Experimental infection of brown‐marbled grouper,Epinephelus fuscoguttatus (Forskal), with Vibrio parahaemolyticus identifies parvalbumin beta‐2 subunit I,alpha‐2‐macroglobulin,nattectin and immunoglobulin light chain,differentially expressed in resistant grouper

The mechanisms through which brown‐marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown‐marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two‐dimensional gel electrophoresis (2‐DE). The results showed that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.     

What causes cannibalization‐associated suffocation in cultured brown‐marbled grouper,Epinephelus fuscoguttatus (Forsskål, 1775)?

During culture of grouper, cannibalism is a frequent phenomenon that usually causes economic loss. Grouper culture often requires grading to prevent size‐dependent cannibalism. In comparison with orange‐spotted (Epinephelus coioides) and giant grouper (E. lanceolatus), failure to swallow prey during cannibalism is frequently observed in brown‐marbled grouper (E. fuscoguttatus). The cannibal cannot engulf the entire prey and the two fish ultimately end up dying together. Herein, we attempted to compare morphometric differences among orange‐spotted, giant and brown‐marbled grouper. We established a cannibal–prey total length (TL) relationship for brown‐marbled grouper: TL_prey=0.71 TL_cannibal−1.75. According to the equation, a difference of approximately 50% in TL is a threshold to use for grading the grouper. This value is much larger than that used (30%) in orange‐spotted and giant grouper, and this size difference results in a higher incidence of failed cannibalism attempts in brown‐marbled grouper. It is our belief that the standard grading method will fail to produce as good an effect in brown‐marbled grouper as is seen in orange‐spotted and giant grouper. Therefore, in addition to grading, other manipulations such as satiation feeding, nutritional supplementation and optimal stocking densities should be applied to reduce cannibalism of brown‐marbled grouper.     

Soy protein concentrate as an alternative in replacement of fish meal in the feeds of hybrid grouper,brown‐marbled grouper (Epinephelus fuscoguttatus) × giant grouper (E. lanceolatus) juvenile

Hybrid grouper juveniles (body weight, 6.1 ± 0.7 g) (brown‐marbled grouper, Epinephelus fuscoguttatus × giant grouper, E. lanceolatus) were fed with six isoproteic (50% crude protein) and isolipidic (12% crude lipid) feeds containing different levels of soy protein concentrate (SPC) in replacement of fish meal (SPC at 20%, 30%, 40%, 50% and 60% protein) and control feed (SPC0) for 6 weeks. Hybrid grouper juveniles were cultured in 100‐L fibreglass tank equipped with flow‐through water system and fed twice a day to apparent satiation level. The highest and lowest growth was recorded in fish fed SPC20 and SPC60 respectively. However, growth of SPC20 was not significantly higher than those fed SPC0, SPC30, SPC40 and SPC50 (p > .05). A decreasing growth trend was observed with the increasing level of SPC from feed SPC40 to SPC60. A noticeable better feed utilization was also observed in fish fed SPC0, SPC20, SPC30, SPC40 and SPC50 compared to fish fed SPC60 (p < .05). The fish condition factor, hepatosomatic index, viscerosomatic index and whole body proximate content of the fish were not affected by the graded levels of SPC. However, the body lipid content was significantly lower in fish fed SPC40 to SPC60 (p < .05). The apparent digestibility coefficient (ADC) of protein and lipid was significantly higher in fish fed SPC0 and SPC20 compared to other dietary treatments (p < .05). Based on the regression analysis on specific growth rate, the study suggests that the hybrid grouper grow best at 21.4% and can utilize up to 50% inclusion level of SPC in protein without significantly affect their growth and its body condition.     

Characterization and viral susceptibility of a brain cell line from brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål) with persistent betanodavirus infection

A continuous cell line designated BMGB (brown‐marbled grouper brain) was established from the brain tissues of the brown‐marbled grouper Epinephelus fuscoguttatus and characterized. BMGB cells were identified as astroglial progenitor cells because they expressed glial fibrillary acidic protein and keratin and were persistently infected by betanodavirus, as confirmed through immunocytochemistry, polymerase chain reaction and immunoblot analyses. Because few intact virions were present in the BMGB cell culture fluid, the cytopathic effect (CPE) was not observed when the culture fluid was inoculated with GBC1 cells. However, BMGB cells displayed typical CPE after infection with additional betanodavirus, megalocytivirus and chum salmon reovirus. BMGB cells showed low myxovirus resistance (Mx) protein expression, which increased following betanodavirus and reovirus infection. Because the cells contained several unusual or degraded viral proteins, the persistent infection of betanodavirus in the BMGB cells may have resulted from a mechanism that destroys the viral proteins rather than the result of Mx protein expression. Despite the persistent betanodavirus infection, BMGB cells proliferated in a manner similar to other normal tropic fish cells and supported the propagation of several piscine viruses; however, the yield was lower than that of normal cells. The BMGB cells will be useful for investigating virus and host cell interaction.     

Cloning,expression of Vibrio alginolyticus outer membrane protein‐OmpU gene and its potential application as vaccine in crimson snapper,Lutjanus erythropterus Bloch

The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein‐OmpU was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant OmpU, and the reaction of the antibody was confirmed by Western blotting using the isolated OmpU and the recombinant OmpU of V. alginolyticus. To analyze the immunogenicity of the recombinant OmpU, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme‐linked immunosorbent assay (ELISA). The results demonstrated that the recombinant OmpU produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant OmpU is an effective vaccine candidate against V. alginolyticus in L. erythropterus.     

Development of novel aptamer‐based enzyme‐linked apta‐sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 10⁴/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.     

Occurrence of trypanosomiasis in net‐cage cultured groupers (Cromileptes altivelis and Epinephelus fuscoguttatus) in Nanshan port of Sanya,Hainan province,China

In the present study, reported was an unidentified trypanosome that caused massive mortalities of cultured humpback grouper, Cromileptes altivelis (Valenciennes, 1828) and brown‐marbled grouper, Epinephelus fuscoguttatus (Forsskål, 1775) in the net‐cages in Sanya, China, and some general pathological features of E. fuscoguttatus after experimental infection of the trypanosome. Before dying in the net cages of diseased fish, listlessness and anorexia were the observable symptoms. The blood of the diseased fish was light‐coloured in comparison with that of healthy one. E. fuscoguttatus experimentally infected with this trypanosome are with different degrees of engorgement in gill lamella, liver, spleen and kidney. Tissue lesion and necrosis were observed in liver and spleen of diseased fish.     

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange‐spotted grouper (Epinephelus coioides)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable Montanide^TM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.     

Parasites of cultured and wild brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) in Lampung Bay,Indonesia

A total of 210 Epinephelus fuscoguttatus (brown‐marbled grouper) was examined for parasites. During three consecutive seasons (two rainy and one dry season from 2002 to 2004), 35 specimens each taken from floating net cages of the National Sea Farming Development Centre (Balai Budidaya Laut) and from wild catches in Lampung Bay, South Sumatra, Indonesia were studied. Twenty‐five (cultured grouper) and 30 (wild grouper) parasite species/taxa were identified, with an infracommunity ranging from one to nine (cultured) and three to 14 parasite species (wild), demonstrating a species‐rich parasite fauna even in the cultured fish. Protozoans (1 species), microsporeans (1), myxozoans (1), digeneans (8), monogeneans (5), cestodes (3), nematodes (8), acanthocephalans (2) and crustaceans (6) were found. The most abundant parasites were the monogeneans Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus lantauensis for both, cultured and wild grouper during all seasons. For the cultured fish, the prevalence of monoxenous ectoparasites (e.g. P. epinepheli, P. lantauensis, Capsalidae gen. et sp. indet., Benedenia epinepheli) was in most cases higher than that of heteroxenous endoparasites. This contrasts the wild grouper, where heteroxenous parasites such as Allopodocotyle epinepheli and Raphidascaris sp. occurred at a similar prevalence compared with the fairly abundant Pseudorhabdosynochus spp. No seasonality of infestation was observed for both cultured and wild fish. The high levels of infestation of potentially pathogenic monogeneans throughout the year could result in significant parasite outbreaks at the locality studied.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

Identification of novel sRNAs involved in oxidative stress response in the fish pathogen Vibrio alginolyticus by transcriptome analysis

Vibrio alginolyticus as an important pathogen in aquaculture can encounter the oxidative stress produced by the immune system during infection. Previous studies showed that sRNAs have important functions in response to oxidative stress in bacteria; however, less of sRNAs related to oxidative stress response were identified in V. alginolyticus. In this study, a total of 749 novel sRNAs were identified by RNA sequencing; among them, 128 sRNAs were up‐ or downregulated in response to oxidative stress. In addition, 1,870 genes exhibited variation on mRNA levels in oxidative stress response. By analysing the target genes of the sRNAs, we concluded that these sRNAs could regulate expressions of genes responsible for iron transport, catalase, GSH‐dependent defence system, electron transferred and stress response. Moreover, the functions of the sRNAs are also seemed related to the pathogenicity in V. alginolyticus. Based on the results, we constructed the oxidative stress model in V. alginolyticus. This study provides us the first outlook of sRNAs function in oxidative stress response in V. alginolyticus. Furthermore, this study can help us to prevent and control this important opportunistic pathogen in aquaculture.     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.     

Nano zinc vis‐à‐vis inorganic Zinc as feed additives: Effects on growth,activity of hepatic enzymes and non‐specific immunity in rohu,Labeo rohita (Hamilton) fingerlings

The effects of dietary administration of inorganic zinc (zinc sulphate, ZnSO₄) and nano zinc (zinc oxide nanoparticles, ZnO‐NP) were evaluated in rohu, Labeo rohita fingerlings. Fish were fed with a basal diet (Control) supplemented with ZnSO₄ (T1, T2 and T3) and ZnO‐NP (T4, T5 and T6) at 10, 20 and 30 mg/kg, respectively, for a duration of 45 days. The results revealed that fish fed diet containing 20 mg ZnO‐NP per kg (T5) had the highest weight gain and specific growth rate (SGR, % per day), which was significantly different (p < .05) from the other experimental diets. Significantly (p < .05), higher activities of the digestive and metabolic enzymes were recorded in the fish fed ZnO‐NP containing diets as compared to the diets containing inorganic Zn or control diet. The maximum serum glucose and protein levels were noted in fish reared on diet T5. Both SGOT and SGPT activities were significantly increased in fish fed Zn‐supplemented diets (T1 to T6), as compared to the control group. Similarly, innate immune parameters were improved with feeding Zn incorporated diets. The highest phagocytic (40.74 ± 0.65%) and respiratory burst (0.33 ± 0.001, OD 630nm) activities were recorded in the fish fed diet containing ZnO‐NPs at 20 mg/kg (T5). The maximum superoxide production and serum peroxidase activity were detected in the fish fed T5 and T6 diets. Overall, results indicated that short‐duration feeding (≤45 days) of dietary ZnO‐NP (20 mg/kg) improved growth, enzyme activity, serum biochemical parameters and immune function in rohu fingerlings.     

Development of biofilm of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> for oral immunostimulation of shrimp

Biofilm (BF) of Vibrio alginolyticus was developed on chitin flakes. BF formation was studied at various nutrient concentrations and incubation time. Highest colony-forming units of BF cells were obtained with 0.15% trypticase soya broth and at 3 days of incubation. The BF cells could be completely inactivated at 80°C in 10 min and with 10% formalin in 24 h. SDS–PAGE profile of BF cells revealed repression of four proteins and expression of three new proteins compared to free cells (FC). The preliminary immune response studies showed that BF cells were superior to FC in stimulating the non-specific immune response of Penaeus monodon.     

Non‐specific immune responses and intestinal immunity of common carp (Cyprinus carpio) fed Jujube (Ziziphus jujube) fruit extract

This study investigated the effects of Jujube (Ziziphus jujube) fruit extract (JFE) as an excellent source of nutrients and phytochemicals on the innate immune responses and expression of genes involved in intestinal immunity of common carp (Cyprinus carpio) fingerlings. Three hundred healthy carps (10.78 ± 0.05 g) fingerlings were randomly allocated to 12 fibre glass tanks (300L; 15 fish per tank) and fed for 8 weeks with experimental diets which contained different levels of JFE (0%, 0.25%, 0.5%, or 1%). Thereafter, serum non‐specific immune parameters [total immunoglobulin (Ig) as well as alternative complement (ACH50) and lysozyme activity (SL)] and expression of genes involved in intestinal immunity (lyz, tnf‐alpha, il1b, il8, il10, and tgfb) were measured. The results revealed a significant elevation of Ig level in the JFE‐fed fish (p < 0.05) over the control fish. However, there was no significant difference among treated groups in case of serum total Ig. The highest levels of SL and ACH50 were observed in fish fed 1% of JFE. Nonetheless, no significant difference was noticed regarding SL and ACH50 of control and other supplemented groups (0.25% and 0.5% JFE). Also, expression of lyz, tnf‐alpha, and il1b genes increased significantly (p < 0.05) in JFE‐fed fish, in a dose‐dependent manner. On the other hand, significant (p < 0.05) down‐regulation of il110 and tgfb were noticed in treated groups. The present findings suggested that extraction of Jujube, Z. jujube fruit, possesses beneficial effects on immune responses of common carp.     

Carbon sources affect water quality and haemato‐biochemical responses of Labeo rohita in zero‐water exchange biofloc system

Biofloc technology degrades waste into useful resources exploiting microbes and can be used in zero‐water exchange systems. To study the effect of different biofloc systems on haematological and metabolic response of Labeo rohita fingerlings, a 60‐days experiment was conducted using four long lasting carbon sources. Seven hundred and fifty fingerlings having mean weight of 4.80 ± 0.12 g were randomly distributed into 15 tanks (n = 50 per tank). Five experimental groups were set in triplicate; T1 (Tapioca), T2 (Wheat), T3 (Corn) T4 (Sugar bagasse) and control (clear water). In‐situ biofloc was developed in 300 L fibre‐reinforced plastic (FRP) tanks and a C/N ratio of 15 was maintained. Water quality variables indicated ammonia immobilization by heterotrophic bacteria, as the dominant mechanism for the removal of toxic‐nitrogenous compounds in the biofloc systems. Results exhibited significantly higher floc volume (53.33 ± 7.88 ml/L), haemoglobin content (6.61 ± 0.03 g/dl) and total leucocyte count (109.66 ± 0.06 thousand cells/mm³) in tapioca biofloc system. Furthermore, the digestive and anti‐oxidative enzymes activities were also significantly higher in tapioca biofloc system. The lactate dehydrogenase and malate dehydrogenase enzyme assays showed a decreased level in tapioca biofloc system as compared with other biofloc systems and control group. Our observations indicate that tapioca biofloc system could improve the water quality, haematological and anti‐stress responses of L. rohita fingerlings in biofloc systems and thus can effectively replace other carbohydrate sources for the biofloc system.     

Growth performance and disease resistance against Vibrio vulnificus infection of novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus)

Groupers are economically important for aquaculture in Thailand. A novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) has been successful cross‐bred; therefore, the present work aimed to assess the hybrid's traits. The growth performance, strength and tolerance to a pathogenic bacterial infection of this hybrid were compared with its parent species, tiger grouper and giant grouper. The results of all measured growth parameters indicated that the hybrid strain grew fastest followed by giant and tiger grouper respectively. The expressions of the growth‐related genes, insulin‐like growth factor (IGF) I and II, were also analysed in fish muscle and liver which are the main target organs in fish growth regulation. Among tested species, similar expression patterns of IGF‐I and IGF‐II were detected in both organs. The levels of these genes in liver and muscle of hybrid and giant grouper were higher than those of tiger grouper comparable with the growth manner. After challenge with Vibrio vulnificus, the immunological parameters, clearance time of Vibrio in haemolymph and survival was measured to verify the fish immunity. Leucocyte number, lysozyme activity and the ability to eliminate the pathogen were very high in hybrid and giant grouper while these parameters were lower in tiger grouper. Correspondingly, the mortality rate of tiger grouper was higher than others and % survival at the end of observation time (15 days post challenge) was lowest in infected tiger grouper. Altogether, the results suggested that the hybrid grouper has desirable traits that will improve cultured grouper.     

Osteological development and deformities in hatchery‐reared longtooth grouper (Epinephelus bruneus): Vertebral column,dorsal‐fin supports and caudal‐fin skeleton

Skeletal deformities are one of the serious problems in hatchery‐reared longtooth grouper (Epinephelus bruneus). In this study, seed production of longtooth grouper was carried out in four large hatchery‐grade tanks. Osteological development of the vertebral column, caudal‐fin skeleton and dorsal‐fin supports in larvae and juveniles was described. The vertebral ontogeny started at 4.8 mm total length (TL) with the formation of neural spines 2 and 3, and the vertebral centra began forming at 5.3 mm TL. By 16 mm TL, all vertebral bony elements were formed except for the dorsal ribs. The onsets of the ontogeny of the caudal‐fin skeleton and dorsal‐fin supports occurred at approximately 5.1 mm and 3.9 mm TL, respectively, and the completion of these bony elements occurred at approximately 20 mm TL. The incidence of deformity was examined; some type of deformity was observed in 57%–68% of the specimens. These deformities mainly occurred as three types: lordosis, around vertebrae 1–5; saddleback‐like syndrome, around vertebrae 7–11; and vertebrae fusion, around vertebrae 22–24. The incidence of prehaemal lordosis was significantly higher among individuals with an uninflated swim bladder than among those with an inflated swim bladder.     

Supplementation of heat‐inactivated Bacillus clausii DE5 in diets for grouper,Epinephelus coioides,improves feed utilization,intestinal and systemic immune responses and not growth performance

In recent years, more and more attentions have been paid to the development and application of probiotics in aquaculture, and viable probiotics have been extensively studied, while rare information was available about inactivated probiotics in aquaculture. Therefore, in this study, a feeding trial was designed to investigate the effect of dietary supplementation of heat‐inactivated probiotic Bacillus clausii DE5 on growth performance, immune response and key immune genes expression in head kidney and intestine in grouper Epinephelus coioides. Fish were fed for 60 days with control diet (C) and two experimental diets containing 1.0 × 10⁸ CFU/g live (T1) and heat‐inactivated (T2) B. clausii DE5, respectively. The probiotic treatments did not affect the final weight, weight gain (WG) and specific growth rate (SGR) of E. coioides at days 30 and 60 (p > .05), while both heat‐inactivated and live B. clausii DE5 significantly decreased the feed intake and feed conversion ratio (FCR) at day 60 (p < .05). Serum lysozyme activity and complement C3 level in the two probiotic treatments were significantly higher than those in the control (p < .05). The lysozyme activity and complement C3 level at day 60 were significantly higher than those at day 30 (p < .05), while no significant interaction effect between diet and administration date was observed. Moreover, the heat‐inactivated B. clausii DE5 significantly improved the expression of TLR5, pro‐inflammatory cytokines (IL‐8 and IL‐1β) and TGF‐β1 in head kidney and intestine (p < .05), while the live probiotic did not show any significant effect on the expression of these key immune‐related genes in head kidney and intestine. These results indicate that dietary supplementation of heat‐inactivated B. clausii DE5 effectively improved feed utilization and both the local and systemic immune responses of E. coioides.