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.     

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.     

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.     

Immune responses of Asian sea bass, Lates calcarifer Bloch, against an inactivated betanodavirus vaccine

Asian sea bass, Lates calcarifer (Bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (RGNNV), a betanodavirus originally isolated in Japan. Fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). When fish were challenged with the homologous RGNNV at day 54 post-vaccination, there were no mortalities in both the vaccinated and unvaccinated control fish. However, a rapid clearance of the virus was observed in the brains and kidneys of vaccinated fish, followed by a significant increase in neutralizing-antibody titres. Furthermore, the vaccine-induced antibodies potently neutralized Philippine betanodavirus isolates (RGNNV) in a cross-neutralization assay. The present results indicate the potential of the formalin-inactivated RGNNV vaccine against viral nervous necrosis (VNN) of Asian seabass.     

Evaluation of techniques for marking juvenile barramundi, Lates calcarifer (Bloch), for stocking

To monitor the success of stockings of hatchery-reared barramundi, Lates calcarifer (Bloch), in tropical Australian waters a series of laboratory and field experiments were undertaken to evaluate a range of marking techniques. Juvenile fish of stocking size 30-85 mm total length were first used in laboratory experiments and then some were stocked into a freshwater impoundment for field evaluation. Marking techniques evaluated were fin crimping, fin removal, cold branding, polyethylene streamer (ags and coded-wire tags. The results showed that dorsal fin crimping and pelvic fin removal were relatively ineffective methods of marking, with only 53% and 63% of marks in each respective group being recognizable after 3 months. The polyethylene streamer tags were quickly shed, with 100% loss after only 77 days. Cold brands faded rapidly and were not considered to be of value as long-term marks. Coded-wire tags, inserted into the cheek musculature, were considered to be the most successful of the techniques assessed. Coded-wire tags were implanted at a rate of 250-270 tags per hour with an immediate success rate of 95-97%. These tags had no significant detrimental effect on either growth or survival and tag retention in the 2-month laboratory trials was 93%. Longer-term field studies, where fish had been stocked for about a year, also suggested little effect of coded-wire tags on growth or survival and no evidence of shedding.     

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.     

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.     

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.     

Replacement of fishmeal with commercial soybean meal and EnzoMeal in juvenile barramundi Lates calcarifer

This study investigates the impact of fishmeal replacement by soybean meal (SM) and improved SM known as EnzoMeal (EZ) on fish growth performance. The SM and EZ were used in five experimental diets: fishmeal (600 g/kg) as the control diet, 50% fishmeal replaced with soybean meal (SM50), 50% fishmeal replaced with EnzoMeal (EZ50), 100% fishmeal replaced with soybean meal (SM100) and 100% fishmeal replaced with EnzoMeal (EZ100). Fish in the control group gained the most weight, followed by those under the SM50 and EZ50 diets, while the least weight gain was observed in fish fed the SM100 and EZ100 diets. The specific growth rate (SGR) was higher in the control, but was not different between fish fed SM50 and EZ50, or between SM100 and EZ100. The highest feed intake occurred in the control but decreased sequentially from fish fed SM50 to fish fed EZ50, EZ100 and SM100. Protein digestibility was best in SM50, poorest in EZ100, but no difference between the control, EZ50 and SM100 diets. This study indicates that EnzoMeal, containing high crude protein (56%), is a potential source of plant meal to replace fishmeal in the barramundi diet, but low feed intake is a challenge when EnzoMeal is >300 g/kg.     

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.     

Effects of two commercial microdiets on growth and survival of barramundi (Lates calcarifer Bloch) larvae within various early weaning protocols

Because of high costs and labour requirements along with the highly variable nutritional value of live feeds, we investigated the possibility of early weaning for barramundi (Lates calcarifer Bloch) larvae aimed at reducing the use of Artemia. Two commercial microdiets, Gemma Micro (Skretting, Australia) and Proton (INVE, Belgium) were compared for growth and survival of larvae using three weaning protocols, until 33 days posthatch (dph). Enriched rotifers were fed to larvae in all protocols through mouth opening until 21, 18 and 30 dph (protocols 1, 2 and 3, respectively). At 13 dph, enriched Artemia metanauplii were introduced to weaning protocols 1 and 2, and continued until 29 and 24 dph, respectively, whereas protocol 3 did not receive Artemia. Microdiet was initiated at 20, 16 and 13 dph in protocols 1, 2 and 3, respectively. Barramundi larvae grew successfully to 33 dph when co‐fed rotifers and microdiet, and significantly larger larvae resulted from feeding Gemma Micro rather than Proton, when Artemia were not used. However, larvae weaned onto Proton using a longer period of Artemia provision were significantly larger than larvae reared according to all other protocols. Survival was significantly higher in all Gemma Micro protocols when compared with Proton protocols. This was in part due to higher cannibalism when using Proton compared with Gemma Micro (22.8 ± 0.9% and 9.2 ± 0.6%, respectively). Cannibalism became more noticeable in all protocols when the larvae reached 7–8 mm standard length and further increased after the cessation of live feed. Tank biomass production was the highest when larvae were weaned onto Gemma Micro including a short period of Artemia provision as a result of a combination of high growth and survival. However, similar biomass production resulted when larvae were weaned directly from rotifers onto Gemma Micro and/or from a prolonged Artemia period onto proton. The success of weaning barramundi larvae directly to microdiet from rotifers, thus eliminating the need for Artemia, was influenced by the microdiet. Relatively higher levels of free amino acids and lipids were believed to contribute to increasing larval growth and survival. Larvae that were fed Gemma Micro showed higher growth when Artemia were utilized for a shorter period, while Proton‐fed larvae benefited from an extended Artemia feeding period.     

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.     

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg)

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.     

Immune Response and Disease Resistance of Barramundi,Lates calcarifer (Bloch), Offered Diets Supplemented with Replete Levels of Tryptophan or Whey

A basal diet containing 45% protein, 10% lipid, and 1.95% tryptophan (Trp) was formulated, and then supplemented with crystalline Trp (0, 0.1, 0.5, and 1% of the diet) or whey (16.9%) to evaluate the physiological effects of juvenile (initial weight: 0.57 ± 0.15 g) barramundi over a 58‐d rearing period. Significant reductions in growth and feed intake were observed in fish offered diets supplemented with Trp as compared to those offered the basal diet or the whey diet. Brain serotonin increased in fish fed the 1% Trp and whey diets albeit a decrease in cannibalism was not observed. Phagocytic activity, and Mx gene expression in spleens were not significantly changed among all groups. There were significant increases in lysozyme activity in the 1% Trp and whey groups, respiratory burst in the whey group, and Mx gene expression in head kidneys in the 1% Trp group 12 h after nerve necrosis virus (NNV) injection and in the whey 12 group and 24 h after NNV injection. Cumulative mortalities of fish fed the whey diet were significantly lower than those of fish fed the basal diet after Aeromonas hydrophila infection, but not Trp groups. Based on these results, diets supplemented with whey had reduced mortality when challenged with A. hydrophila and consequently function as an immunostimulant.     

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.     

Partial utilization efficiencies of protein and methionine by barramundi (Lates calcarifer) in response to dietary methionine source and form

An experiment was conducted with barramundi (Lates calcarifer) juveniles (initial weight 10.3 g ± 0.3 g) to examine the partial efficiency of utilization of methionine (Met) from intact protein (fishmeal or lupin protein concentrate) and a crystalline DL‐Met source. Fish were fed at one of three ration levels: Low (0.4 g/fish/day), Moderate (0.8 g/fish/day) and High (1.6 g/fish/day). Those fed the fishmeal‐based diet (Diet FML) at the highest ration level grew to an average weight of 37.3 ± 0.46 g, whereas those fed the Lupin Protein Concentrate (LPC)‐based diet fortified with all EAA (Diet LPCM) at the highest ration level grew to 25.4 ± 2.27 g. The weight of the fish fed the LPC diet with no additional Met (Diet LPC) even at the highest ration level declined over the course of the experiment resulting in a final weight of 9.2 ± 0.88 g, clearly demonstrating the impact of dietary Met deficiency. The partial efficiency of protein utilization was also significantly reduced when Met was limiting (a coefficient of 0.06, compared to 0.39 in the Met‐supplemented LPC diet). The results suggested that the partial efficiencies (coefficients) of both Met and protein utilization in diets where crystalline Met is the primary source of Met (Diet LPCM: 0.26 and 0.39 respectively) were significantly poorer than from an intact protein source (Diet FML: 0.89 and 0.67 respectively) when Met is provided in excess.     

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.