Dietary administration of natural immunostimulants on growth performance,haematological, biochemical parameters and disease resistance of Asian Sea bass Lates calcarifer (Bloch, 1790)

This study was aimed to address the promising evaluation of Cissus quadrangularis plant (stem) and lipopolysaccharide (LPS from bacteria) supplemented diets on innate immune response in Lates calcarifer fingerlings against Aeromonas hydrophila infection. Fingerlings were fed supplemented diets containing four different concentrations of C. quadrangularis (0.5, 1.0, 1.5 and 2.0 g kg^?1 feed), LPS (25, 50, 75 and 100 mg kg^?1 feed) and control (normal formulated diet) for 60 days. The fish fingerlings fed supplemented diet displayed significant differences (P < 0.05) in specific growth rate (SGR) and relative percentage survival compared to the control group fed without C. quadrangularis and LPS‐supplemented diet. Fingerlings were injected intraperitoneally with 100 μL lethal dose of A. hydrophila containing 1 × 10⁶ CFU g^?1. Supplementation of C. quadrangularis and LPS diet significantly increased biochemical profile such as protein, lipid and carbohydrate content, haematological parameters of L. calcarifer fingerlings in different experimental periods when compared with the control group. Dietary doses of C. quadrangularis and LPS‐supplemented diet significantly influenced growth performance and increased survival rate in L. calcarifer fingerlings against A. hydrophila infection.     

Growth trials for polyculture of hatchery-reared juvenile spotted babylon (Babylonia areolata) and sea bass (Lates calcarifer) in a flow-through sea water system

Juvenile spotted babylon (Babylonia areolata) and sea bass (Lates calcarifer) were cultured in 11.5‐m³ indoor rearing tanks supplied with flow‐through of ambient natural sea water over a 120‐day experiment. Each species, stocked at the following densities for the following treatments, was tested with three replicates per treatment: 5000 B. areolata per tank (Treatment 1); 200 L. calcarifer per tank (Treatment 2); and 5000 B. areolata plus 200 L. calcarifer per tank (Treatment 3),. The average growth (length and weight), feed conversion ratio and total production of spotted babylon and sea bass from Treatment 3 were not significantly different from those of Treatment 1 and 2 (P > 0.05). Average survival rates for both spotted babylon and sea bass exceeded 95% for all treatments.     

Modelling selective breeding in protandrous,batch‐reared Asian sea bass (Lates calcarifer,Bloch) using walkback selection

A bioeconomic simulation model for Lates calcarifer predicted that a strategy involving crossing current generation males with previous generation females would be a practical, effective and profitable way of dealing with protandry when batch rearing for selective breeding to improve the growth rate. The strategy allowed earlier initialization and more frequent ongoing rounds of selection, and resulted in a 16–19% higher overall response, than an alternative where each generation's males were crossed with the same generation's females. The strategy also yielded the highest short‐term benefit–cost ratio (13:1 versus 7:1 after 8 years of selective breeding) and the highest short‐ and long‐term value for participants in a breeding cooperative (a net present value of AU$28 million and an internal rate of return of 144% over 10 years), due to higher yields per fixed costs of production per unit area and due to savings in feed costs per kilogram of production. Breeding facilities of scale producing 50 full‐sibling families per generation were found to be more profitable than those producing 100 families.     

Nutrient and amino acid profiles of egg and larvae of Asian seabass, Lates calcarifer (Bloch)

Fertilized eggs and developing larvae of hatchery reared Asian seabass, Lates calcarifer (Bloch), were analyzed to determine the changes occurring in their proximate and amino acid (AA) composition. The fertilized dry egg weighed 31 μg and contained 13.71 μg (44%) protein, 8.48 μg (27%) lipid and 0.657 J of gross energy. Dry weight decreased by 39% during hatching. The protein, lipid and carbohydrate nutrients decreased by 4.86, 4.15 and 0.09 μg, respectively from egg to 2-days post hatching (dph) larvae (pre-feeding). The protein content of the spawned eggs and larvae were hydrolysed to AA in the laboratory. The fertilized eggs had a total AA content of 42% of their dry weight. The egg contained 1.287 μg, 1.132 μg, 0.964 μg, 0.942 μg, 0.787 μg and 0.713 μg of leucine, lysine, arginine, valine, threonine and phenylalanine, respectively and these six indispensable amino acids (IAA) constituted approximately 78% of the total IAA. In the early feeding stages of L. calcarifer larvae, the ratio of IAA/DAA increased from 0.797 in the pre-feeding stage to 1.632 after 2 days of feeding. During larval growth of L. calcarifer, the percentage contribution of isoleucine and leucine to total IAA contents increased, while it decreased for lysine, phenyl alanine and arginine. L. calcarifer larvae were found to have proteins, which are rich in glutamic acid, leucine and lysine, and poor in threonine and histidine, suggesting high dietary leucine and lysine IAA requirement. This revised version was published online in July 2006 with corrections to the Cover Date.     

Age dependency of nervous necrosis virus infection in barramundi Lates calcarifer (Bloch)

Age‐dependent susceptibility to nervous necrosis virus (NNV) was demonstrated for barramundi (Lates calcarifer). The experiment used juvenile barramundi produced from a single spawning that were challenged consecutively by immersion with a redspotted grouper nervous necrosis virus (RGNNV) isolate. The dose and environmental conditions (35 ppt salinity and 30 °C) were constant. Fish and water were sampled longitudinally for histopathology and RT‐qPCR analysis to examine the evolution of the disease, virus replication, immune response and release of virus into water. Viral nervous necrosis (VNN) disease occurred in barramundi challenged at 3 and 4 weeks of age while fish challenged at 5, 7 and 9 weeks of age developed subclinical infection. Replication of NNV occurred faster and the concentration of virus reached higher concentrations in the younger fish with clinical disease. Virus isolation and qPCR tests indicated that infectious NNV was released from carcasses into water when fish were affected with clinical disease but not when NNV infection was subclinical. Based on these observations, we consider that carcasses from clinically infected fish have a potentially important role in the horizontal transmission of NNV, and barramundi juveniles should be protected from exposure to NNV until they are 5 weeks of age and reach the disease resistance threshold.     

Effects of sodium diformate on growth performance,gut microflora,digestive enzymes and innate immunological parameters of Asian sea bass (Lates calcarifer) juveniles

This study was conducted to evaluate the effects of dietary sodium diformate (NaDF) on growth performance, gut microflora, digestive enzyme activities and immune response parameters of Asian sea bass (Lates calcarifer) juveniles. Fish with initial weight of 12.5 ± 0.4 g were fed with five experimental diets contained 0.0 (control), 5, 10.0, 15.0 and 20 g NaDF kg^?1 in triplicate for 6 weeks. Fish fed diet containing 5 g NaDF kg^?1 had significantly the highest final body weight and feed intake among different treatments. The gut total viable bacterial counts gradually decreased with increasing dietary NaDF level. Specific activity of chymotrypsin improved in fish fed diets administered with NaDF compared to the control group. Fish fed 5 g NaDF kg^?1 diet showed the highest serum lysozyme level among different treatments. The serum classical pathway activity of complement showed higher level in fish fed diets contained 5 and 10 g NaDF kg^?1 than other groups. According to break‐point regression method analysis, the optimum inclusion of dietary NaDF in L. calcarifer juveniles was estimated between 4.6 and 5.1 g/kg, when specific growth rate and feed conversion ratio were plotted against dietary NaDF levels.     

Effect of dietary prebiotic inulin on histology,immuno‐haematological and biochemical parameters of Asian seabass (Lates calcarifer)

The present study was carried out to investigate the effect of supplementation of inulin on histology, immuno‐haematological and biochemical parameters of Asian seabass (Lates calcarifer) fingerlings with an initial average body weight of 7.14 ± 0.05 g. Inulin was supplemented at four different concentrations (control) 0, 5, 10, 15, and 20 g/kg in the L. calcarifer diet containing 400 g/kg protein and 90 g/kg lipid. At the end of the 60 days feeding trial, the absorptive surface area of the intestine and glycogen deposition in liver were increased in fish fed inulin supplemented diets. The immune parameters lysozyme, alternative complement pathway, superoxide dismutase and nitroblue tetrazolium assay showed significant (p < 0.05) difference between control and treatment groups. Haematological parameters showed that red blood cells, white blood cells, haemoglobin, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration levels increased significantly (p < 0.05) in the fish fed with inulin supplemented diets. The analysis of biochemical parameters revealed that glucose, urea, cholesterol, triglyceride, aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase, and lactate dehydrogenase showed significant differences (p < 0.05) between control and treatments groups. No significant difference (p > 0.05) was observed for total protein among different treatments. The results of the study revealed that 15–20 g/kg inulin supplementation has a beneficial effect in the histology, immuno‐haematological, and biochemical parameters in L. calcarifer juveniles.     

Mortality from scale drop disease in farmed Lates calcarifer in Southeast Asia

In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2–1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density‐gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral‐like particles (~100–180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%–99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow‐out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread.     

中国明对虾C 型凝集素基因(Fclectin)的重组表达及活性分析

研究拟通过分析对虾C型凝集素的活性特点,探讨其在对虾先天免疫应答过程中的潜在功能以及在养殖生产实践中的应用。实验利用原核表达系统对中国明对虾C-型凝集素基因的两个串联的糖识别结构域(carbohydrate recognition domain,CRD)进行了重组表达,并通过纯化复性获得了重组目的蛋白(rFclectin-CRD1和rFclectin-CRD2)。活性分析结果显示,重组目的蛋白对多种病原菌有凝集和抑制生长的作用,并且具有Ca2+依赖活性;其凝集活性可被半乳糖、肽聚糖、脂多糖等多种病原相关分子模式所抑制,研究结果证实,Fclectin是一种典型的C-型凝集素,它可能作为中国明对虾先天免疫中重要的模式识别受体,在一定程度上参与了机体应答病原微生物的防御过程。     

Evaluation of mosquito fish Gambusia holbrooki (Girard) as a partial and complete pellet replacement for juvenile barramundi Lates calcarifer (Bloch)

Barramundi (Lates calcarifer, Bloch) fed live mosquito fish (Gambusia holbrooki, Girard) exhibited faster growth and a better food conversion ratio (on a dry‐weight basis) than those fed an iso‐calorific ration of commercial barramundi pellets and a 50:50 ration of mosquito fish and pellets. The improved performance of barramundi fed mosquito fish was attributed to their higher protein content, higher protein to energy ratio and lower carbohydrate content. The essential amino acid indices and fatty acid profiles of the two diets were similar, suggesting that imbalances or deficiencies in these nutrients were not the limiting factor to the slower growth of barramundi fed on pellets. The use of mosquito fish as barramundi food offers a potential use of this noxious species and may allow barramundi to be cultured without the use of conventional fish meal.     

Effect of dietary chitosan on haematology,innate immunity and disease resistance of Asian seabass Lates calcarifer (Bloch)

This study was performed to evaluate the effect of dietary chitosan on haematology, innate immunity and protection against Vibrio anguillarum in Asian seabass, Lates calcarifer. A basal diet supplemented with 0, 5, 10 and 20 g chitosan kg^?1 diet was fed to the four different groups for 60 days. The haematological (total erythrocyte count, total leucocyte count, total serum protein, albumin, globulin and albumin‐globulin ratio) and innate immune parameters (phagocytic ratio, respiratory burst, serum lysozyme and serum bactericidal activities) were monitored at fortnight interval to assess the effect of chitosan feeding in Asian seabass. All the studied haematological and innate immune parameters were increased significantly (P ≤ 0.05) in chitosan‐fed groups in comparison with control. However, the group fed diet containing 10 g chitosan Kg^?1 feed showed highest haematological and innate immune parameters on 45^th day in comparison with other groups. Moreover, the fish fed the diet containing 10 g chitosan Kg^?1 feed had significantly higher post‐challenge survival (75.56 ± 4.44%) on the 30th day following V. anguillarum challenge. Therefore, this study suggests that chitosan at 10 g kg^?1 diet could be used as prophylactic in Asian seabass culture to enhance the protection against any possible infection by V. anguillarum.     

Use of neem oil (Azadirachta indica) to control caligid copepod infestation on Asian seabass (Lates calcarifer)

Asian seabass (Lates calcarifer) culture in marine cages have been developed rapidly in Malaysia. The high intensity of culture might facilitate several disease infection including caligid infestation. This study was conducted to evaluate the effect of neem oil (Azadirachta indica) against caligid parasites on seabass. The prevalence and clinical pathogen of Caligus infection has been described through a sampling of 150 fish from floating cage at different cultured periods. Acute toxic tests with different concentrations of neem oil were carried out on the parasite and fish host. Results revealed that the 96‐hr median lethal concentration (LC₅₀) of Caligus and fish (10.3 ± 2.5 g of body weight) were 2 and 20 ppm respectively. The in vivo test indicated that 10 ppm of neem oil could result in 100% antiparasitic efficacy within 96 hr. These results therapeutically demonstrated the efficacy of neem oil in caligid control.     

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL‐8 and COX‐2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL‐1β, IL‐8 and COX‐2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)‐induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation‐related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.     

The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

Susceptibility of freshwater rearing Asian seabass (Lates calcarifer) to pathogenic Streptococcus iniae

Asian seabass (Lates calcarifer) has been recognized as an economically important aquaculture species which can be adapted to and cultivated in wide range of salinities. The number of freshwater intensive seabass farms in Thailand is increasing annually. Here, we first describe the susceptibility of Asian seabass, which were cultured in freshwater, to Streptococcus inae (SI) and their pathological changes. Three isolates of putative SI were identified using a combination of standard biochemical assays and species‐specific PCR prior subjected to in vivo challenge. Accumulated mortalities of the fish which received 10⁷ CFU fish^?1 of either SI1J, SI SGSA or SI2J were 90%, 90% and 100% at 7 days‐post infection (dpi), respectively, and mortalities increased sharply between 3 and 5 dpi. Clinical signs such as erratic swimming and opaque eyes were identified from a few infected fish, while most died rapidly without any abnormal signs. Histopathological manifestations were observed in the multiple organs (kidney, liver and brain). Haemorrhage, hyperhemia, cellular degeneration and inflammatory cells infiltration were commonly found within the internal organs. Notably, the formation of numerous encyst‐like lesion aggregated by eosinophilic cells, resembling macrophages, were typically found in the brain of the infected fish. Summarily, this study first revealed that freshwater reared Asian seabass is highly susceptible to SI infection and haemorrhagic septicaemia was a major pathological change that could be found in the infected fish.     

Draft genome sequence of scale drop disease virus (SDDV) retrieved from metagenomic investigation of infected barramundi,Lates calcarifer (Bloch, 1790)

Scale drop disease virus (SDDV) is a novel viral pathogen considered to be distributed in farmed barramundi (Lates calcarifer) in South-East Asia. Despite the severity of the disease, only limited genomic information related to SDDV is available. In this study, samples of SDDV-infected fish collected in 2019 were used. The microbiome of brain tissue was investigated using Illumina HiSeq DNA sequencing. Taxonomic analysis showed that SDDV was the main pathogen contained in the affected barramundi. De novo metagenome assembly recovered the SDDV genome, named isolate TH2019, 131 kb in length, and comprised of 135 ORFs. Comparison between this genome and the Singaporean SDDV reference genome revealed that the nucleotide identity within the aligned region was 99.97%. Missense, frameshift, insertion and deletion mutations were identified in 26 ORFs. Deletion of four deduced amino acid sequence in ORF_030L, identical to the SDDV isolate previously identified in Thailand, would be a potential biomarker for future strain classification. Interestingly, the genome of SDDV TH2019 harboured a unique 7,695-bp-long genomic region containing six hypothetical protein-encoded genes. Collectively, this study demonstrated that the SDDV genome can be sequenced directly, although with limited coverage depth, using metagenomic analysis of barramundi sample with severe infection.     

Effects of nutritional enhancement of live food organisms on growth and survival of barramundi, Lates calcarifer (Bloch), larvae

Abstract Larval and juvenile barramundi, Lates calcarifer (Bloch), were reared intensively on test diets comprising nutritionally supplemented and unsupplemented rotifers, Brachionus plicatilis Muller, and brine shrimp, Artemia salina L. Both growth and survival of barramundi larvae fed on nutritionally supplemented brine shrimp were superior to those of larvae fed on untreated brine shrimp. Barramundi larvae fed diets incorporating untreated brine shrimp exhibited a mortality syndrome which commenced from 20 to 30 days after hatching and resulted in almost total mortality within the next 10 days. Analyses of the proximate, fatty acid and amino acid composition of the live food organisms used in the test diets, and reference samples comprising barramundi egg yolk and extensively reared juvenile barramundi, suggest that this mortality syndrome was primarily associated with the fatty acid composition of the food organisms, particularly the relative amount of 20:5n-3 in the brine shrimp fed to the larvae. These results, and the work of other authors, indicate that there are two mortality syndromes which affect intensively cultured L. calcarifer larvae.     

Development of two cell culture systems from Asian seabass Lates calcarifer (Bloch)

Two new cell culture systems namely epitheloid cells of Lates (LCE) and fibroblastic cells of Lates (LCF) have been developed from fry and fingerling of the economically important brackishwater fish Lates calcarifer. Primary cultures were initiated by explant technique using caudal fin of fingerling and whole body tissue of the fry. The nutritional requirements and the growth pattern in response to different culture environment were similar for the two cell cultures. The culture medium used was Leibovitz‐15 supplemented with 20% fetal bovine serum (FBS) and 1% fish serum. The LCE comprised of epithelioid cells and LCF cells were fibroblastic. With a split ratio of 1:2, the confluency of cells was attained in 8–10 days at an incubation temperature of 28°C. The cells were found to grow well in a wide range of temperature (24–32°C) and stable at 20 and 36°C. The growth rate of LCF and LCE cells increased proportionately with the concentration of FBS from 5% to 20%. A decrease of serum level to 10% after eight subcultures produced no apparent change in cell morphology and growth rate. The viability of cells was found to be 70% when revived after a month of storage in liquid nitrogen (?196°C).     

Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass, <Emphasis Type="Italic">Lates calcarifer</Emphasis>

Lates calcarifer, commonly known as the Asian sea bass or barramundi, is an interesting species that has great aquaculture potential in Asia including Malaysia and also Australia. We have investigated essential fatty acid metabolism in this species, focusing on the endogenous highly unsaturated fatty acid (HUFA) synthesis pathway using both biochemical and molecular biological approaches. Fatty acyl desaturase (Fad) and elongase (Elovl) cDNAs were cloned and functional characterization identified them as ∆6 Fad and Elovl5 elongase enzymes, respectively. The ∆6 Fad was equally active toward 18:3n-3 and 18:2n-6, and Elovl5 exhibited elongation activity for C_18–20 and C_20–22 elongation and a trace of C_22–24 activity. The tissue profile of gene expression for ∆6 fad and elovl5 genes, showed brain to have the highest expression of both genes compared to all other tissues. The results of tissue fatty acid analysis showed that the brain contained more docosahexaenoic acid (DHA, 22:6n-3) than flesh, liver and intestine. The HUFA synthesis activity in isolated hepatocytes and enterocytes using [1-¹⁴C]18:3n-3 as substrate was very low with the only desaturated product detected being 18:4n-3. These findings indicate that L. calcarifer display an essential fatty acid pattern similar to other marine fish in that they appear unable to synthesize HUFA from C₁₈ substrates. High expression of ∆6 fad and elovl5 genes in brain may indicate a role for these enzymes in maintaining high DHA levels in neural tissues through conversion of 20:5n-3.     

病毒感染过程中青鳉irf2基因过表达的双面性

为了探究干扰素调节因子2 (interferon regulatory factor,IRF2)如何通过调控干扰素(IFN)表达影响鱼类的免疫,实验从青鳉中克隆了irf2 (Olirf2),发现该基因在青鳉各个组织中均有表达;将构建的真核表达载体pTol2/CMV-IRF2/IE1-pr转染到胖头鱥肌肉细胞系(FHM)后,发现瞬时过表达Olirf2能够显著促进鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的复制,并抑制抗病毒相关基因mx1、ifn和irf3的表达。进一步通过双荧光素报告系统发现,Olirf2能够显著抑制NF-κB和ISRE的活性,说明Olirf2可能通过抑制细胞的天然免疫应答进而促进病毒的增殖。然而持续过表达Olirf2则增强了细胞的抗病毒能力,同时促进干扰素相关基因mx1、ifn和irf3的表达。因此,Olirf2基于表达的持续时间不同而具有抗病毒或者促病毒的双面效果。实验通过研究Olirf2在抗病毒信号通路中发挥的作用,为通过基因编辑或者转基因手段来构建抗病毒的鱼类提供了理论基础。