Marine Food Fish Research Articles

Molecular cloning, characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides): Serum amyloid A

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.

Isolation and characterization of microsatellite DNA loci from Sillago sihama

The silver sillago, Sillago sihama Forsskal is naturally distributed in Indo–West Pacific: Red Sea and Knysna, South Africa to Japan and south to Australia (Wantiez 1993), and is common along beaches, sandbars, mangrove creeks and estuaries. Adults bury themselves in the sand when disturbed (McKay 1992), and feed mainly on polychaete worms, small prawns (Penaeus), shrimps and amphipods (Allen et al. 2002). This fish is found in all Chinese waters including East China Sea, Yellow Sea, Bohai Sea, South China Sea, and the Northern Bay. In China, it is a common marine food fish species, and the supply of this fish relies on capture from the wild. So far, studies of this species have focussed on ecology (Lee et al. 1981; Lu et al. 2008), morphology of early development (Wan 1996), discrimination of the genus Sillago with sagittal otolith shape (Pan and Gao 2010), and phylogenetic relationships of the genus Sillago (Xue et al. 2010; Gao et al. 2011). In the present study, we developed 66 microsatellite loci isolated from a dinucleotide-enriched genomic library of S. sihama using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol of Zane et al. (2002). These markers should provide a tool for assessing genetic diversity and population structure of wild stocks.

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.

Estimating reproductive success of brooders and heritability of growth traits in Asian sea bass (Lates calcarifer) using microsatellites

Asian sea bass (Lates calcarifer) is one of the most important marine food fish species in Asia and Australia. To estimate the reproductive success of broodstock and heritabilities of growth-related traits, two independent full-factorial crosses (PI and PII) were created by crossing 10 males and 10 females. At 90 days post hatch (dph), the body weight (BW) and total length (TL) of 804 individuals from PI and 900 individuals from PII were measured and tissues samples of each fish were collected. Parents and offspring were genotyped with nine polymorphic microsatellites. Out of 1704 offspring from the two crosses, 98.7% were assigned to their parents. In PI, 19 of 20 parents produced offspring, while in PII, only five parents contributed to offspring. Low contribution of parents to offspring could lead to reduced genetic variation in the next generation. Heritabilities for growth-related traits were estimated using the pedigrees reconstructed using microsatellite genotypes. The estimates of heritability were 0.22±0.16 and 0.25±0.18 for BW, 0.31±0.14 and 0.24±0.21 for TL and 0.22±0.22 and 0.15±0.09 for Fulton's condition factor in the two crosses respectively. Body weights at 90 dph and at harvest (289 dph) were significantly correlated (r=0.601, P<0.01). Therefore, growth-related traits could be improved by exploiting additive genetic effects through selective breeding, and broodstock candidates could be selected early in the production cycle.

Molecular mechanisms underlying sex change in hermaphroditic groupers

Groupers are widely distributed throughout the tropical and subtropical waters of the world and are regarded as a favourite marine food fish. However, their large-scale aquaculture has been hindered by the rarity of natural males. Being protogynous hermaphrodites, groupers have been considered as study model for development and reproduction, especially for sex determination or sex differentiation, owing to the advantage that grouper gonad development undergoes transition from ovary to intersexual gonad and then to testis, and primordial germ cells and different stages of gametic cells during oogenesis and spermatogenesis are synchronously observed in the transitional gonads. Recently, a series of genes related to the reproduction regulation or sex differentiation have been identified in the groupers, mainly by researchers in China. One important finding was that the grouper gene, doublesex/male abnormal 3-related transcription factor 1 (DMRT1), is not only differentially expressed in gonads at different stages, but that it is also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, no introns were found in the grouper DMRT1, and no duplicated DMRT1 genes were detected. The finding implies that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. Additionally, we have found that grouper expression of sex-determining region Y-related high-mobility group-box gene 3 (SOX3) is a significant time point for enterable gametogenesis of primordial germ cells, because SOX3 is obviously expressed and localized in primordial germ cells. As SOX3 continues to express, the SOX3-positive primordial germ cells develop toward oogonia and then oocytes, whereas, when SOX3 expression is ceased, the SOX3-positive primordial germ cells develop toward spermatogonia. Therefore, we suggest that SOX3, as a transcription factor, might have more important roles in oogenesis than in spermatogenesis. Based on the findings, a hypothetic molecular mechanism underlying sex change is proposed in the hermaphroditic groupers, and some candidate genes related to the grouper sex change are also suggested for further research.

Construction of a BAC library and mapping BAC clones to the linkage map of Barramundi, Lates calcarifer

BackgroundBarramundi (Lates calcarifer) is an important farmed marine food fish species. Its first generation linkage map has been applied to map QTL for growth traits. To identify genes located in QTL responsible for specific traits, genomic large insert libraries are of crucial importance. We reported herein a bacterial artificial chromosome (BAC) library and the mapping of BAC clones to the linkage map.ResultsThis BAC library consisted of 49,152 clones with an average insert size of 98 kb, representing 6.9-fold haploid genome coverage. Screening the library with 24 microsatellites and 15 ESTs/genes demonstrated that the library had good genome coverage. In addition, 62 novel microsatellites each isolated from 62 BAC clones were mapped onto the first generation linkage map. A total of 86 BAC clones were anchored on the linkage map with at least one BAC clone on each linkage group.ConclusionWe have constructed the first BAC library for L. calcarifer and mapped 86 BAC clones to the first generation linkage map. This BAC library and the improved linkage map with 302 DNA markers not only supply an indispensable tool to the integration of physical and linkage maps, the fine mapping of QTL and map based cloning genes located in QTL of commercial importance, but also contribute to comparative genomic studies and eventually whole genome sequencing.

A Microsatellite Linkage Map of Barramundi, Lates calcarifer

Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its compact genome (approximately 700 Mb) is among the smallest genomes of food fish species. We established a first-generation genetic linkage map of Barramundi with a mapping panel containing three parents (two males and one female) and 93 progeny. A total of 240 microsatellite markers were mapped into 24 linkage groups. Among these markers, 10 were located in ESTs and known genes. The total lengths of the female and male maps were 873.8 and 414.5 cM with an average marker spacing of 6.20 and 4.70 cM, respectively. Comparing the flanking sequences of the 240 Barramundi microsatellites with the assembled whole-genome sequences of Tetraodon nigrovidiris revealed 55 homologous sequences located in 19 of the 21 chromosomes of T. nigrovidiris. The map will not only enable the mapping of quantitative trait loci, but also provide new resources for understanding the evolution of fish genomes.

Isolation, characterization, and linkage analyses of 74 novel microsatellites in Barramundi (Lates calcarifer)

Barramundi (Lates calcarifer) is an important marine food fish species in Southeast Asia and Australia. Seventy-four novel microsatellites were isolated from a genomic DNA library enriched for CA repeats and were characterized in 24 unrelated individuals. Among the 74 microsatellites, 71 were polymorphic, with an average allele number of 7.0 +/- 3.6/locus. The average expected heterozygosity of these polymorphic markers was 0.66. Sixty-three of the 71 polymorphic microsatellites conformed to Hardy-Weinberg equilibrium. Linkage analyses were conducted in a reference family, leading to the assignment of 34 novel microsatellites and 16 published markers in 16 linkage groups. The novel microsatellites developed in this study will contribute significantly to the construction of a first-generation linkage map for mapping of quantitative trait loci in Barramundi, and supply a large choice of markers for studies on population genetics, stock management, and pedigree reconstruction.

Twelve novel polymorphic microsatellites in a marine fish species, yellow croaker Larimichthys polyactis

AbstractWe isolated 12 polymorphic microsatellites from an important marine food fish Larimichthys polyactis and characterized them in 32 unrelated individuals. Among the 12 microsatellites, four were tetranucleotide repeats and eight were dinucleotide repeats. The allele number ranged from five to 25 with an average of 15.4/locus; average expected heterozygosity was 0.81, ranging from 0.57 to 0.95, whereas the observed heterozygosity ranged from 0.34 to 1.00 (average: 0.78). Nine of the 12 markers conformed to Hardy–Weinberg equilibrium and showed no sign of linkage. These microsatellites will be useful for population genetic studies and selective breeding programs of this species.

Isolation and characterization of microsatellites in a marine food fish species, golden trevally Gnathanodon specious

AbstractThe first set of six polymorphic microsatellites was isolated from a partial genomic DNA library of an important marine food fish Gnathanodon specious, and was characterized in 28 unrelated individuals. The allele number ranged from three to 12 with an average of seven/locus; average expected heterozygosity was 0.57 ranging from 0.10 to 0.87, whereas the observed heterozygosity ranged from 0.11 to 0.96 (average: 0.57). All six markers conform to Hardy–Weinberg and linkage equilibria. These markers will be useful for accessing genetic population structure of wild populations and for selective breeding program in fish farms.

Isolation and characterization of polymorphic microsatellites from red coral grouper (Plectropomus maculatus)

AbstractThe red coral grouper, Plectropomus maculatus, is a high value marine food fish species. We isolated the first set of 10 polymorphic microsatellites and examined their polymorphism in two related species: the Malabar grouper Epinephelus malabaricus and the brown‐marbled grouper Epinephelus fuscoguttatus. The average allele number of these 10 microsatellite DNA loci is 9.1/locus with a range of four to 17, whereas the expected heterozygosity ranged from 0.18 to 0.94, averaging 0.76. Five of the 10 markers amplified polymorphic and specific products in E. malabaricus, whereas only four were amplified in E. fuscoguttatus. These microsatellites could be applicable to captive breeding and to the studies on genetic diversity and population structure of grouper wild stocks.

Novel Optical Biotest for Determination of Cyanide Traces in Marine Fish Using Microbial Cyanide Hydratase and Formate Dehydrogenase

A novel and simple enzymatic method for the determination of trace cyanide in marine fish is presented. This method is based on the conversion of metal-cyanide complexes by a fungal enzyme extract containing cyanide hydratase (E.C. 4.2.1.66; CyHT) and formamidase (E.C. 3.5.1.49) into formate and ammonia. The formate produced in the sample pretreated with the fungal enzymes was measured by adding formate dehydrogenase (E.C. 1.2.1.2; FDH) and excess NAD+. The NADH formed accordingly was monitored at 340 nm. The cyanide calibration curve was found to be linear in the range of 10–100 µM, and the detection limit was 1.1 µM (0.0286 ppm). The proposed biotest was successfully applied to the determination of trace cyanide in a tropical marine food fish species (Russell’s Snapper (Lutjamus russellii)) which had been exposed to cyanide.

The Pathology of Systemic Iridoviral Disease in Fish

Iridoviruses have been associated with severe disease and economic loss in farmed food fish and ornamental fish, with mortality often reported to reach 50% or more. In the present study, three tropical marine food fish species and four tropical freshwater ornamental fish species with systemic iridovirus infections were examined histopathologically and ultrastructurally. Light microscopy consistently revealed pale to intensely basophilic hypertrophied virus-infected cells in spleen, kidney and intestine from all seven species. Ultrastructural examination showed changes in the vascular endothelium overlying hypertrophied virus-infected cells suggestive of pressure necrosis. Viral isolation was improved by the use of fibroblastic cell lines. This, together with the sub-endothelial location of infected cells in all infected species examined, suggests that systemic iridoviruses are mesotheliotropic.

Application of Marine Foodfish Techniques in Marine Ornamental Aquaculture: Reproduction and Larval First Feeding

The long-term sustainability of the marine ornamental industry is being threatened by environmental pressures that are severely degrading the health of coral reef ecosystems. There is now a compelling need to practice resource conservation through the development of 'reef friendly' aquaculture technologies as an alternative to wild collection practices and to restore degraded wild populations. The commercial culture of marine ornamental finfish is very much in its infancy, but advances can be made more rapidly using insights from years of research and development with marine foodfish species. Many of the bottlenecks and constraints to developing marine ornamental fish culture are those now being addressed with the more challenging species of foodfish being attempted. The two key bottlenecks that currently limit expansion of the marine ornamental industry are the control of captive maturation and spawning and the identification of appropriate first-feed items for marine ornamental fish larvae. This paper highlights basic principles and recent achievements in marine foodfish culture that might be applicable to rapid development of controlled reproduction and propagation techniques for marine ornamental finfish.

Artificial Propagation of Mutton Snapper Lutjanus analis, A New Candidate Marine Fish Species for Aquaculture

Abstract Wild‐caught mutton snapper Lutjanus analis, a high‐value marine food fish species, matured in flow‐through seawater (36 g/L) tanks after 3 yr in captivity. On 31 May 1995, a female with a mean oocyte diameter of 382 μm was injected with human chorionic gonadotropin (HCG) (500 IU/kg body wt.) followed 24 h later by a second injection (1,000 IU/kg body wt.). At the time of the second injection, three males were injected with HCG (500 IU/ kg body wt.). Voluntary spawning occurred 33 h after the first injection, with a total of 534, 781 eggs released. Fertilization rate was 75.7%, while average diameter of fertilized eggs was 783 μm. Embryos were stocked in a 30‐m3 outdoor tank at a density of 10.5/L. On day 2 post‐hatching (d2ph), larval density was 8.61 larvae/L, and average notochord length was 2.6 mm. Larvae were fed ss‐type rotifers from dl‐d28ph, Artemia nauplii from 0–08ph, and artificial diets (52–48% protein) from d24‐d38ph. On d38ph, fish averaged 0.308 g and 22.2 mm standard length. Survival (from d2ph) was 14.3%, with a total of 36,900 post‐metamorphic juveniles produced. On d97ph, 1,390 hatchery‐reared juveniles (avg. wt. = 10.5 g) were stocked into two 14.5‐m3 recirculating seawater tanks (695 fish/tank; 48 fish/m3) and fed a 56% protein pellet. After 168 d, fish averaged 140.8 g, with a survival rate of 97.8% and a feed conversion ratio (dry wt./wet wt.) of 1.2. These preliminary results reveal the mutton snapper to be a prime, new candidate species for commercial cultivation.

Nutrient requirements of marine food fish cultured in Southeast Asia

The culture of marine and brackish-water fish has gained increased importance in the aquaculture industry, particularly in Southeast Asia. As feed accounts for the major portion of rearing costs, nutritional adequacy and cost-effectiveness is critical to the industry. This review will focus on the nutritional requirements of tropical marine food fish that include seabass ( Lates calcarifer), grouper ( Epinephelus sp.), milkfish ( Chanos chanos) and rabbitfish ( Siganus sp.). Culture practices of marine fish in Southeast Asia with special reference to Thailand will also be described.

Preliminary Observations on the Marketability of Saltwater‐Cultured Florida Red Tilapia in Puerto Rico

AbstractA preliminary assessment of the marketability of saltwatercultured Florida red tilapia was conducted at a farm outlet store and at nine restaurants in Puerto Rico. Florida red tilapia were grown in six 0.2‐ha ponds at 22.7 ± 1.2 (mean ± SE) ppt salinity by a commercial aquaculture enterprise in Dorado, northeastern Puerto Rico. Customers purchased 4,683kg of dressed‐out fish (gilled, gutted and scaled) at $7.70/kg and 826kg of whole fish at $6.60/kg from a retail outlet store located at the aquaculture farm, and nine restaurants purchased 1,071kg of dressed‐out fish at wholesale prices ranging from $4.96 to $5.18/kg. Farm outlet customers and restaurants preferred dressed‐out fish between 454–567g and 567–580g, respectively. Prices paid per serving by restaurant customers for red tilapia ($7.00–$25.00) were similar to that paid for silk snapper Lutjanus vivanus, a popular marine food fish in Puerto Rico. Market surveys of customers at these outlets showed that 75–81% of the respondents were new consumers of red tilapia and that they rated red tilapia highly in taste, texture, freshness, and presentation and equal to or better than silk snapper. There was no effect (P &gt; 0.05) of age group or economic level on consumer responses. Survey participants stressed the need for product promotion, including a more markehriented name that emphasized the red color and that it was farm‐raised in saltwater, to distinguish it from darkcolored, wild‐caught freshwater tilapia. Local saltwater production of Florida red tilapia could help supply the market demand for fresh, marine fish in Puerto Rico if producers can meet the demand for quality, availability and price.

ON THE LARVAL REARING OF THE WHITE‐SPOTTED GREEN GROUPER, Epinephelus amblycephalus (BLEEKER), WITH A DESCRIPTION OF LARVAL DEVELOPMENT

ABSTRACTThe white‐spotted green grouper, Epinenephelus amblycephalus (Bleeker), a popular and cmercially important marine food fish in Hong Kong, has been used for intensive cage culture in coastal water for many years. The major constraints to its large‐scale development are, however, the shortage and uncertain supply of fingerlings from the wild. Research efforts have therefore been directed at producing large supplies of fingerlings, either for culture to marketable size or for restocking.Larvae of E. amblycephalus were reared through metamorphosis in 1000‐liter rectangular tanks covered with a large translucent roof. The newly hatched larvae measured 2.0 mm in total length and possessed a large yolk sac. Effects of temperature and salinity on the yolk sac larvae were examined in laboratory experiments. Recommended temperature and salinity for rearing of the early larvae are 24°C and 25–32 Ppt. In the laboratory larvae were fed fertilized oyster eggs, rotifers, Artemia nauplii, copepods and shrimp meat. The developnent stages from hatching through metamorphosis are described. Three mortality peaks were observed during the experiment. The highest larval survival rate through metamorphosis (i.e., 40 days) after hatching was 25%.

Social Organization and Foraging Ecology in the Arctic Skua Stercorarius Parasiticus: A Test of the Food Defendability Hypothesis

Colonial and solitary breeding in birds have been hypothetically explained as adaptations to different types of food. Evenly distributed food with stable occurrence can be defended, which favours solitary nesting in feeding territories. Patchily distributed food at unpredictable locations is not economically defendable, which selects against feeding territoriality. Colonial breeding may then be adaptive. We test this hypothesis in arctic skuas, which show both forms of social system in NE Norway. As predicted, colonial pairs mainly forage far from the nest, at sea, whereas solitary pairs often hunt in large (1 km2) defended feeding territories around the nest. The diets differ accordingly, with highest proportions of terrestrial food (birds, eggs, rodents, insects, berries), and lowest proportions of marine food (fish, Polychaeta and Crustacea), in droppings from solitary pairs. These results therefore support the food defendability hypothesis.

MUSCLE CATHEPSINS IN THREE SPECIES OF PACIFIC SOLE

The cathepsins in saline muscle extracts of three species of Pacific sole were investigated. A buffered hemoglobin solution (pH 3.0) served as substrate for the enzyme assay. No apparent change in catheptic activity in muscle homogenates of English sole was observed during 25 days of frozen (−26 °C) storage. Partial characterization of the cathepsins indicated a pH activity optimum of 3.0–3.5 for rex and petrale sole but a slightly higher PH optimum of 3.2–3.8 for English sole. The three species showed a temperature optimum of 45 °C for hemoglobin splitting. A wide variation in catheptic activity within the species was observed for rex sole while English and petrale sole showed more uniform catheptic activity. The mean activities for the three species varied significantly (P &lt; 0.05). Results of sensory evaluation of muscles containing high and low levels of catheptic activity from the three species of sole did not support the assumption that cathepsins have a substantial influence upon the quality of marine food fish.