Detection of betanodavirus in juvenile barramundi, Lates calcarifer (Bloch), by antigen capture ELISA

Betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. Betanodaviruses possess a bipartite single-stranded RNA genome consisting of the 3.1 kb RNA1 encoding an RNA-dependent RNA polymerase and the B2 protein, while the 1.4 kb RNA2 encodes the viral nucleocapsid protein, alpha. A panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (GGNNV) was developed for use in diagnostics. All antibodies reacted with native and recombinant alpha in immunoblot and indirect immunofluorescence assays. Each of the monoclonal antibodies reacted against discrete regions of the alpha protein, though none reacted with the extreme C-terminal region of the protein. One of the monoclonal antibodies, specific for the K151-T246 region of alpha, was used for the development of an antigen capture ELISA. In this assay we could detect 10(3)-10(4) TCID(50) units of virus derived from infected tissue culture supernatants. Head tissue extracts prepared from experimentally infected barramundi, Lates calcarifer, juveniles were assayed for GGNNV using the antigen capture assay and a clear increase in alpha antigen was detected from 5 to 15 days post-challenge. The assay thus represents a useful method for field-based detection of betanodavirus in fish hatcheries.     

Skeletal myopathy in juvenile barramundi, Lates calcarifer (Bloch), cultured in potassium-deficient saline groundwater

Saline groundwater is being pumped from a number of locations in rural Western Australia to prevent secondary salinity impacting farmland, rural infrastructure and areas with high conservation value. Aquaculture may offset the costs of groundwater pumping, and the suitability of groundwater for finfish aquaculture is being assessed through bioassays. There are marked spatial variations in the ionic composition of saline ground water in Western Australia and this paper describes two bioassays investigating a saline, potassium-deficient water source that resulted in mortalities in juvenile barramundi, Lates calcarifer (Bloch). Histopathological examination revealed severe degeneration and necrosis of skeletal muscles, marked hyperplasia of branchial chloride cells and renal tubular necrosis. Clinical chemistry findings included hypernatraemia and hyperchloridaemia of the blood plasma and lowered muscle potassium levels. It is concluded that the principal cause of death was skeletal myopathy induced by low water potassium levels.     

Cost benefit of hatchery-reared barramundi, Lates calcarifer (Bloch), in Queensland

Abstract. Recreational fishing for barramundi, Lates calcarifer (Bloch), is a major contributor to the economy of Queensland, generating an estimated $A 8 to $A 15 million per annum. The Queensland Department of Primary Industries is investigating the feasibility of enhancing barramundi populations with fish produced in fish hatcheries. Cost benefit analysis of the Department's stocking of baaamundi in Lake Tinaroo indicates that each dollar spent on the programme generates a potential $A 31 of economic benefit to the Queensland economy.     

Cytophaga johnsonae: a putative skin pathogen of juvenile farmed barramundi, Lates calcarifer Bloch

Abstract Cytophaga johnsonae , a gliding filamentous Gram-negative rod, was isolated from diseased farmed juvenile barramundi, Lates calcarifer Bloch. Infected fish had extensive but superficial erosion of the skin, mainly involving the posterior flanks, but also affecting the pectoral fins and lower jaw in some individuals. Minimum inhibitory concentrations of a number of antibacterial agents for C. johnsonae were determined; acriflavine and oxolinic acid inhibited growth between 0.78 and 1.5 μg ml⁻¹, while polymyxin B, trimethoprim and sulphamethazine failed to inhibit growth at 100 μg ml⁻¹.     

Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus

We recently reported that grouper iridovirus (GIV) can induce apoptosis in barramundi, Lates calcarifer , muscle (BM) and swim bladder (BSB) cell lines. In this paper, we further characterize the molecular mechanism underlying apoptotic death in BM cells triggered by GIV. DNA-laddering and apoptotic cells were observed in BM cells infected with UV-irradiated or untreated GIV but was absent in cells infected with heat-inactivated GIV, indicating the involvement of viral protein in the apoptosis event. In GIV-infected BM cells, the conversion of procaspase-3 to caspase-3 was evident and the level of caspase-8 and -9 increased as early as 30 min post-infection. When treated with a pancaspase inhibitor, the GIV-induced apoptosis event was abolished. These observations indicate that GIV-induced apoptosis is caspase-dependent, and that both the external and internal routes in the caspase-dependent pathway are likely involved in the apoptosis process.     

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.     

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.     

Dietary composition manipulation to enhance the performance of juvenile barramundi (Lates calcarifer Bloch) reared in cool water

Barramundi Lates calcarifer reared in cool water (20–22°C) grow slowly and feed is used poorly compared with fish in warm water (28–32°C). Two comparative slaughter growth assays were carried out with juvenile barramundi to see if increasing the digestible energy (DE) and/or the n‐3 highly unsaturated fatty acid (n‐3 HUFA) content of the feed would improve growth of fish raised in cool water. Increasing the DE content of the feed from 15 to 17 or 19 MJ kg⁻¹ while maintaining a constant protein to energy ratio in Experiment 1 brought about significant improvements in feed conversion ratio (FCR) (from 2.01 to 1.19) and daily growth coefficient (DGC; from 0.69 to 1.08%/day) for fish at 20°C. For fish at 29°C, improvements, while significant, were of a lesser magnitude: from 1.32 to 0.97 for FCR and from 3.24 to 3.65%/day for DGC. Increasing the absolute amount of dietary n‐3 HUFA, expressed as the sum of eicosapentaenoic and docosahexaenoic fatty acids, from 0.5% to 2.0% in Experiment 2 improved DGC linearly and FCR curvilinearly for fish at 29°C whereas at 20°C, DGC was not affected while FCR improved slightly (from 1.83 to 1.68). Feed conversion ratio was optimized with a dietary n‐3 HUFA of about 1.5%. Providing barramundi with a feed that is high in DE (viz 19 MJ kg⁻¹) and a digestible protein to DE ratio of 22.5 g MJ⁻¹ is a practical strategy for improving the productivity of barramundi cultured in cool water whereas increasing dietary n‐3 HUFA conferred very little additional benefit.     

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.     

Evaluating options for fishmeal replacement in diets for juvenile barramundi (Lates calcarifer)

Two experiments were conducted to examine critical thresholds to fishmeal inclusion in diets for barramundi and also the suitability of a range of different raw materials as alternative protein sources for this species. The first experiment used two diets formulated to the same digestible protein and energy specifications, which were then used to create a series of blended experimental diets that varied in fishmeal content from 0 to 770 g kg^?1. An additional diet containing sodium sulfamerazine was used as a negative control. Feed intake was unaffected with diets containing as little as 11% fishmeal, although broken‐line regression suggests that an inclusion of ～150 g kg^?1 fishmeal is a more likely threshold value. In a second experiment, a further series of diets was formulated for juvenile barramundi according to digestible protein and energy specifications predicted by existing bio‐energetic models. Each of the test raw materials was substituted for fishmeal at either 200 or 300 g kg^?1 (dependent on formulation or extrusion limitations), and two additional diets were included to examine two practical formulations. A diet with only fishmeal as the protein source was included as a reference. Each diet was produced using an APV19 twin‐screw extruder and then vacuum infused with the specified fish oil allocation. Each of the diet pellets produced was also characterized for a range of physical parameters. Fish of an initial weight of 70 ± 0.6 g fish^?1 were randomly allocated across 24 tanks with three replicates per treatment. After 6 weeks, average weight gain across all treatments was 73 ± 12.7 g fish^?1 and feed conversion across all treatments averaged 0.94 ± 0.08 g fish^?1. None of the diets using alternative raw materials had poorer growth or feed conversion than the fishmeal‐based reference diet. The inclusion of either the lupin kernel meals or canola meal significantly improved both weight gain and feed conversion compared to the reference diet. The results from this study demonstrate that there is clear potential to replace the fishmeal content of diets for barramundi without loss of fish performance, up to and including diets with as little as 150 g kg^?1 fishmeal inclusion.     

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.     

A determination of the quantitative requirements for docosahexaenoic acid for juvenile barramundi (Lates calcarifer)

A series of diets with varying docosahexaenoic acid (DHA; 22:6n‐3) inclusion levels (1 g kg^?1 3 g kg^?1, 6 g kg^?1, 10 g kg^?1, 15 g kg^?1 and 18 g kg^?1) were fed to juvenile barramundi (Lates calcarifer) for 6 weeks. Two additional diets examined the addition of arachidonic acid (ARA; 20:4n‐6) or eicosapentaenoic acid (EPA; 20:5n‐3) to the diets at 10 g kg^?1 when DHA was also included at 10 g kg^?1. Fish were fed the diets on a pair‐fed feeding regime to eliminate feed intake variability. Fish were weighed, and blood and tissue samples were collected after 6 weeks. Behavioural parameters were also assessed. Improvement in growth was seen with increasing inclusion of DHA up to a maximum at 10 g kg^?1 inclusion, albeit the response was minor. However, the addition of ARA to the diet reduced the growth response, while the addition of EPA improved the growth response. An improvement in feeding behaviour was also seen with increasing DHA up to a peak at 10 g kg^?1, while those animals fed diets low in DHA showed increasingly cryptic behaviour. With the increasing inclusion of DHA, a range of pathologies were observed, but the addition of an EPA component to the diet limited these pathologies, while the addition of ARA made little improvement and in some cases exacerbated the pathologies.     

Influence of fish size and water temperature on the metabolic demand for oxygen by barramundi, Lates calcarifer (Bloch), in freshwater

Oxygen demand by all animals is driven primarily by their needs for sustaining metabolism. Typically, larger animals require more oxygen and cellular fuel to carry out respiration than smaller animals. This relationship in most cases is not linear and is usually described by a coefficient and exponent (e.g. ax^b): the exponent b showing the relationship between live‐weight and energy/oxygen demand and is often termed the metabolic body weight (MBW) exponent, while the coefficient (a) tends to be temperature specific and describes the relationship between MBW and maintenance metabolic energy and oxygen demand at that specific temperature. Across all temperatures (range 26.0–32.0°C), the relationship between barramundi (Lates calcarifer) live‐weight (x; g) and relative oxygen consumption as standard metabolic rate (y; mg O₂ kg^?1 h^?1) at 29.4±1.5°C (mean±SD) was described by the exponential curve: y=710.19 x^?0.3268, R²=0.6875 (n=222 assessments). Examination of the same data but on a gross oxygen consumption (mg O₂ h^?1) basis showed a relationship between live‐weight (x; g) and gross oxygen consumption (y; mg O₂ h^?1) that was described by the exponential curve: y=0.710 x^0.6732, R²=0.9033. Evaluation of the combined relationship between fish live‐weight (y; g) and water temperature (x; °C) on gross oxygen consumption rate (z; mg O₂ h^?1) was described by the equation: z=(?20.7818+1.4017x?0.0227x²) ×y^0.673.     

Use of circuli spacing on scales to discriminate hatchery and wild barramundi, Lates calcarifer (Bloch)

Abstract. A study was conducted to determine if hatchery-reared and wild barramundi, Lates calcarifer (Bloch), could be distinguished by the patterns of circuli spacing on the scales. Proprietary software and digitizing equipment was used to obtain measurements of circuli spacing within one millimetre of the focus of the scales. Discriminant analyses separated the groups with up to 83% accuracy. As the technique utilizes innate tags laid down in response to the rearing environment, it has considerably more potential for evaluating the efficacy of large-scale enhancement programmes than do traditional physical tagging techniques.     

Proteolytic enzyme activity of juvenile Asian sea bass, Lates calcarifer (Bloch), is increased with protein intake

The effect of high dietary protein intake on proteolytic enzyme activity of feeding juvenile Asian sea bass, Lates calcarifer (Bloch) was studied. Ninety fish [mean body weight ± standard error (SE) 304.62 ± 34.84 g] were randomly assigned to two dietary treatments, each with three replicates. In treatment 1, fish were fed by‐catch (Thunnus albacares) and in treatment 2, a formulated diet containing 50% protein. Proteolytic enzyme activity was determined in pyloric caecae and intestine at day 0, 7, 15, and 30. Initial proteolytic enzyme activity in sea bass ranged from 174 to 232 azocasein units (U^AC.) per mg of protein. After 7 days there was no significant difference in proteolytic enzyme activity of fish fed the two diets. However, a marked increase was observed in fish fed the formulated diet at day 15. After 30 days, the proteolytic enzyme activity in fish fed the formulated diet was threefold higher than that in fish fed the by‐catch diet. Fish fed the formulated diet had significantly higher total protein intake at day 7 than did fish fed by‐catch. Thereafter, a twofold weekly increase in the total protein intake was observed in both fish fed the by‐catch and formulated diets until day 30. These results suggest that a high dietary protein intake induces increased proteolytic enzyme activity in Asian sea bass.     

Effect of dietary saturated and monounsaturated fatty acids in juvenile barramundi Lates calcarifer

Barramundi (Lates calcarifer), a catadromous teleost of commercial interest, perform well when fed a wide range of dietary oils. However, the range of alternative oils now being explored is typically rich in saturated and monounsaturated fatty acids (SFA and MUFA). In this study, the response of juvenile barramundi (47.0 g per fish initial weight) fed isolipidic and isoenergetic diets with 82 g kg^?1 added oil was tested. The experimental test diets had a 2 : 1 or 1 : 2 ratio of SFA to MUFA (SFA‐D and MUFA‐D, respectively) compared to a control diet (CTRL‐D) fed for 8 weeks. The diets containing mostly olive oil (dietary MUFA‐D) and mostly refined palm oil (dietary SFA‐D) did not impact the growth performance or feed utilization parameters of the barramundi. The in vivo beta‐oxidation activity was consistent with the dietary fatty acid composition, with the most dominant FA being heavily beta‐oxidized. Together, the in vivo whole‐body mass balance of fatty acids showed that n‐3 long‐chain polyunsaturated fatty acids (LC‐PUFA) were most efficiently utilized in the SFA‐D‐ and MUFA‐D‐fed fish. This study provides evidence that additional dietary MUFA and SFA are suitable lipid classes for juvenile barramundi and they are both equally efficient at sparing LC‐PUFA from an oxidative fate.     

A compendium of raw material digestibilities for barramundi,Lates calcarifer

A series of experiments were conducted to examine the nutrient and energy digestibility of a suite of diets and specific test raw materials when fed to juvenile (179 to 439 g) barramundi, Lates calcarifer. Each of the diets was prepared using a twin‐screw extruder to mimic modern aquafeed manufacturing processes. Each of the diets was fed to juvenile barramundi for a minimum of a week to allow acclimation to the diet before the faeces were collected using stripping methods. A broad range of digestible nutrient and energy values among the different raw materials were observed, with protein digestibilities ranging from 36% to 106% and energy digestibilities ranging from 36% to 93%. This range in nutritional values of the different raw materials provides substantial utility in allowing the formulation of diets on a digestible nutrient and energy basis across the Asia Pacific region. These results also provide critical data to help underpin the replacement of both fishmeal and fish oil in barramundi diets.