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.     

A quantitative determination of deformities in barramundi (Lates calcarifer; Bloch) fed a vitamin deficient diet

Cultured barramundi, Lates calcarifer, suffer from abnormalities affecting the jaw, opercula and spine. The aim of this study was to quantify for the first time the effects of supplemented dietary vitamin C, vitamin D₃ and ultraviolet (UV) light on the development of jaw, opercula and spinal deformities. Four diets were formulated to contain (i) no vitamin C or vitamin D₃, (ii) only vitamin D₃, (iii) only vitamin C and (iv) both vitamin C and vitamin D₃. In addition, two commercial diets (diets 5 and 6) were also tested. These diets were replicated in the presence, and in the absence, of ultraviolet (UV) light as this may also affect skeletal development. Diets formulated with 170 ± 1 mg kg^?1 and 195 ± 0.5 mg kg^?1 of vitamin C (diets 3 and 4, respectively) and the commercial diets (diets 5 and 6) had significantly lower incidences of spinal deformities (0–2.5%; P < 0.01) and opercula deformities (nil detected). Spinal deformities were ‘broken back’ syndrome, which was found only in the precaudal vertebrae, and lordosis which was only in the caudal vertebrae. UV light and vitamin D₃ did not affect spinal or opercula deformities. There was no change in the occurrence of jaw deformities in vitamin C, vitamin D₃ or UV light treatments.     

Detection of nodavirus in barramundi, Lates calcarifer (Bloch), using recombinant coat protein-based ELISA and RT–PCR

The coat protein encoded by the nodavirus RNA2 gene originally isolated from greasy grouper, Epinephelus tauvina , was cloned, expressed as a recombinant polyhistidine-tailed fusion protein and characterized by immunoblot analysis. The purified recombinant protein was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) to detect body exudate and plasma antibodies against the coat protein in both experimentally infected and commercial barramundi. In addition, the nucleotide sequence was employed to develop a RT–PCR detection assay based on the T4 region. The results showed that the virus could be detected as early as 3 days post-infection by RT–PCR while antibodies against the recombinant coat protein were detectable on day 6 post-infection. Among 112 commercial barramundi samples collected from October 1999 to April 2000, 9% showed positive ELISA results which were further verified by Western blot.     

CRISPR-based platform for rapid,sensitive and field-deployable detection of scale drop disease virus in Asian sea bass (Lates calcarifer)

Scale drop disease virus (SDDV) is a major pathogen of Asian sea bass that has emerged in many countries across the Asia Pacific since 1992 and carries the potential to cause drastic economic losses to the aquaculture sector. The lack of an approved vaccine for SDDV necessitates timely prevention as the first line of defence against the disease, but current diagnostic platforms still face challenges that render them incompatible with field applications, particularly in resource-limited settings. Here, we developed a novel detection platform for SDDV based on a CRISPR-Cas12a-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA-Cas12a). Using the viral adenosine triphosphatase (SDDV-ATPase) gene as a target, we achieved the detection limit of 40 copies per reaction and high specificity for SDDV. The coupling with fluorescence and lateral flow readouts enables naked-eye visualization and straightforward data interpretation requiring minimal scientific background. Compared with semi-nested PCR in field sample evaluation, our RPA-Cas12a assay is more sensitive and capable of detecting SDDV in asymptomatic fish. Importantly, the entire workflow can be carried out at a constant temperature of 37°C within an hour from start to finish, thus removing the need for an expensive thermal cycling apparatus and long turnaround times associated with PCR-based methods. Therefore, owing to its high accuracy, rapidity and user-friendliness, the developed RPA-Cas12a platform shows the potential for diagnosis of SDDV at point of need and could be a valuable tool to help protect fish farming communities from large-scale epidemics.     

Immune responses of barramundi, Lates calcarifer (Bloch), after administration of an experimental Vibrio harveyi bacterin by intraperitoneal injection, anal intubation and immersion

Barramundi, Lates calcarifer (Bloch), were immunized with an experimental Vibrio harveyi bacterin via intraperitoneal injection, immersion and anal intubation. Both specific and non-specific immune parameters were measured to compare responses to bacterin after delivery by various methods. Elevated antibody activities in sera were found in all treatment groups with barramundi injected intraperitoneally displaying significantly higher antibody activity than the other groups. In addition, there was evidence of memory induction with a heightened antibody response in the intraperitoneally injected group only. Bacteriostatic assays indicated activity against V. harveyi in the sera of all bacterin-treated groups; again this activity was significantly higher in the intraperitoneally injected groups. There was no enhancement noted in head kidney macrophage phagocytic activity or in serum lysozyme levels.     

Effects of dietary fish oil replacement on growth and carcass proximate composition of juvenile barramundi (Lates calcarifer)

An experiment was carried out to determine the effects of dietary fish oil replacement at an optimal level on the growth and carcass proximate composition of juvenile barramundi or sea bass (Lates calcarifer). Ten fish feeds were formulated to contain iso‐ingredients with theoretically identical dietary lipid levels but with different sources of lipids (fish oil, soybean oil, canola oil and linseed oil) replacing dietary fish oil. Three hundred fish were equally divided into 30 70‐L plastic tanks (three replicate tanks for each treatment). Fish were fed with experimental diets for 40 days within closed recirculating freshwater systems (70 L h^?1, 28°C and 12L:12D). Growth, specific growth rate, food conversion ratio (FCR) and carcass proximate composition were determined. Dietary lipids significantly affected (P<0.05) the growth of the fish. Good growth and low FCR were observed in all treatments. Therefore, fish oils can be partially replaced by vegetable oils to reduce the feed cost. There were no significant differences (P>0.05) in lipid, moisture and protein content in fish carcasses among the group. However, juvenile barramundi showed increasing lipid and moisture content in muscle, whereas decreasing protein content when compared to the initial fish.     

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.     

Genetic relatedness and differentiation of hatchery populations of Asian seabass (Lates calcarifer) (Bloch, 1790) broodstock in Thailand inferred from microsatellite genetic markers

After more than 20 years of hatchery production of Asian seabass in Thailand, genetic information is still lacking for effective genetic management and a selective breeding programme. This study aimed to evaluate genetic status of existing hatchery populations and genetic consequences of a selective breeding attempt. We examined genetic relatedness in seven hatchery samples, including a selectively bred population (RACF‐F1), compared with three wild samples using 11 microsatellite loci. Genetic diversity and relatedness values within most hatchery samples, except for RACF‐F1, did not differ from those of wild populations (P > 0.05). RACF‐F1 had the lowest allelic diversity and effective population size (A_r = 6.99; N_e = 7.8) and highest relatedness values (mean r_xy = 0.075–0.204). Pairwise Φ_ST values, principal component analysis and model‐based cluster analyses revealed three genetically distinct hatchery groups: Eastern Thailand (CHN, RACF, NSCF and SKCF), Southern Thailand (NICA) and the Andaman Sea (STCF). Results suggest that exiting domestic populations capture reasonable amount of genetic variation and can be useful for a base population for genetic improvement programmes. In addition, given the rapid increase in relatedness that we observed in one selectively bred population, we recommend using selection methods and hatchery practices that reduce variability in family contribution in the subsequent generations.     

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.     

The influence of dietary protein and energy levels on growth, survival and thyroid hormone (T3 and T4) composition of Lates calcarifer larvae

Two growth trials were conducted to determine the effects of different dietary protein (450–550 g kg^?1) and energy contents (18–22 MJ kg^?1) on growth, survival and carcass thyroid hormone (T3 and T4) levels of barramundi (Lates calcarifer) larvae. Larvae fed diets containing 21 and 22 MJ kg^?1 dietary energy performed consistently better than those fed diets containing 18 and 19 MJ kg^?1 dietary energy in terms of final dry weight and total length, while those fed 20 MJ kg^?1 had intermediate values for both the parameters. No effects of dietary protein level were discernable from the physical parameters measured; however, larvae fed diets containing the lowest protein and energy combination (450 g kg^?1 protein/18 MJ kg^?1 energy) had significantly lower carcass T4 levels than larvae in all other treatments, except for those fed the 500 g kg^?1 protein/18 MJ kg^?1 diet, which had an intermediate value. The results indicate that the optimum diet for L. calcarifer larvae from 14 to 28 days after hatch should contain 500 g kg^?1 protein and a minimum of 21 MJ kg^?1 dietary energy. Carcass T4 content was influenced by macronutrient inclusion level, and correlated significantly with growth, described by the total length. Reduced T4 levels may indicate a depressed larval status in this species.     

光合细菌PS2对尖吻鲈的生长、消化酶及非特异性免疫酶的影响

研究了光合细菌对尖吻鲈（Latescal carifer）的生长、消化酶及血清免疫酶活性的影响。把浓度为8×10^8cfu·mL^-1的荚膜红假单胞菌（Rhodopseudomonas capsulate）的液体制剂分别以0．0％（对照组）、0．5％、1．0％和1．5％的比例添加到饲料中,投喂初始体质量为（10．95±0．25）g的尖吻鲈50d。鱼的增重率、特定生长率、成活率及饲料系数在组间均没有显著性差异（P〉0．05）。肝蛋白酶、肠淀粉酶及胃淀粉酶在1．0％组显著高于对照组（P〈0．05）;肠蛋白酶在1．5％组显著低于1．0％组（P〈0．05）,但试验组与对照组间没有显著性差异（P〉0．05）;幽门垂的消化酶在1．5％组显著高于其他各组（P〈0．05）。血清碱性磷酸酶（AKP）、过氧化物酶（POD）和超氧化物歧化酶（SOD）的活性在组间均没有显著性差异（P〉0．05）。结果表明,该株光合细菌对尖吻鲈的生长及非特异性免疫力没有显著影响,但在1．5％组能显著促进其幽门垂消化酶的活性,在1．0％组显著促进肝蛋白酶及肠和胃淀粉酶的活性。     

Characterization of apoptosis induced by grouper iridovirus in two newly established cell lines from barramundi,Lates calcarifer (Bloch)

Two new cell lines have been established from the muscle and swim bladder tissues of barramundi, Lates calcarifer, and designated as BM (barramundi muscle) and BSB (barramundi swimbladder), respectively. The cells multiplied well at 28 °C in Leibovitz’s L‐15 medium supplemented with 10% foetal bovine serum, and have been continuously subcultured more than 100 times to date. Morphologically, BM cells were mostly fibroblastic, whereas BSB were mostly epithelial. Both cell lines were susceptible to grouper iridovirus (GIV) and displayed characteristics of apoptosis after viral infection. The induction of apoptosis was further assayed in GIV‐infected BM and BSB cells by various methods. The inhibition of cell growth by GIV was demonstrated by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. Morphological observations revealed typical apoptotic features in the infected cells, including cell shrinkage and rounding, chromosome condensation and formation of apoptotic body‐like vesicles. Chromosome fragmentation was detected by DNA laddering and TUNEL assays. Finally, the appearance of phosphotidylserine on the outer leaflet of apoptotic cell membranes was confirmed by annexin V staining. This is the first report of apoptosis induced by GIV in fish cells.     

Growth,enzymatic glutathione peroxidase activity and biochemical status of juvenile barramundi (Lates calcarifer) fed dietary fermented lupin meal supplemented with organic selenium

To investigate the effects of high level of lupin meal (LM) supplemented with organic selenium (OS) on the growth and blood biochemistry of barramundi (Lates calcarifer), four isocaloric and isonitrogenous diets were prepared, containing either non‐fermented or fermented LM, and either supplemented with 2 mg OS/kg (LM, LM_OS, FLM and FLM_OS), or not. A fishmeal (FM)‐based diet formulated for juvenile barramundi was used as a control diet. Fish (initial mean weight of 5.88 g) were triplicated and fed the test diets for 75 days. The findings demonstrated that growth performance of fish fed with the FLM and FLM_OS diets were similar to fish fed with the FM diet (p > .05). The antioxidant glutathione peroxidase (GPx) activity, and haemoglobin (Hb) of fish fed with the FLM_OS diet were significantly higher than that of FM‐fed fish (p < .05). Plasma alanine aminotransferase (ALT) activity was significantly increased in fish fed with non‐fermented diets (LM and LM_OS) than in those fed with fermented LM diets (FLM and FLM_OS) (p < .05). However, there were no significant differences in ALT activity among LM_OS, FLM, FLM_OS and FM diets. There was an interaction between the LM and OS on plasma CK activity; the CK of fish fed with diets supplemented with OS was higher in non‐fermented LM diets but lower in fermented LM diets (p < .05). This study suggests that fermented LM have an obvious potential to substantially replace 75% FM protein in the diets of barramundi.     

Dietary administration of astaxanthin improves feed utilization,growth performance and survival of Asian seabass,Lates calcarifer (Bloch, 1790)

This study was undertaken to assess the impacts of dietary astaxanthin supplementation on growth performance, feed utilization, survival, and serum growth hormone (GH) availability of Asian seabass, Lates calcarifer, with special reference to dose–response relationships and variations during different feeding phases (short‐term, medium‐term and long‐term). Fish were fed the following diets in triplicate for 90 days: the control (CD), AX50 (50 mg astaxanthin/kg diet), AX100 (100 mg astaxanthin/kg diet) and AX150 (150 mg astaxanthin/kg diet). The findings revealed that fish exhibited significant linear increments (p < .05) in specific growth rate (SGR), weight gain, feed utilization efficiency and survival when fed various diets with escalating levels of astaxanthin. Supplementation with dietary astaxanthin significantly augmented (p < .05) GH levels in fish. Significant positive associations (p < .05) were observed between circulating serum GH levels and SGR of fish from all groups following three consecutive feeding phases, denoting a robust cause‐and‐effect relationship. Circulating GH concentrations were considered as a sensitive biomarker of growth performance in Asian seabass. This study illustrated that supplemental astaxanthin could be administered in culture protocols to improve the growth rate and commercial hatchery production of Asian seabass, and possibly other teleost species.