Methodology in the study of seabass (Lates calcarifer Bloch): nutritional requirements in Singapore

This paper describes the effort of the Marine Aquaculture Section of the Primary Production Department in Singapore and the Institut Francais de Recherche pour l'Exploitation de la Mer in developing adequate facilities for fish nutritional requirement studies in Singapore, and in introducing appropriate nutritional methodology so that results are reliable and accurate. One of the first achievements was the demonstration of homogeneous fish (tropical seabass, Lates calcarifer Bloch) growth response in a newly established tank system before any nutritional experiments were initiated. The application of statistical procedures that take into consideration the power of a test and number of replications required has allowed for more accurate interpretation of results. This can be seen in the results of the first nutritional requirement experiment on the optimal dietary protein level of the seabass at constant energy.     

A standard panel of microsatellites for Asian seabass (Lates calcarifer)

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 ± 0.71 (range: 6–19), and the expected heterozygosity averaged 0.76 ± 0.02 (range: 0.63–1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM‐T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3–5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 × 10 and 20 × 20) demonstrated a high power of these markers for revealing parent‐sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.     

Economic assessment of commercial hatchery production of milkfish (Chanos chanos Forsskal) fry

The economic viabilities of two types of commercial hatchery milkfish ( Chanos chanos Forsskal) fry operations were assessed and compared. Based on the actual cost of input, the physical facilities, and the potential production yields, four commercial hatcheries previously used for shrimp ( Penaeus monodon Fabricius) fry production were classified as either large- or small-scale operations. Cost–return analysis revealed high profits for both types of operation. The return on investment (54–61%) and the payback period (≈ 1.5 years) were comparable between the two types, although a large-scale operation (476%) had double the working capital return of a small-scale hatchery (221%). Benefit–cost analysis over a 5-year period also revealed positive and above-baseline discounted economic indicators [net current value = 0.2–2.2 million Philippine Pesos (1 US Dollar = 25 Philippine Pesos); internal rate of return = 88–107%]. The net benefit–cost ratio of a large-scale operation (2.0) was higher than that of a small-scale hatchery (1.4), suggesting a slight edge in the investment viability of a large-scale hatchery. Compared with a large-scale operation, a small-scale hatchery was more sensitive to changes in the acquisition price of eggs or newly-hatched larvae and in the price of selling hatchery fry. Both types of operation are viable nonetheless when the acquisition cost is P6000 per million eggs or larvae and hatchery fry are sold at P0.50 each. Together, profit and investment in milkfish hatchery fry production appear viable, making milkfish an alternative commodity for production in many abandoned shrimp hatcheries. The limited availability of spawned eggs and larvae for rearing and the quality of hatchery fry are issues requiring urgent attention.     

Cloning and characterization of the calreticulin gene in Asian seabass (Lates calcarifer)

Calreticulin (CRT) is a Ca2+-binding molecular chaperone in the endoplasmic reticulum. We cloned and characterized the CRT gene in an important marine food fish species Asian seabass (Lates calcarifer). The full-length DNA of the CRT gene was 2194 bp, including a complete open reading frame encoding 420 amino acid residues, a 113 bp 5'-untranslated region and an 818 bp 3'-untranslated region. The CRT gene contained nine exons and eight introns covering a total of 2772 bp genomic DNA from the start to stop codon. Ten single nucleotide polymorphisms (SNPs) were detected in introns and an exon in six individuals collected from five different locations. The CRT gene was assigned to linkage group 4 of the linkage map of Asian seabass. Quantitative real-time PCR revealed that the CRT gene was highly expressed in liver at the age of 1, 3 and 7 months under normal conditions, whereas its expression in liver reduced sharply after 0.5 to 2 h cold challenge at 16°C, and then increased slowly. A preliminary association analysis showed a significant (P < 0.001) association between the SNP6 in the CRT gene and the mortality after cold challenge at 16°C. Our results suggest that the CRT gene is associated with cold tolerance of Asian seabass and further investigation will be necessary to illustrate the underlying mechanisms.     

Environmental capacity of receiving water as basis for regulating intensity of milkfish (Chanos chanos Forsskal) culture

This paper presents the results of the assessment of the environmental condition of a receiving water and demonstrates how the environmental capacity of the system can be estimated based on effluent discharge from milkfish ponds and water criteria from scientific literature and other studies. An estuary (average volume, 295 333 m³; average depth, 1.9 m) in Punta Pulao, Dumangas, Iloilo, Philippines served as discharge and irrigation system for commercial milkfish ponds and experimental/verification ponds owned by the Southeast Asian Fisheries Development Center. Total ammonia nitrogen, nitrite, nitrate, phosphate, and chlorophyll a (chl a) were determined monthly (during spring tide) for 4 months. Samples were taken at low and high tides that coincided with the draining and flooding of ponds, respectively. Monthly concentrations of these water quality variables generally increased from the first to fourth month of sampling (April–July 2001) during low tide. Very slight increases were observed during high tide. The magnitude of changes in the diurnal patterns of phosphate, chl a, and dissolved oxygen appeared to be higher at spring tide than at neap tide. This indicates that the inflowing river at low tide (during spring tide) brought effluents containing high amounts of nutrients from ponds located upstream. The water, salt, dissolved inorganic nitrogen (DIN), and phosphate (DIP) budgets of the entire estuary were determined following the one‐box model by LOICZ‐IGBP (2000, LOICZ biochemical budgeting procedure: a tutorial pamphlet. L. T. David, M. L. San Diego‐McGlone, C. J. Crossland and S. V. Smith (Eds). Publ. for LOICZ International Project Office, the Netherlands, 29 pp.). The budgets indicate that the system is net heterotrophic and nitrogen (N) fixing during the dry months, but that there could be no environmental impact during the rainy months because of short water exchange time. Results further suggest that the system is a source of N and phosphorus (P) during the dry and rainy months; the condition is largely influenced by the high amount of nutrient inputs in to the river. Linear regression analysis was performed to determine relationship between nutrient concentrations in the system and total DIN or DIP input to the system at low tide. Environmental capacity in terms of the maximum amount of DIN or DIP input to the system was predicted using regression analysis and following set criteria for nutrients, i.e. nitrite, nitrate, and phosphate. At present, the estuarine water quality has already reached its environmental capacity during the dry months. About 945 ha of commercial milkfish ponds are operating upstream, mostly as extensive systems. If these ponds are converted to semi‐intensive or intensive systems, it is recommended that the pond area be reduced to 122 ha if the DIP criterion is to be followed so as not to exceed the environmental capacity. Exceeding this environmental capacity may affect production through reduction of fish growth, occurrence of diseases, and fish mortalities.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

Effect of inland water salinity on growth performance and nutritional physiology in growing milkfish, Chanos chanos (Forsskal): field and laboratory studies

To investigate the effect of inland groundwater salinity on growth performance, feed conversion efficiency, nutrient retention and intestinal enzyme activity in milkfish, two experiments were conducted. In the first experiment (Expt I), a 100‐day monoculture of Chanos chanos [mean body weight (BW): 2.2 g] at different salinities (0, 10, 15, 20 and 25‰) was carried out in ponds fertilized with cowdung (about 10 000 kg ha^?1 year^?1) and poultry droppings (about 3000 kg ha^?1 year^?1). The fish were fed a compounded supplementary diet (containing 40% protein) at 5% BW day^?1. Studies have revealed that growth increased with each increase in the salinity level; the highest values in weight gain and energy assimilated were observed in ponds maintained at 25‰ salinity [weight: 322.2 g and specific growth rate (SGR): 8.3]. Highest values of condition factor (0.7) and exponential value (n) of the length–weight relationship (LWR; n = 3.25) were also observed in ponds maintained at 25‰ salinity. Dissolved oxygen (DO), biological oxygen demand (BOD), pH and nutrient release remained at the optimal level during the culture period. High values of chlorophyll a, net primary productivity (NPP), phytoplankton and zooplankton population coincided with the highest values of alkalinity and turbidity in ponds maintained at 25‰ salinity. Multivariate analysis revealed a significant positive correlation of chlorides (r = 0.91), conductivity (r = 0.89) and hardness (r = 0.96) with fish growth. Productivity indicating parameters viz. NPP (r = 0.45), nitrate (r = 0.94) and o‐PO₄ (r = 0.52) also showed a significant positive correlation with fish weight gain. In the second experiment (Expt II), milkfish (mean BW: 3.7 g) fry were exposed to different levels of salinity (0.0, 10, 15, 20, 25 and 30‰), and maintained for 90 days in the laboratory. Significantly (P < 0.05) high growth (percentage increase in BW: 183.1 and SGR: 1.2), feed conversion efficiency (64.5%) and intestinal enzyme activity (protease 5.1, amylase 4.1 and cellulolytic 3.2) were observed in the group maintained at 25 ppt salinity in comparison with other groups similarly maintained at low or high salinity levels. Carcass composition, muscle and liver glycogen levels were also significantly (P < 0.05) affected by salinity changes. The significance of these findings is discussed in this paper.     

Parasite fauna of seabass (Lates calcarifer) under mariculture conditions in Lampung Bay, Indonesia

Fish parasites have been repeatedly reported to be a major threat to the developing industry of finfish mariculture in Indonesia, due to severe parasite and disease outbreaks. The aim of this study was to identify the metazoan parasite fauna and trichodinid ciliates that infect Lates calcarifer in a representative mariculture farm in Indonesia. Examined were 105 L. calcarifer (seabass) for the metazoan parasite fauna and trichodinid ciliates. Thirty‐five specimens each from the net cages of the National Sea Farming Development Centre (Balai Budidaya Laut, BBL) in Lampung Bay, South Sumatra, Indonesia were investigated in three consecutive seasons (two dry and one rainy season from 2002 to 2003). Nineteen parasite species were identified; all fish specimens were infected with two to 10 parasite species, demonstrating a species‐rich parasite fauna. Protozoans (1 species), myxozoans (1), digeneans (3), monogeneans (5), cestodes (3), nematodes (5) and acanthocephalans (1) were found, including 11 new host records in cultured L. calcarifer from Indonesia. Larval and adult parasite stages were isolated, demonstrating that this fish species, although kept inside the net cages, still functions as an intermediate and final host for marine fish parasites. During all seasons, the six detected monoxenous (single host life cycle) parasite species showed a higher prevalence than the 13 heteroxenous (multiple hosts) species. Most abundant were the fish pathogenic monogeneans Pseudorhabdosynochus epinepheli, Pseudorhabdosynochus lantauensis, Benedenia epinepheli and Neobenedenia melleni with a high prevalence. Most heteroxenous parasites (Digenea, Cestoda, Nematoda and Acanthocephala) occurred with a low prevalence below 26%, caused by the specific culture conditions. Diversity of the heteroxenous parasites was higher in the dry seasons than in the rainy season. Though some seasonality could be observed for the fish pathogenic monogeneans, severe disease outbreaks of these ectoparasites cannot be excluded in either the dry or rainy season.     

Development of larval diets for milkfish (Chanos chanos)

This study aimed to develop nutritionally balanced and cost-effective processed diets for milkfish larvae ( Chanos chanos Forsskal). Two larval diets (feed A and feed B) were formulated and prepared to contain 45&percnt| protein and 10&percnt| lipid. Several larval diet preparations were tried such as microbound/ unpelleted (freeze-dried), microbound/pelleted (oven-dried) and microbound/flaked (drum-dried) and assessed in terms of feed particle size and buoyancy, water stability and feed acceptability. The preparation that gave the best particle size and buoyancy as well as good water stability was prepared as the microbound diet (using K -carrageenan as a binder) and flaked using a drum drier. A series of feeding experiments were conducted to determine the growth and survival of milkfish larvae reared on various feeding schemes using these processed larval diets which were fed either solely or in combination with live feed. Larvae in control treatments were reared on live foods such as Brachionus plicatilis and Artemia nauplii. Larvae were observed to ingest the diets, indicating that the feeds had suitable physical characteristics and were attractive to the larvae. The overall results of the feeding trials showed that the artificial diets could be fed to milkfish larvae in combination with Brachionus rotifers starting on day 2 or day 8, and could be fed alone starting from day 15. These promising results would reduce the dependence of milkfish larvae on live feed and would have significant economic benefits in the form of simplified milkfish hatchery procedures.     

Characterization of two parvalbumin genes and their association with growth traits in Asian seabass (Lates calcarifer)

Parvalbumins are extremely abundant in fish muscle and play an important role in muscle relaxation. In this study, two parvalbumin genes (PVALB1 and PVALB2) were cloned from Asian seabass (Lates calcarifer). The cDNAs for PVALB1 and PVALB2 were 840 and 667 bp respectively. Both genes consisted of five exons and four introns, encoded 109 amino acids, and were of beta lineage. Using real-time polymerase chain reaction, expression of PVALB1 was detected in all 10 tissues tested, with expression in brain, kidney, muscle and small intestine being 15- to 322-fold higher than in the other tissues. Expression of PVALB2 was detected only in muscle, brain and intestine, and was up to 10-fold lower than PVALB1 expression. A (CT)(17) microsatellite in the 3'-untranslated region of PVALB1 and three single nucleotide polymorphisms (SNPs) in the third intron of PVALB2 were identified. The microsatellite in PVALB1 was significantly associated with body weight and body length at 90 days post-hatch (P < 0.01), whereas the SNPs in PVALVB2 were not associated with these traits.     

Significant associations of polymorphisms in the prolactin gene with growth traits in Asian seabass (Lates calcarifer)

Prolactin (encoded by PRL) is a multifunctional hormone involved in osmoregulation, reproduction, growth, development, immunomodulation, endocrine and metabolic regulation. We cloned the full-length cDNA of Asian seabass PRL, searched for polymorphism in the DNA sequence, and conducted association analyses. Twelve SNPs and one 4-bp deletion were identified in PRL. The SNP c.264+127C>G was used for linkage mapping, and this gene was mapped to linkage group 11. The c.264+980_983delTTGT, c.264+127C>G, c.264+138T>G, c.264+269T>C and c.330C>G polymorphisms were genotyped in 521 individuals with growth trait records. Association analyses between single markers and growth traits revealed that the c.264+269T>C SNP was significantly associated with body weight (BW), total length (TL), standard length (SL) and Fulton's condition factor (KTL and KSL), while the other four were not. Analysis of haplotypes showed that there were 10 haplotypes and 22 haplotype combinations in the population. The differences of BW, TL, KTL and KSL among different haplotype combinations were significant.     

Bioactivity of stored luteinizing hormone-releasing hormone analogue (LHRHa) in sea bass, Lates calcarifer Bloch

The spawning induction activity of dissolved and pelleted (D-Ala⁶, Pro⁹ N ethylamide) luteinizing hormone-releasing hormone analogue (LHRHa) stored for various periods was assessed in mature female sea bass. The spawning response of mature fish was reduced significantly after injection of dissolved LHRHa (20 μg kg⁻¹) stored for more than 90 days in a refrigerator (4–10°C) or for more than 30 days at room temperature (28–30°C). Similar to fish administered fresh preparations of LHRHa, fish spawned successfully after injection of a solution of LHRHa previously frozen, subjected to alternate freezing and thawing, exposed to sunlight or implanted pelleted LHRHa (50 ng kg⁻¹) stored at room temperature for 30–120 days. Loss of hormone bioactivity after prolonged storage may have been due to bacterial growth in solubilized preparations. Injection or implantation of stored LHRHa did not influence egg production among treated sea bass. These results demonstrated the relatively prolonged shelf life of stored LHRHa.     

Ontogenetic eye development and related behavioural changes in larvae and juveniles of barramundi Lates calcarifer (Bloch)

Larvae and juveniles of barramundi Lates calcarifer (Bloch) were examined for the development of the retina, occurrence of the retinomotor response and retinal tapetum and change in eye size with age in days. The barramundi hatched with unpigmented non-functional eyes in which retinal cells had not yet differentiated into the various elements. Soon it was followed by rapid changes in the histology of the retina. Two-day-old larvae had a well-pigmented retina with area temporalis which would allow acute vision and prey attack in the nasal direction. At 10 days, rod cells and the retinal tapetum first appeared in the retina and the retinomotor response first occurred; these would allow feeding in dim light. The retinal tapetum moved in unison with the cones and the pigment epithelium during the retinomotor response. At 26 days, the horizontal cells were divided into two layers and the twin cones appeared. These changes in the eyes occurred concurrently or in anticipation of behavioural changes, such as the onset of the first feeding at 2 days, the shift of habitat from coastal waters to swamps at the notochord flexion stage at 7–15 days, the abrupt change in feeding behaviour from roving zooplanktivore to lurking predator at 25–30 days and a later shift of habitat from turbid swamps to open coastal or lake areas at the early juvenile stage.     

The use of sectioned otoliths to age barramundi (Lates calcarifer) (Bloch, 1790) [Centropomidae]

The relationship between the number of rings present in sagittal otoliths and the age of barramundi, Lates calcarifer (Bloch, 1790) [Centropomidae], was investigated by examining cross sectioned otoliths of 37 tagged fish of known age between 1 and 5 years from the Johnstone River, north Queensland. Concentric rings were clearly visible in all otolith sections and were validated as annual marks. The technique was then used to estimate the age and calculate von Bertalanffy growth parameters for 70 barramundi from the Fitzroy River, central Queensland. Growth appeared to be rapid but variable in the first year; the von Bertalanffy growth parameters for length versus age were L =690 mm, K=0.53, t ₀= 0.003 years. October 1 was designated as the birth date. Whole otolith length, width and thickness were also approximated well by the von Bertalanffy equation. We suggest that examination of otoliths is a useful technique for ageing barramundi but note that further validation of the ageing method is still needed for fish older than six years.