Effects of Dietary Bovine Lactoferrin on Growth Performance and Immuno-physiological Responses of Asian Sea Bass (Lates calcarifer) Fingerlings

Probiotics and Antimicrobial Proteins - The aim of this study was to evaluate the effects of lactoferrin (Lf) on growth and feeding performance, biochemical and immune parameters in Asian sea bass...     

Characterization of Microsatellites in the IGF-2 and GH Genes of Asian Seabass (Lates calcarifer)

Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene (IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species. Received April 10, 2000; accepted July 13, 2000.     

Effects of Probiotic Bacteria Bacillus on Growth Performance,Digestive Enzyme Activity,and Hematological Parameters of Asian Sea Bass,Lates calcarifer (Bloch)

Probiotics and Antimicrobial Proteins - This study was conducted to evaluate different doses of two species of Bacillus (Bacillus licheniformis and Bacillus subtilis), on growth parameters,...     

Lysine and arginine requirements of juvenile Asian sea bass (Lates calcarifer)

Two separate experiments were conducted to determine the dietary requirements of juvenile Asian sea bass Lates calcarifer Bloch for lysine and arginine. Fish (average initial weight: lysine experiment, 13.12 ± 0.12 g; arginine experiment, 2.56 ± 0.13 g) were given amino acid test diets for 12 weeks containing fish meal, zein, squid meal, and crystalline amino acids. Each set of isonitrogenous and isocaloric test diets contained graded levels of L ‐lysine or L ‐arginine. The feeding rate in the lysine experiment was at 4–2.5% of the body weight day^?1, while in the arginine experiment it was at 10–4% of the body weight day^?1. The fish (20 per tank, lysine experiment; 15 per tank, arginine experiment) were reared in 500‐L fibreglass tanks with continuous flowthrough sea water at 27 °C and salinity of 31 ppt in the lysine experiment and at 29 °C and salinity of 29 ppt in the arginine experiment. The experiments were in a completely randomized design with two replicates per treatment. Survival was high in fish given adequate lysine or arginine. Mean percentage weight gains were significantly different in fish fed varying levels of lysine or arginine. Fish fed high levels of L ‐arginine suffered high mortalities. No significant differences were obtained in the feed efficiency ratios (FER, g gain g^?1 feed) of fish fed graded lysine, although the values tended to increase as the dietary lysine level was increased up to the requirement level. In contrast, in the arginine experiment, significant differences in FER of fish among treatments were obtained; the highest FER was observed in fish fed the diet containing an optimum arginine level. On the basis of the growth response, survival, and FER, the lysine and arginine requirements of juvenile Asian sea bass were estimated to be 20.6 g kg^?1 dry diet (4.5% protein) and 18.2 g kg^?1 dry diet (3.8% protein), respectively. These data will be useful in the further refinement of practical diet formulations for the Asian sea bass.     

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.     

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.     

微卫星DNA标记技术及其在植物研究中的应用

微卫星DNA(或简单重复序列,simplesequencerepeats,SSR)是继RFLP、RAPD等分子标记后出现的第二代分子标记技术。随着分子生物学的发展,微卫星标记技术在植物基因组中的应用越来越广泛。由于SSR具有多态性高,呈共显性遗传,遵守孟德尔式分离,在数量上没有生物学的限制,实验操作简单,对样品质量要求不高等特点,因此被广泛用于植物遗传图谱的构建、基因定位、构建指纹图谱、遗传多样性及物种进化与亲缘关系的研究等方面。     

Microsatellites from kousa dogwood (Cornus kousa)

Microsatellite loci were identified from Cornus kousa'National'. Primer pairs for 86 loci were developed and of these, eight were optimized and screened using genomic DNA from 22 kousa cultivars. All optimized loci were polymorphic and the number of alleles per locus ranged from three to 17. Observed heterozygosity ranged from 0 to 0.3 and expected heterozygosity ranged from 0.38 to 0.91. These microsatellites will be useful in population studies, and a breeding programme for cultivar development of Cornus species.     

The Acclimation of European Sea Bass (Dicentrarchus labrax) to Temperature: Behavioural and Neurochemical Responses

Studies on fish behavioural and neurophysiological responses to water temperature change may contribute to an improved understanding of the ecological consequences of global warming. We investigated behavioural and neurochemical responses to water temperature in European sea bass (Dicentrarchus labrax) acclimated to three temperatures (18, 22 and 28°C). After 21 d of acclimation, three groups of 25 fish each were exposed to four behavioural challenges (foraging, olfactory, aversive and mirror tests). The expression of choline acetyltransferase (ChAT) was then analysed by Western blotting in CNS homogenates (from a subset of the same fish) as a marker for cholinergic system activity. In both foraging and olfactory tests, fish acclimated to 28°C exhibited significantly higher arousal responses than fish acclimated to lower temperatures. All specimens showed fright behaviour in the aversive test, but the latency of the escape response was significantly less in the fish at 28°C. Finally, the highest mirror responsiveness was exhibited by the fish acclimated to 22°C. As in the case of cholinergic neurotransmission, significantly higher ChAT levels were detected in the telencephalon, diencephalon, cerebellum and spinal cord of fish acclimated to 22 or 28°C in comparison with those maintained at 18°C. Lower ChAT levels were detected in the mesencephalon (optic tectum) at 22 and 28°C than at 18°C. These data indicate that neuronal functions are affected by water temperature. Increases or decreases in ChAT expression can be related to the functional modulation of brain and spinal cord centres involved in behavioural responses to temperature change. Overall, the results of this study suggest that the environmental temperature level influences behaviour and CNS neurochemistry in the European sea bass.     

DNA barcoding reveals a likely second species of Asian sea bass (barramundi) (Lates calcarifer)

DNA barcoding – the sequencing of a c. 650 base pair region of the mitochondrial cytochrome c oxidase I gene – strongly suggests that barramundi ( Lates calcarifer ) from Australia and from Myanmar are different species (Kimura 2 parameter distance of c. 9·5%). Cytochrome b sequence data support this conclusion (distance c. 11·3%). Further examination, both genetic and morphological, of L. calcarifer throughout its range is recommended.     

Isolation and characterization of Pseudomonas sp. KUMS3 from Asian sea bass (Lates calcarifer) with fin rot

Bacteria were isolated from Asian Sea bass, Lates calcarifer kept in a farm, on the South-east coast of India. During an outbreak of fin rot, the affected fish had hemorrhages at the base of fins, mouth and skin muscles and faded pigments. Pure colonies were isolated on NA and ZMA from internal organs of the fish and the bacterial morphology was identified as gram-negative rod-shaped bacteria. Based on different biochemical tests and sequence of 16S rDNA, the causative bacteria were identified as Pseudomonas sp. KUMS3. Bacterial cells were isolated from liver and kidney of all artificially infected moribund fish and confirmed as Pseudomonas sp. KUMS3 by morphological and biochemical characteristics. During the experimental infection, the first incidence of dead fish was observed on 2nd day after exposure to Pseudomonas sp. KUMS3 and no fish died after 12 days post exposure and the cumulative percent of mortality was 70. Histological lesions were observed in the spleen, liver and kidney of the infected fish. Pseudomonas sp. KUMS3 could be considered as an opportunistic pathogen, which can survive on the fish surface or in water or in the gut and may cause disease when unfavorable conditions develop.     

Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi (Lates calcarifer)

In Streptococcus iniae, lactate metabolism is dependent upon two proteins, lactate permease that mediates uptake and lactate oxidase, a flavin mononucleotide-dependent enzyme that catalyzes oxidation of α-hydroxyacids. A novel variant of the lactate oxidase gene, lctO, in Australian isolates of S. iniae from diseased barramundi was found during a diagnostic screen using LOX-1 and LOX-2 primers, yielding amplicons of 920 bp instead of the expected 869 bp. Sequencing of the novel gene variant (type 2) revealed a 51-nucleotide insertion in lctO, resulting in a 17-amino-acid repeat in the gene product, and three-dimensional modeling indicated formation of an extra loop in the monomeric protein structure. The activities of the lactate oxidase enzyme variants expressed in Escherichia coli were examined, indicating that the higher-molecular-weight type 2 enzyme exhibited higher activity. Growth rates of S. iniae expressing the novel type 2 enzyme were not reduced at lactate concentrations of 0.3% and 0.5%, whereas a strain expressing the type 1 enzyme exhibited reduced growth rates at these lactate concentrations. During a retrospective screen of 105 isolates of S. iniae from Australia, the United States, Canada, Israel, Réunion Island, and Thailand, the type 2 variant arose only in isolates from a single marine farm with unusually high tidal flow in the Northern Territory, Australia. Elevated plasma lactate levels in the fish, resulting from the effort of swimming in tidal flows of up to 3 knots, may exert sufficient selective pressure to maintain the novel, high-molecular-weight enzyme variant.Streptococcus iniae is a major pathogen of farmed fish, resulting in severe economic losses globally estimated at U.S. $150 million annually (27). S. iniae is essentially a blood pathogen, with infection resulting in a generalized septicemia and meningitis (1). During infection the pathogen avoids phagocytosis by means of an antiopsonic capsule (4, 18, 22) and by binding host serum components including immunoglobulins (3) and fibrinogen (2). Little is known, however, about the metabolism of S. iniae during infection although lactic acid bacteria may produce l-lactate from fermentation of glucose. In S. iniae a lactate oxidase gene, lctO, has been characterized previously (11). The product of the lctO gene in S. iniae is a flavin enzyme (l-lactate 2-monooxygenase, EC 1.13.12.4), which catalyzes the oxidation of lactate to pyruvate, coupled with reduction of O₂ to H₂O₂ (11). Lactate oxidase has been extensively characterized, both structurally and functionally, in the cold-water marine pathogen Aerococcus viridans (9, 30, 35, 36); thus, the catalytic activities of these enzymes are relatively well understood. In S. iniae, lactate can be utilized as an energy source through an aerobic but nonrespiratory mode of metabolism (11), a mechanism that is coupled to hydrogen peroxide production in Streptococcus pyogenes (26).Since the discovery of the lactate oxidase gene in S. iniae, its presence has been routinely used for PCR-based diagnosis, overcoming the lack of specificity of commercial biochemical diagnostic kits and other molecular methods. Confirmation of isolate identity as S. iniae by commercial bacterial identification kits is problematic because the biochemical profile is absent from databases supplied with the kits or because the databases are unable to identify atypical strains with confidence (25). Identification of isolates by molecular methods such as PCR is more reliable since isolates with atypical biochemical profiles can confidently be identified. PCR has been used to amplify sections of the 16S rRNA gene (37), the chaperonin HSP60 (12), and the 16S-23S rRNA gene intergenic spacer region (5) for identification of S. iniae. The development of the lactate oxidase gene (lctO) PCR assay by Mata et al. (21) reported that the primer pair LOX-1/LOX-2 could be used successfully to aid in the identification of S. iniae via the generation of a specific 870-bp product. Moreover, the LOX-1/LOX-2 primer pair overcame the problem of nonspecific amplification of Streptococcus difficilis that had previously been reported with the 16S rRNA gene primer pair described previously (21, 37).In Australia, S. iniae causes major economic loss in farmed barramundi (Lates calcarifer, Bloch) (1, 6). Barramundi, also known as Asian sea bass, are perciform euryhaline fish native to Australia and tropical southeast Asia. In Australia, barramundi have both cultural and commercial significance in terms of their iconic status among indigenous populations and the recent rapid growth of commercial farming. The value of intensive barramundi culture in Australia increased from Australian $15.5 million in 2004 to Australian $23.5 million in 2006 (34). There is also increasing farmed output of L. calcarifer in Malaysia, Indonesia, Taiwan, and Vietnam (33) and small to medium recirculating aquaculture ventures in the United States and United Kingdom using imported fingerlings.During routine diagnostic screening of S. iniae isolated throughout Australia from diseased barramundi, a novel variant of the lctO gene was found that resulted in amplicons of 920 bp following PCR using the LOX-1/LOX-2 primer pair. Isolates expressing the novel lactate oxidase gene were isolated only from a single site in the Northern Territory, Australia. In the present study, the novel lctO variant is investigated genetically and phenotypically in order to better understand how the larger gene product may have arisen from this single site.     

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.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

Identification and Functional Analysis of Novel Bradykinin-Related Peptides (BRPs) from Skin Secretions of Five Asian Frogs

In recent decades, various types of bioactive substances have been identified from amphibian skin and its secretions. Bradykinin-related peptides (BRPs) are among these compounds that make up the host defence system of amphibians. In the present study, we identified six novel BRPs, amolopkinin-GN1, amolopkinin-RK1, amolopkinin-TR1, amolopkinin-LF1, ranakinin-MS1, and ranakinin-MS2, from five East Asian amphibians, Amolops granulosus, Amolops ricketti, Amolops torrentis, Amolops lifanensis, and Hylarana maosonensis. This is the first report on BRPs in the skin of these species. Physiological assays reveal that these peptides have a contractive effect on the smooth muscle of rat ileum.     

Microsatellites from the charcoal rot fungus (Macrophomina phaseolina)

Microsatellite loci were identified from the charcoal rot fungus (Macrophomina phaseolina). Primer pairs for 46 loci were developed, and of these, 13 were optimized and screened using genomic DNA from 55 fungal isolates collected predominantly from two soybean fields in Mississippi. Twelve of the optimized loci were polymorphic and the number of alleles per locus ranged from 6 to 22. These microsatellites will be useful in population and pathogenicity studies to correspond with development of potential disease-resistant soybean and other susceptible crops.