Effects of vitamin C combined with sodium alginate on serum biochemistry, oxidative stress, gill tissue morphology, and muscle quality of pearl gentian grouper during waterless transport.

This research examined the effects of sodium alginate (SA) and vitamin C (Vc) soaking of pearl gentian grouper before waterless transportation from the perspectives of serum parameters, oxidative stress, muscle quality, and gill tissue morphology. After the fish reached semi-dormancy with a cooling rate of 3 °C/h, fish (420 ± 25 g) were distributed to 4 treatments as follows: S1 group (50 mg/L Vc and 0.1% SA were added), S2 group (50 mg/L Vc and 0.3% SA were added), S3 group (50 mg/L Vc and 0.5% SA were added), and control group (without soaking in protective fluid). After oxygenated packaging, samples were taken at 0, 8, and 16 h of waterless transportation and 12 h after rehydration, respectively. It was found that after 16 h of waterless transport, compared with the control group, cortisol, glucose, blood urea nitrogen (BUN), uric acid (UA), creatinine (CREA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels were significantly decreased (p < 0.05), while albumin, lysozyme (LZM), muscle pH, and total free amino acid (TFAA) contents were significantly elevated (p < 0.05) in the S3 group. Moreover, by gill tissue microscopy, it was found that the protective solution of group S3 did not cause serious deleterious morphological changes to the gill epithelium. The results showed that the grouper was soaked by protective fluid before waterless could maintain surface moisture, reduce gill and kidney function and oxidative stress damage, and maintain the stability of muscle quality. This study provides a novel transportation method for waterless preservation, which helps to reduce transportation costs and improve transportation efficiency.

Immune-enhancing effects of Astragalus polysaccharides and Ganoderma lucidum polysaccharides on Vibrio harveyi flgJ DNA vaccine in grouper.

Astragalus polysaccharides (APS) and Ganoderma lucidum polysaccharides (GLP) have been shown to possess strong immunoregulatory properties in aquatic animals. In this study, the fragment containing Vibrio harveyi flgJ gene was ligated into pcDNA3.1(+) vector and pcDNA3.1(+)-flgJ was constructed as DNA vaccine. APS and GLP were used as DNA vaccine adjuvants to evaluate the immunoregulatory effect by intramuscular injection to pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚E. lanceolatus). The results showed that pcDNA3.1(+)-flgJ combined with APS or GLP could significantly up-regulate the innate and adaptive immune response in fish, including serum-specific antibody titres, catalase and lysozyme activities. At the same time, DNA vaccine combined with APS or GLP significantly up-regulated the expression levels of CD8α, IgM, IL-1ß, MHC-Iα, MyD88 and TLR3 genes in thymus, head kidney, spleen and liver of pearl gentian grouper in comparison with those of the pFlgJ group. After 42 days post-vaccination, V. harveyi was used to challenge pearl gentian grouper by intraperitoneal injection. The relative percentage of survival (RPS) of pFlgJ, pFlgJ +APS, pFlgJ +GLP and pFlgJ+APS+GLP groups were 69%, 81%, 77% and 88%, respectively. These results suggested APS and GLP were potential adjuvants for DNA vaccine against V. harveyi infection in pearl gentian grouper.

Identification and whole-genome sequencing analysis of Vibrio vulnificus strains causing pearl gentian grouper disease in China.

Vibrio vulnificus is a pathogenic bacterium that causes disease in marine fish, affecting fish farming and human health worldwide. In May 2021, in the Bohai Bay region, a disease broke out in commercially farmed pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus), causing huge economic losses. The diseased fish had skin lesions, water accumulation in their abdomens, and showed tissue and organ damage. V. vulnificus biotype 2 has been reported in eels and other marine fish, but it is less reported in pearl gentian grouper. In this study, the pathogenic strain isolated from diseased fish was identified as V. vulnificus EPL 0201 biotype 2 on the basis of physiological and biochemical characteristics and the results of 16S rRNA gene and gyrB sequencing, virulence gene detection, and recursive infection experiments. To gain a comprehensive understanding of the pathogenicity and drug resistance of this strain, whole-genome sequencing was performed. Whole-genome analysis showed that the gene map of this strain was complete. The Virulence Factor Database annotation results showed that this strain had the key virulence factor genes vvhA and rtxA, which cause host disease. In addition, this strain had genes conferring resistance against cephalosporins, aminoglycosides, tetracyclines, and sulfonamides. Antimicrobial susceptibility testing confirmed the presence of these resistance genes identified in the genome. The results of this study show that V. vulnificus EPL 0201 biotype 2 is a multi-drug resistant strain with high pathogenicity.

Construction of a phosphodiesterase mutant and evaluation of its potential as an effective live attenuated vaccine in pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus).

Vibrio alginolyticus is a dominant pathogen that causes vibriosis of fish and shellfish. VAGM003125 is a specific phosphodiesterase bearing HD-GYP domain, which extensively regulates multicellular behavior and physiological processes in bacteria. In this study, an in-frame deleted ΔVAGM003125 mutant was constructed and changes of ΔVAGM003125 mutant in physiology and pathogenicity were examined. The potential application of ΔVAGM003125 mutant as a live attenuated vaccine was also assessed. The ΔVAGM003125 mutant displayed no significant differences in the growth rate and morphology in comparison to the wild type strain. However, the ΔVAGM003125 mutant significantly enhanced biofilm formation compared to the wild type strain. Also, the ΔVAGM003125 mutant was noted as being able to attenuate swarming motility, ECPase, and adherence compared to the wild type strain. Moreover, the ΔVAGM003125 mutant induced high antibody titers and provided effective immune protection, which was evidenced with a relative survival rate of 81% without histopathological abnormality. Following ΔVAGM003125 mutant vaccination, immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus) including IgM, MHC-Iα, IL-16, IL-1, and TNF-α was up-regulated. Taken together, the present data suggested that the ΔVAGM003125 mutant might be applied as an attenuated live vaccination against V. alginolyticus during fish culture.

Functional characterization of VscCD, an important component of the type â ¢ secretion system of Vibrio harveyi.

Vibrio harveyi is a Gram-negative bacterium that occurs widely in the ocean and a kind of pathogenic bacteria associated with vibriosis in grouper. We investigated whether the VscCD protein of the type Ⅲ secretion system (T3SS) was important for pathogenicity of V. harveyi. Mutations to the vscC and vscD genes (ΔvscCD) and complementation of the ΔvscCD mutant (C-ΔvscCD) were created. Moreover, the biological characteristics of the wild-type (WT) and mutant strains of V. harveyi 345 were compared. The results showed that deletion of the vscCD genes had no effect on bacterial growth, swimming/swarming ability, secretion of extracellular protease, or biofilm formation. However, as compared with the V. harveyi 345: pMMB207 (WT+) and complementary (C-ΔvscCD) strains, the ΔvscCD: pMMB207 (ΔvscCD+) mutant displayed decreased resistance to acid stress, H2O2, and antibiotics. In addition, infection of the pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) showed that as compared with the WT+ and C-ΔvscCD strains, the ΔvscCD+ strain significantly reduced cumulative mortality of the host. The colonization ability of the ΔvscCD+ mutant in the spleen and liver tissues of the pearl gentian grouper was significantly lower than that of the WT+ and C-ΔvscCD strains. In the early stage of infection with the ΔvscCD+ strain, the expression levels of IL-1ß, IL-16, TLR3, TNF-α, MHC-Iα, and CD8α were up-regulated to varying degrees. As compared with the WT+ and C-ΔvscCD strains, luxR expression was significantly up-regulated in the ΔvscCD+ strain, while the expression of vcrH and vp1668 was significantly down-regulated. As an important component of the T3SS, VscCD seemed to play a significant role in the pathogenesis of V. harveyi.

Establishment and characterization of a gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀) and its application in cadmium toxicology.

A novel gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀, PGGG cell line) was established, its application in cadmium (Cd) toxicology was demonstrated in this study. Primary cultures and PGGG subcultures were carried out at 25 °C in Dulbecco's Modified Eagle medium/F12 medium (1:1; pH 7.2) supplemented with 15% fetal bovine serum (FBS). Primary PGGG cells were spindle-shaped, proliferated into a confluent monolayer within two weeks and were continuously subcultured over passage 60. The growth of cells at passages 20, 40, and 60 was examined. Chromosome analysis revealed that the chromosomal number of normal PGGG cells was 48, but the number of cells with the normal chromosomes number decreased during the passaging process. Cadmium is one of the most toxic metals in aquatic systems and has been associated with multiple animal and human health problems. To interpret the cytotoxicity and related mechanisms of cadmium, PGGG cells were used as an in vitro model. After treatment with cadmium at concentrations ranging from 1 µM to 500 µM, PGGG cells demonstrated dose- and time-dependent cytotoxicity, manifested as morphological abnormalities and a viability decline. Further, it was found that the reactive oxygen species (ROS) and malondialdehyde (MDA) levels were elevated following cadmium exposure, and related genes involved in the antioxidant system, including those encoding catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and Kelch-like- ECH-associated protein 1 (Keap1), were regulated differently. In addition, PGGG cells treated with cadmium had the typical features associated with apoptosis, including phosphatidylserine (PS) externalization; upregulated expression of caspase-3, -8, and -9; and apoptotic body formation. In general, the PGGG cell line may serve as a useful tool for studying the toxic mechanisms of cadmium or other toxicants or for toxicity testing and environment monitoring.

Construction and evaluation of Vibrio alginolyticus ΔclpP mutant, as a safe live attenuated vibriosis vaccine.

Vibrio alginolyticus is a common and serious pathogen threatening the progress of coastal aquaculture. ClpP protease has been proved to be closely associated with biofilm formation, stress tolerance, autolysis and virulence in several pathogens. Hence, targeting ClpP may be a potentially viable, attractive option for the preparation of vaccine in preventing vibriosis. In this study, an in-frame deleted mutant strain (ΔclpP) was constructed by allelic exchange mutagenesis to investigate physiological role of clpP in pathogenicity of V. alginolyticus and evaluate its potential as a live attenuated vaccine. The results exhibited that ΔclpP showed no differences in external morphology, growth, swarming motility and ECPase activity. However, ΔclpP represented an increment in biofilm formation, and a decrement in adherence to CIK cells. In addition, virulence of ΔclpP was examined in pearl gentian grouper and was found to be seriously attenuated. ΔclpP induced high antibody titers and provided a valid protection with a relative percent survival value of 83.8% without histopathologic abnormality. Our results indicated ΔclpP showed a great potential to be a live attenuated vaccine.

Protective effects of ß-glucan as adjuvant combined inactivated Vibrio harveyi vaccine in pearl gentian grouper.

Vaccination is one of the strategies for preventing Vibrio harveyi infection in marine-cultured animals. In this study, we prepared a formalin-killed cells of V. harveyi ZJ0603 vaccine (FKC) combined with ß-glucan to immune pearl gentian grouper. The results indicated that the expression levels of IgM, TNF-α, MHC-Iα, IL-1ß and IL-16 significantly increased in the spleen of the vaccinated fish. Antibody titers, activities of lysozyme and superoxide dismutase were significantly prompted in blood of the vaccinated fish. After 35 d post-vaccination, all fish were challenged intraperitoneally by virulent V. harveyi, and the relative percentage of survival (RPS) of FKC+ß-glucan, FKC, ß-glucan and PBS were 68 ± 5.7%, 55 ± 8.5%, 42 ± 7.5% and 32 ± 6.9%, respectively. These results demonstrated that ß-glucan could be as a potential adjuvant of FKC and provide good protective effect against V. harveyi infection in the pearl gentian grouper culture.

Single-walled carbon nanotubes enhance the immune protective effect of a bath subunit vaccine for pearl gentian grouper against Iridovirus of Taiwan.

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-ß, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines.

Protection against Vibrio alginolyticus in pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) immunized with an acfA-deletion live attenuated vaccine.

Vibrio alginolyticus is well-known as an opportunistic Gram-negative pathogen, which endangers the development of global aquaculture as well as human health. In this study, a ΔacfA mutant strain and complementation of the ΔacfA mutant (C-acfA) were constructed. The ΔacfA mutant was tested in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the ΔacfA mutant caused a high antibody titer and a significant reduction in the ability to colonize the intestine of pearl gentian grouper. Grouper vaccinated with ΔacfA mutant were more tolerant of the infection by virulent V. alginolyticus HY9901 without inducing clinical symptoms and obvious pathological changes. The relative percent survival value of pearl gentian grouper vaccinated with ΔacfA mutant intraperitoneal injection reached 81.1% after challenging with V. alginolyticus HY9901. The specific antibody titers immunized with ΔacfA was significantly higher than that in the PBS group. The antibody titer of ΔacfA group displayed the tendency of rising up from the first to fourth week and declining from fifth to eighth week and reached the peak at the fourth week. In the meanwhile, the expression level of genes associated with immunity, including IL-1ß, TNF-α, IL-16, IgM, CD8α and MHC-Iα, was up-regulated after vaccination, indicating that the ΔacfA can induce effective and durable immune response in pearl gentian grouper and it may be an effective attenuated live vaccine candidate for the prevention of infections by V. alginolyticus.

Effects of Ocimum basilicum essential oil and ginger extract on serum biochemistry, oxidative stress and gill tissue damage of pearl gentian grouper during simulated live transport.

This study explored the effects of the essential oil of Ocimum basilicum (EOOB) and ginger extract (GE) during the transportation of pearl gentian grouper from water quality, serum biochemistry, oxidative stress, meat flavor, and gill tissue morphology. Fish (450 ± 50 g) were allocated to the following 5 treatments: control group (fish transported in water only), 5 mg/LEOOB, 10 mg/LEOOB, 3 mg/LGE, and 6 mg/LGE and transported in insulation boxes (66 × 51 × 37.8 cm) for 72 h. Samples were taken at 0, 12, 36, 60, and 72 h immediately after transport. It was found that 10 mg/LEOOB and 6 mg/LGE could reduce the levels of total ammonia nitrogen (TAN), dissolved oxygen (DO), water pH, serum glucose (GLU), cortisol (COR), liver superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPX), increase the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), as well as significantly increase the total free amino acid (TFAA) content in muscle compared to the control group (P < 0.05). In addition, by observing the microstructure of gill tissue, it was found that compared with untreated grouper, the morphological damage of gill tissue in EOOB and GE treatment was alleviated. These results indicated that adding appropriate amounts of EOOB and GE to transport water could improve the water quality, relieve stress, and lower energy metabolism of grouper during transport. The results of this research will help to improve the survival rate of grouper after transportation and decrease economic losses to fishery.

The analysis of complete genome sequence and comparative genomics of Vibrio parahaemolyticus LF1113 in Hainan.

Vibrio parahaemolyticus is a Gram-negative, halophilic and polymorphic coccobacillus. It is world-widely distributed and has resulted in great economic losses since its first appearance. In this study, a pathogenic strain was isolated from diseased pearl gentian grouper and identified as V. parahaemolyticus based on the sequencing results of 16S rDNA gene. In order to gain a comprehensive understanding of this isolation, the whole genome sequencing was conducted. Phylogenetic analysis of the complete genomes of 16 Vibrio species showed that LF1113, ATCC17802, ATCC33787, 2210633, FORC 004, and 160807 were the most closely related. Animal experiments demonstrated that the isolated LF1113 strain was pathogenic in a fish model. This study is the first study to describe the complete genome sequence of a V. parahaemolyticus isolate, which infected pearl gentian grouper from an outbreak in a fish factory farm in Hainan. The results will expand our understanding of genetic characteristics, pathogenesis, diagnostics and disease prevention of V. parahaemolyticus, and lay the foundation for further study.

Effects of Fishmeal Replacement by Clostridium Autoethanogenum Protein Meal on Cholesterol Bile Acid Metabolism, Antioxidant Capacity, Hepatic and Intestinal Health of Pearl Gentian Grouper (Epinephelus Fuscoguttatus ♀ × Epinephelus Lanceolatus ♂).

In this study, we present data from an eight-week growth trial with pearl gentian grouper fed either a reference diet (FM) with a fishmeal level of 50%, or test diet wherein 15% (CAP15), 30% (CAP30), 45% (CAP45), and 60% (CAP60) fishmeal was replaced by Clostridium autoethanogenum protein meal (CAP). Results showed that the weight gain and daily feed intake ratio of CAP60 were significantly lower than the FM group. In the serum, compared to the FM group, the content of malondialdehyde (MDA), the activities of alanine aminotransferase in CAP60 and CAP45 groups, and acid phosphatase in the CAP60 group were significantly higher, while the content of total cholesterol in CAP60 and CAP45 groups was significantly lower. In the liver, compared to the control group, the content of MDA in the CAP60 group was significantly higher. 3-hydroxy-3-methylglutaryl coenzyme A reductase in CAP30 to CAP60 groups and farnesoid X receptor in CAP60 were significantly upregulated. In distal intestines, the activities of trypsin and superoxide dismutase of CAP30 to CAP60 groups were significantly lower than the FM group. In conclusion, for pearl gentian grouper, CAP could replace up to 45% of the fishmeal in the feed, while a 60% replacement level will affect cholesterol bile acid metabolism and health.

Development and Evaluation of a Bicistronic DNA Vaccine against Nervous Necrosis Virus in Pearl Gentian Grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus).

Nervous necrosis virus (NNV) can cause enormous economic losses in mariculture. Vaccines are promising ways to control the disease. In this study: the interferon regulatory factor 3 (IRF3) gene of pearl gentian grouper was cloned and functionally analyzed; then a bicistronic DNA vaccine encoding both capsid protein (CP) and IRF3 was constructed; then the cellular, humoral, and local immune responses in the grouper after immunization were investigated; and then the protective effects after the NNV challenge were investigated. The results showed that the vaccine successfully expressed CP and IRF3. After immunization, the lymphocytes were recruited at the injection site in the muscles. The percentage of sIgM+ lymphocytes in the head, kidney, and spleen significantly increased and peaked at 28.8 ± 3.1% and 42.6 ± 4.2% at the 3rd to 4th weeks. Six immune-related genes were significantly up-regulated. In the meantime, the total antibodies, anti-NNV specific antibodies, and neutralizing antibody titers in serum increased. After the challenge with 105, 106 or 107 TCID50/fish, the relative percent survival rate was 81.25%, 73.91%, and 66.67%, respectively. In 106 TCID50/fish groups, the percentages of sIgM+ lymphocytes, antibodies, and the viral load were investigated. In conclusion, the bicistronic vaccine significantly induced humoral and cellular responses in pearl gentian grouper and provided effective protection against NVV infection.

Characterization and functional analysis of hybrid pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) complement C3 against Cryptocaryon irritans infection.

Pearl gentian grouper is a new aquacultural hybrid resulted from breeding of female tiger grouper (Epinephelus fuscogutattus) and male giant grouper (E. lanceolatus). Our preliminary study found that pearl gentian grouper exhibits less susceptible to the primary infection of Cryptocaryon irritans, which is an important parasitic ciliate in marine aquaculture, indicated that pearl gentian grouper might own a strong innate immune system. Complement system play key roles in innate immunity, whether pearl gentian grouper's complement component contribute for the defensing against the C. irritans infection remain unclear. In the present study, we found that C. irritans can be immobilized by untreated serum but not heat-treated serum from pearl gentian grouper, suggested that the heat-labile components in serum are responsible for the immobilization of C. irritans. Moreover, we cloned and characterized the encoding sequence of pearl gentian grouper complement C3 (PGC3), a key component in complement system. We also found that the expression level of PGC3 was increased in infected grouper serum when compared with that of control grouper. Furthermore, the binding of PGC3 on the surface of C. irritans trophonts located on the grouper skin was detected. These data suggested that pearl gentian grouper's complement system indeed play roles in the immune response against the C. irritans infection.

Gut-Liver Immune Response and Gut Microbiota Profiling Reveal the Pathogenic Mechanisms of Vibrio harveyi in Pearl Gentian Grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀).

Vibrio harveyi causes vibriosis in nearly 70% of grouper (Epinephelus sp.), seriously limiting grouper culture. As well as directly inhibiting pathogens, the gut microbiota plays critical roles in immune homeostasis and provides essential health benefits to its host. However, there is still little information about the variations in the immune response to V. harveyi infection and the gut microbiota of grouper. To understand the virulence mechanism of V. harveyi in the pearl gentian grouper, we investigated the variations in the pathological changes, immune responses, and gut bacterial communities of pearl gentian grouper after exposure to differently virulent V. harveyi strains. Obvious histopathological changes were detected in heart, kidney, and liver. In particular, nodules appeared and huge numbers of V. harveyi cells colonized the liver at 12 h postinfection (hpi) with highly virulent V. harveyi. Although no V. harveyi was detected in the gut, the infection simultaneously induced a gut-liver immune response. In particular, the expression of 8 genes associated with cellular immune processes, including genes encoding inflammatory cytokines and receptors, and pattern recognition proteins, was markedly induced by V. harveyi infection, especially with the highly virulent V. harveyi strain. V. harveyi infection also induced significant changes in gut bacterial community, in which Vibrio and Photobacterium increased but Bradyrhizobium, Lactobacillus, Blautia, and Faecalibaculum decreased in the group infected with the highly virulent strain, with accounting for 82.01% dissimilarity. Correspondingly, four bacterial functions related to bacterial pathogenesis were increased by infection with highly virulent V. harveyi, whereas functions involving metabolism and genetic information processing were reduced. These findings indicate that V. harveyi colonizes the liver and induces a gut-liver immune response that substantially disrupts the composition of and interspecies interactions in the bacterial community in fish gut, thereby altering the gut-microbiota-mediated functions and inducing fish death.