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.     

Dietary influence of Fucoidan supplementation on growth of Lates calcarifer (Bloch)

Sulphated polysaccharides exhibit a range of activities which promote health and growth benefits to aquaculture species. One class of sulphated polysaccharides derived from marine algae is fucoidan, which is suggested to exhibit an affinity to the negative muscle growth inhibitory protein myostatin. Addition of fucoidan to the diet of fish therefore may lead to enhancements in growth through inactivation of the myostatin protein. To examine the growth enhancing potential of this sulphated polysaccharide, fucoidan extracts from Undaria pinnatifida were added to the diet of juvenile barramundi, Lates calcarifer. The fucoidan extract was of 86% purity and included in two treatment diets at 5 g kg^?1 (0.5%) and 10 g kg^?1 (1%) respectively. Fish were fed the diet for 52 days. At the end of the feeding trial, barramundi fed with the 1% fucoidan inclusion diet exhibited enhanced growth with significantly increased lengths, weights and muscle fibre area (hypertrophy) when compared to the 0.5% fucoidan inclusion diet and the non‐fucoidan control diet. This research suggests that addition of fucoidan to the diet of fish may have growth promoting effects.     

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 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.     

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.     

Combined or individual effects of dietary magnesium and selenium nanoparticles on growth performance,immunity, blood biochemistry and antioxidant status of Asian seabass (Lates calcarifer) reared in freshwater

This study was conducted to investigate effects of and interactions between nanoselenium (NanoSe) and nanomagnesium (NanoMg) on growth, humoral immunity, serum biochemistry and antioxidant capacity of juvenile Asian seabass Lates calcarifer reared in freshwater. Four groups of fish with an average weight of 32.78 ± 2.23 g were fed one of the experimental diets for 6 weeks: (a) control (basal diet); (b) NanoSe (basal diet + 4 mg NanoSe/kg diet); (c) NanoMg (basal diet + 500 mg NanoMg/kg diet); and (d) combination (basal diet + 4 mg NanoSe/kg diet + 500 mg NanoMg/kg diet). Fish fed with NanoSe‐supplemented diets (NanoSe and combination) showed higher weight gain, specific growth rate and feed intake. The combination of NanoSe and NanoMg enhanced the immune response. Also, fish fed on combination diet showed higher serum alanine aminotransferase and aspartate transaminase levels but lower glucose concentration. The activities of liver superoxide dismutase, glutathione peroxidase and catalase were not different among all groups, but liver malondialdehyde level was lower in fish fed diets supplemented with NanoSe and/or NanoMg. It could be concluded that NanoSe improved growth performance; the combination of both NanoSe and NanoMg enhanced humoral immunity; and NanoSe and/or NanoMg improved antioxidant capacity in Asian seabass.     

Dietary butyric acid improved growth,digestive enzyme activities and humoral immune parameters in Barramundi (Lates calcarifer)

A six‐week feeding trial was conducted to determine the effect of butyric acid (BA) inclusion in diet on performance of Barramundi (Lates calcarifer). Fish (12.0 ± 0.2 g) were fed with four experimental diets contained 0.0 (control), 2.5, 5.0 and 10.0 g BA/kg in triplicate. Fish fed on 5 and 10 g BA/kg diets had higher growth and total alkaline protease and lipase activities than the other treatments. Fillet protein content in BA‐supplemented groups was higher than the control. Liver catalase activity was highest in 2.5 g BA/kg group compared with the other groups. The values of liver superoxide dismutase activity and serum total protein concentration in 5 and 10 g BA/kg treatments were higher than those in the control (p < .05). Moreover, the highest and the lowest levels of serum lysozyme activity were noticed in the 10 g BA/kg and the control groups, respectively (p < .05). Also, the highest respiratory burst activity was observed in fish fed 5 g BA/kg feed. Supplementing diet with 2.5 or 5 g BA/kg resulted in higher serum haemolytic activity in fish compared with the other groups. The number of red and white blood cells increased in fish fed BA‐supplemented diets compared with the control (p < .05). In conclusion, the results of this study showed inclusion of BA at 5 g/kg diet in L. calcarifer juveniles improved growth and upgraded the general health condition by enhancing fish antioxidant enzyme activities and haemato‐immunological responses.     

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.     

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.     

Effect of temperature and salinity on sex inversion in Asian Seabass (Lates calcarifer): relationship with plasma sex steroids concentration and aromatase activity of gonad and brain

Asian Seabass which shows precocious sex inversion in cultured environment is a major impediment to maintain a proper broodstocks. Seabass grown commercially in freshwater were transported to the research facility and held in freshwater at 29°C for 4 weeks and acclimatized to the experimental conditions. Fish were daily fed with a commercial pellet (50% protein, 18 MJ kg^?1) to satiety. Blood, brain and gonad collected before and at the end of the experiment were analysed for sex steroids level and aromatase activity. There was an increase in Plasma E₂ levels with temperature in fish at 34°C whereas no significant difference was observed at 24 and 29°C although the highest plasma T level was detected in fish at 34°C which had significantly lower level than at the beginning, except those held at 24°C in freshwater. Plasma 11‐KT was significantly greater in fish at 24°C compared with 29 or 34°C which had clear opposite to the E₂. Aromatase activity in brain was higher at 29°C than at either 24 or 34°C, whereas gonadal aromatase recorded highest at 34°C. These findings concluded that high temperature in culture facility induces sex inversion of Asian Seabass.     

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.     

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.     

Ocular bacterial signatures of exophthalmic disease in farmed turbot (Scophthalmus maximus)

As is the case at other sites in the body (e.g. the gut, skin and mouth), the ocular microbiota plays a crucial role in their host, as disturbances of the composition and function of the ocular microbiota are known to be associated with ocular disorders. Exophthalmic disease (ED) is a significant cause of high mortality in fish species, including farmed turbot (Scophthalmus maximus). However, the relationship between alterations in the ocular microbiota and ED in turbot is unclear. In this work, we collected turbot samples from farmed ponds with ED and healthy samples to understand changes in the ocular microbiota of turbot suffering from ED. We compared the structural and metabolic differences of ocular bacterial communities from farmed turbot with exophthalmic disease and those of healthy controls. Besides less microbial diversity found in turbot with ED regarding the control group, we also found that Aeromonas was the dominant bacteria both in controls and ED samples, but the abundance of Aeromonas was significantly greater in ED individuals. Moreover, the results of correlation test further suggest that Aeromonas overgrowth was correlated with the progress of the disease and shifts in ocular microbiota functional pathways in turbot. These findings emphasize that an increased abundance of Aeromonas serves as an ocular bacterial signature associated with ED in turbot, which provide basic information useful for diagnoses, prevention and treatment of ocular diseases occurring in cultured fish.     

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.     

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S‐1 and S‐2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP‐PCR and ERIC‐PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 10⁴ and 10⁶ CFU per fish with the S‐1 isolate, while 100% mortality rates were recorded in zebrafish at 10² and 10⁴ CFU per fish with the S‐2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.