeruginosa cells to L. vannamei was examined, and the toxic components in the cells were analyzed through high-pressure liquid chromatography (HLPC). In addition, the immune response of shrimp to the M. aeruginosa cell extract was assessed by measuring the activity of immune-related enzymes, as well as the transcription of the relevant genes. The results showed that M. aeruginosa cells, extract and cell-free cultured medium resulted in a 100%, 98.3%, and 1.7% mortality rate in shrimp, respectively. HPLC analysis results revealed the presence of microcystin-LR (MC-LR) at a concentration of 190.40 mg/kg of cells. In addition, the activity and gene transcription of two immune related enzymes, SOD and LZM, were both significantly reduced in shrimp hepatopancreas (p < 0.05) after injection with extract. However, reduced glutathione (GSH) content was slightly increased, but the ratio of GSH to GSSG decreased. The transcription of gst gene function as detoxification, was significantly downregulated (p < 0.05). The results demonstrated that M. aeruginosa cell extract was highly toxic to L. vannamei, and exerted a negative effect on shrimp immunity including reduction of antioxidant capacity, antibacterial activity and detoxification activity, due to toxins including microcystin-LR.

Alleviating effects of essential oil from Artemisia vulgaris on enteritis in zebrafish via modulating oxidative stress and inflammatory response

Artemisia vulgaris (A. vulgaris) is a traditional Chinese medicine widely distributed in China and contains many bioactive compounds with pharmacological effects. However, the anti-inflammatory effects and mechanism of essential oil from A. vulgaris on enteritis in fish are still unclear. In this study, in order to elucidate the underlying mechanism of essential oil from A. vulgaris on zebrafish enteritis, zebrafish were used for establishing animal models to observe the histopathological changes of intestines, determine the activities of immune-related enzymes and oxidative stress indicators, and the mRNA expression of genes in MyD88/TRAF6/NF-KB signaling pathways. The results showed that different doses of A. vulgaris essential oil could effectively alleviate zebrafish enteritis in a dose- and time-dependent manner by improving the intestinal histopathological damage, decreasing the intestinal oxidative stress, repairing the intestinal immune ability, changing the expression levels of IL-1β, IL-10 and genes in MyD88/TRAF6/NF-κB pathway. In addition, co-treatment with oxazolone and MyD88 inhibitor could alleviate the morphological damage, the induction of oxidative stress, and the levels of immune-related enzymes and the mRNA expression of genes in MyD88/TRAF6/NF-κB signaling pathway. Moreover, essential oil from A. vulgaris had more significantly therapeutic effects on enteritis of male zebrafish than that of female zebrafish. This result will clarify the therapeutic effect and anti-inflammatory mechanism of essential oil from A. vulgaris on zebrafish enteritis, and provide a theoretical basis for further research on the rationality of A. vulgaris to replace feed antibiotics.

Comparative study of immunological biomarkers in the carpet shell clams (Ruditapes decussatus) from metal-contaminated sites in the South Lagoon of Tunis (Tunisia)

The South Lagoon of Tunis (Tunisia) is a Mediterranean lagoon adversely affected by industrial contaminants, harbour activity and untreated urban sewage. In this lagoon, the clam Ruditapes decussatus has been widely used as a biomonitor of seawater pollution through measurements of parameters related to oxidative stress and neurotoxicity. However, few studies have considered parameters of the immune system of this species in the South Lagoon of Tunis. Therefore, the aim of the present work was to evaluate several immune-related parameters in the cell-free haemolymph of carpet shell clams sampled during August and February from three polluted sites in the South Lagoon of Tunis (S1, S2 and S3) and one less polluted site as a reference site (RS) in order to identify suitable biomarkers for environmental quality assessments of this ecosystem. Concerning the immune-related parameters, seasonal factors modulated phenoloxidase, lysozyme, protease and esterase activity, with lower values measured for samples collected in August than for samples collected in February. In fact, bactericidal activity against two of the pathogenic bacteria tested and the activity of most immune-related enzymes were reduced in the cell-free haemolymph of clams collected from the most sampling sites in August compared to February one. In addition, values of abiotic parameters (temperature, salinity and pH) and metal (cadmium, copper, iron, lead and zinc) concentrations in the clams’ soft tissues, previously obtained and published by the authors, as well as the values of immune-related parameters were integrated using principal component analyses. Results indicated that the values of all measured immune-related parameters were negatively correlated with the temperature values and the variations most of these parameters highlighted that the chemical industrial area (S3) was the most impacted location within the South Lagoon of Tunis. The present study illustrates that the immune-related parameters measured in carpet shell clam cell-free haemolymph represent suitable biomarkers for environmental quality assessments because they provide effective seasonal and spatial discrimination.

Positive effects of replacing commercial feeds by fresh black soldier fly (Hermetia illucens) larvae in the diets of Pacific white shrimp (Litopenaeus vannamei): Immune enzyme, water quality, and intestinal microbiota

This study investigated the effects of replacing commercial feeds with fresh black soldier fly larvae (BSFL) on the immune enzyme activities, water quality in the culturing environment, and intestinal microbiota of Pacific white shrimp (Litopenaeus vannamei). Five different feeding regimes were designed, in which 0% (control group), 25%, 50%, 75%, and 100% commercial feeds were replaced with equal wet weight of fresh BSFL, respectively. The experiment lasted for 45 days, and the results showed that the serum T-NOS (total nitric oxide synthase), AKP (alkaline phosphatase), and ACP (acid phosphatase) activity were significantly increased in the BSFL25% group. However, the immune enzymes of the hepatopancreas decreased significantly in all the BSFL-containing groups compared to the control group. Total ammonia nitrogen concentrations throughout the experimental phase and nitrate concentrations in the middle and later phases of the experiment were negatively correlated with the replacement rate of fresh BSFL in the diet, whereas pH was positively correlated with the replacement rate of fresh BSFL in the diet. Proteobacteria and Bacteroidetes were dominant phyla in the intestines of shrimps, but the relative abundances of Proteobacteria and Firmicutes decreased, with those of Bacteroidetes and Planctomycetes increased in the BSFL-containing groups. Among them, higher relative abundances of potential probiotics such as Motilimonas, Shimia, Pseudoalteromonas, and Shewanella and lower relative abundance of genus Vibrio were observed in the fresh BSFL-containing groups. Furthermore, shrimps fed with BSFL-containing diets had higher bacterial richness and diversity in the intestines. In conclusion, a proper replacement of commercial feed with fresh BSFL had a positive effects on the immune-related enzyme activities, water quality, and intestinal health in the L. vannamei.

Effects of two fish-derived probiotics on growth performance, innate immune response, intestinal health, and disease resistance of Procambarus clarkii

Selection of the appropriate strain for particular aquatic animals is necessary for the desirable benefits of probiotics application in aquaculture, however, it is not very clear whether fish-derived probiotics could provide beneficial effects on invertebrates. In the present study, two fish-derived probiotics, Bacillus amyloliquefaciens, and Lactococcus lactis, were administrated as feed additives for crayfish (Procambarus clarkii) at concentrations of 106, 107, and 108 CFU/g for 28 days, respectively. Then their effects on the growth performance, innate immune enzyme activity, immune-related genes expression, haemocyte phagocytic activity, intestinal microbial diversity, and pathogen resistance of crayfish were evaluated. The results showed that the final weight, weight gain (WG), and specific growth rate (SGR) were significantly higher in 108 CFU/g B. coagulans and L. lactis additive groups. Compared to the control group, the activities of immune-related enzymes (acid phosphatase, alkaline phosphatase, glutathione peroxidase, and lysozyme) in the intestine were significantly increased in most of the probiotics added groups. In almost all experimental groups, the mRNA expression of two AMP genes (PcALF3 and PcAst) was enhanced in the intestinal of crayfish, and the expression level was proportional to the amounts of probiotics used. Moreover, the intestinal of crayfish in probiotics groups have better integrity and a thicker mucosal layer, and higher density granules in epithelial cells were also observed. According to the results of next-generation sequencing technologies, the diversity of intestinal microbiota in crayfish was also increased after supplementation with B. coagulans or L. lactis. Finally, the phagocytosis rate of haemocytes and pathogen resistance has also been improved. Taken together, our results suggested that fish-derived probiotics have beneficial effects on crayfish, and they can be treated as potential feed additives in practical applications.

Histological, physiological, and transcriptomic responses of hepatopancreas to air exposure in asian freshwater clam Corbicula fluminea.

Corbicula fluminea (C. fluminea) is an important freshwater economy shellfish in China, but it often suffers from air exposure during transportation. In this study, we investigated the histological, physiological (mainly including respiratory metabolism, antioxidant capacity, and immune function), and transcriptomic responses of hepatopancreas in C. fluminea to different times of air exposure. At histological level, air exposure caused vacuolation of digestive cells (24–96 h) and enlargement of digestive tubule lumen (6–96 h) in hepatopancreas. At physiological level, the activities of enzymes related to glycolysis (hexokinase and pyruvate kinase) and anaerobic respiration (lactate dehydrogenase) were increased first (6–24 h) of air exposure, then came back to normal level or even decreased. The activity of aerobic respiration-related enzyme (succinic dehydrogenase) began to reduce from 24 h of air exposure. The activities of antioxidant enzymes (superoxide dismutase and catalase) were enhanced during 6–48 h of air exposure and then returned to control level or even inhibited. The content of malondialdehyde (MDA) increased from 96 h of air exposure. The activities of immune-related enzymes (acid phosphatase and alkaline phosphatase) increased during 6–48 h, then returned to normal or began to decline. At transcriptome level, 44 differentially expressed genes (DEGs) in the hepatopancreas were identified after 96-h air exposure. Among these DEGs, 8 were associated with glycolysis, TCA cycle, immune, and antioxidant, and were downregulated after 96-h air exposure. Taken together, these findings illuminated the response of C. fluminea to air exposure at histological, physiological, and transcriptomic levels, which will be beneficial to the aquaculture and transportation of C. fluminea.

Effects of Bacillus subtilis DSM 32315 (Gutcare®) on the growth performance, antioxidant status, immune ability and intestinal function for juvenile Litopenaeus vannamei fed with high/low-fishmeal diets

This study was conducted to investigate the effects of dietary Bacillus subtilis DSM 32315 (Gutcare®) supplementation on the growth performance, antioxidant status, immune ability and intestinal function in juvenile Litopenaeus vannamei fed with high/low-fishmeal diets. A 4 × 2 two-factor design was formulated with graded dietary Gutcare® and fishmeal levels. Specifically, four Gutcare® supplemental levels (0, 0.05%, 0.15% and 0.3%) were added under two fishmeal diets (20% and 5% fishmeal levels). Each diet was randomly assigned to five replicate groups of 30 juvenile Pacific white shrimp that were stocked in fiberglass tanks. The results indicated no significant inhabitation on the growth performance, hepatopancreas antioxidant capacity, immune response and intestinal function at 5% fishmeal compared with 20% fishmeal. Shrimp fed with Gutcare® supplementation regardless of high/low-fishmeal diets exhibited higher growth performance, antioxidant- and immune-related enzyme activities (P < 0.05) compared with that 0% Gutcare® supplementation. However, none of the treatments found significant differences in feed efficiency and survival. The mRNA expression levels of Cu/Zn superoxide dismutase, heat shock protein 70, glutathione peroxidase, prophenoloxidase and lysozyme were significantly up-regulated (P < 0.05) at shrimp fed with Gutcare®. Moreover, the results of mid-intestine morphology showed a significant increase with the addition of Gutcare®, and the highest height and thickness of intestinal epithelium occurred at shrimp fed 0.15% Gutcare®. Bacterial community composition showed that shrimp fed with the graded Gutcare® significantly increased the abundance of Firmicute and reduced the relative abundance of Vibrio (P < 0.05). In conclusion, 5% fishmeal supplementation with well-balanced amino acid composition had no adverse effect on the growth performance of L. vannamei when compared with the 20% fishmeal. Dietary Gutcare® significantly enhanced immunity and antioxidant capacity, and improved intestinal function of L. vannamei. The positive effect of Gutcare® at 5% fishmeal diet provided a possibility for reducing fish meal in the commercial diets of Pacific white shrimp.

Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

Response of Gut Microbiota, Digestive Enzyme Ability, and Immune Function to Starvation in the Oriental River Prawn Macrobrachium nipponense

In order to evaluate the interplay of nutritional status and the digestive capacity, immune function, and gut microbiota in the oriental river prawn Macrobrachium nipponense, a 14-day starvation trial was conducted to detect the effects of starvation on the activities of digestive enzymes and immune enzymes and the structure of prawn gut microbiota. The adult prawns were randomly assigned to a control group (F group) and starvation group (S group) with three replicates. The F group was normally fed with commercial diet, and the S group was starved for 14 days. The result showed that digestive enzyme activities of trypsin and lipase in the hepatopancreas of the S group were significantly lower than those of the F group after 4 d of starvation ( P &lt; 0.05 ), while the activity of amylase significantly increased after 14 d of starvation ( P &lt; 0.05 ). Moreover, the results of the histological analysis of prawn gut showed that both the height of epithelial cells and microvilli of intestine in the S group were obviously decreased than those in the F group ( P &lt; 0.05 ) after 14 d of starvation. The activities of immune-related enzymes including superoxide dismutase, catalase, and acid phosphatase in the hepatopancreas of the S group significantly decreased after 4 d of starvation, and lysozyme activity of the F group was lower than that of the S group after 7 d of starvation ( P &lt; 0.05 ). In addition, the results of Illumina high-throughput sequencing showed that a total of 14,285 OTUs were obtained and classified into 30 phyla, among which Actinobacteria and Proteobacteria were the predominant microflora in the intestinal microbial communities of both groups. However, the relative abundance of opportunistic pathogens was significantly increased in the S group, while the relative abundance of beneficial bacterium was decreased ( P &lt; 0.05 ). The bacterial richness with the Chao estimator was significantly higher in the F group than in the S group ( P &lt; 0.05 ). Both the results of principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) demonstrated that the intestinal microbiota of the F group was separate from those of the S group. The result of functional prediction of the metabolic pathways showed that the intestinal microbiota was enriched in the KEGG pathways of amino acid, carbohydrate, fatty acid, and lipid biosynthesis and degradation at level 2. This result implied that the microbiota of shrimp gut decreased nutrition metabolism under the stress of starvation. Meanwhile, comparing to the F group, the immune-related pathway of enterobacterial common antigen biosynthesis was markedly reduced in the S group ( P &lt; 0.05 ). The result of redundancy analysis (RDA) further confirmed that the activities of digestive and immune enzymes were correlated with the microbial community structure. Finally, the structural equation modeling highlighted that changes in the activities of digestive and immune enzymes were directly related to the gut bacterial community and notably affected prawn growth.

Detection of immune‐related enzyme activity, gene expression and metabolomic profiling in Chinese mitten crab Eriocheir sinensis parasitized by Polyascus gregaria

Polyascus gregaria is a specialized crustacean parasite of Chinese mitten crab; however, only limited information on interactions between parasite and its host is available. This study examined interactions between P. gregaria and Chinese mitten crab with regard to immune-related enzyme activities and expression of respective genes in the hepatopancreas. To further elucidate the pathogen–host interactions, untargeted gas chromatography–mass spectrometry (GC–MS) metabolomics was used to analyse changes in metabolites in response to P. gregaria infection. Superoxide dismutase, acid phosphatase and alkaline phosphatase activities were significantly increased in parasitized crabs, whereas phenoloxidase activity was significantly decreased. Moreover, the expression of three immune-related genes, that is proPO, Toll1 and Toll2, was markedly downregulated in parasitized crabs. In the metabolomic assay, 73 differential metabolites were identified, and they were significantly enriched in 71 pathways. Among these metabolites, 58 were significantly upregulated and 15 were downregulated. Lysolipids lysoPE, lysoPC, lysoPG, LysoPI and lysoPS were the most strongly upregulated metabolites, and 9-cis-retinol, glycerophosphocholine and 5-thymidylic acid were the top three downregulated metabolites. Most of these metabolites were enriched in the glycerophospholipid metabolism pathway, which was one of the most significantly activated pathway responses to P. gregaria infection. This increase in lysoglycerophospholipids caused major structural and functional abnormalities in the membranes of host tissues, especially in the hepatopancreas. We propose that long-term infection with P. gregaria induces silence of the innate immune system, disturbs intracellular homeostasis and causes cell membrane immune-related dysfunction in Chinese mitten crab. These results may help us better understand P. gregaria infection in Chinese mitten crab.

Mucosal immune responses and protective efficacy in yellow catfish after immersion vaccination with bivalent inactivated Aeromonas veronii and Edwardsiella ictaluri vaccine

Mucosal vaccination, which has the potential to induce both mucosal and systemic immune responses, is considered the most suitable method of preventing infectious diseases in farmed fish. Aeromonas veronii and Edwardsiella ictaluri are two pathogenic bacteria found in yellow catfish and often infect the fish through mucosal surfaces. Delivery of a bivalent inactivated vaccine by injection has been shown to induce a strong systemic immune response against both bacterial infections. However, mucosal immune responses and protective efficiency induced by this inactivated vaccine administrated via immersion are yet to be investigated. We developed a bivalent vaccine containing formalin-inactivated A. veronii and E. ictaluri and evaluated the immune response in yellow catfish after immersion vaccination using body fluids biochemistry indices, agglutinating antibody titers, and the expression level of immune-related genes in the skin, gills, spleen, and head kidney. The activities of innate immune-related enzymes and specific agglutination antibody titers in body fluids, as well as the expression of innate and adaptive immune-related genes in both the mucosal and systemic tissues of vaccinated fish, were significantly higher than that in control fish. Next, we assessed the protective efficacy by a challenge model of virulent strains of E. ictaluri and A. veronii. The relative survival percent of vaccinated fish was 80% and 87% after challenging fish with E. ictaluri and A. veronii, respectively, which was higher than unvaccinated control fish (43% and 57%). These results confirm that the bivalent inactivated vaccine administered via immersion induces a strong mucosal immune response and confers good protection against both E. ictaluri and A. veronii. Our results also reinforce the notion that immersion vaccination could stimulate both mucosal and systemic immunity contributing to protection against pathogens.

Control of Edwardsiella tarda infection in turbot Scophthalmus maximus (L.) using phage vB_EtaM_ET‐ABTNL‐9

In recent years, turbot (Scophthalmus maximus L.) ascites caused by Edwardsiella tarda has occurred frequently in China, causing serious economic losses. In this study, with E. tarda ET9 as the host bacteria, a lytic phage was isolated and named vB_EtaM_ET-ABTNL-9 (referred to as PETp9). The effect of using phage to prevent and control E. tarda infection in turbot was discussed. The results showed that the best preventive effect was achieved after 7 days of feeding phage with multiplicity of infection (MOI) = 10 in the phage-feeding experiment; in the phage-injection therapy, treatment was most effective when the phage with MOI = 10 was injected intraperitoneally immediately after the challenge; in the phage-immersion therapy, the best therapeutic effect was obtained when the infected turbot were immersed in phage-seawater mixture (MOI = 10) for 3 h. During the experimental period, the activities of non-specific immune-related enzymes in the blood of turbot were significantly increased in the untreated group, while that of the phage treated group showed a downward trend and gradually reached normal levels. It was also observed that a certain concentration of phages was present in both turbot cultured water and the intestinal tract during the experiment. Therefore, phage may be another effective control agent against E. tarda infection in turbot culture.

Effects of Replacing Fishmeal by Raw or Lactobacillus acidophilus-Fermented Soybean Meal on Growth, Intestinal Digestive and Immune-Related Enzyme Activities, Morphology, and Microbiota in Turbot (Scophthalmus maximus L.)

This study was performed to investigate the fermentation efficiency of Lactobacillus acidophilus and compare the effects of dietary soybean meal (SM) or L. acidophilus-fermented SM (LASM) on turbot. Two hundred and seventy juvenile turbots ( 13.50 ± 0.13 g ) were randomly divided into three treatments fed with a fishmeal-based diet (CNT) and two experimental diets with 45% fishmeal protein replaced by SM or LASM for 8 weeks. The results showed that fermentation significantly improved the essential amino acid profile, increased the lactic acid content, and reduced the antinutritional factor level in SM. Inferior growth of fish was observed in the SM group after the feeding trial, while the LASM group exhibited comparable performance to the CNT group. Activities of intestinal digestive enzymes (trypsin and diastase) and immune-related enzymes (alkaline phosphatase, acid phosphatase, and lysozyme) decreased significantly in the SM group, while no significant reduction was found in the LASM group compared to the CNT group. Dietary LASM alleviated SM-induced intestinal pathological disruption with higher villus and normal lamina propria width. Dietary LASM positively regulated the intestinal microbiota, making the overall profile more coherent with that in the CNT group. Spearman’s correlation analysis revealed that the altered intestinal microbiota was closely linked to the digestive enzyme activities. Collectively, this study indicated that L. acidophilus fermentation significantly improved the nutritional quality of SM and relieved SM-induced adverse effects on turbot, in terms of growth, intestinal digestive and immune-related enzyme activities, morphology, and microbiota.