Uthairat Na-Nakorn

DNA barcoding of catfish: species authentication and phylogenetic assessment.

As the global market for fisheries and aquaculture products expands, mislabeling of these products has become a growing concern in the food safety arena. Molecular species identification techniques hold the potential for rapid, accurate assessment of proper labeling. Here we developed and evaluated DNA barcodes for use in differentiating United States domestic and imported catfish species. First, we sequenced 651 base-pair barcodes from the cytochrome oxidase I (COI) gene from individuals of 9 species (and an Ictalurid hybrid) of domestic and imported catfish in accordance with standard DNA barcoding protocols. These included domestic Ictalurid catfish, and representative imported species from the families of Clariidae and Pangasiidae. Alignment of individual sequences from within a given species revealed highly consistent barcodes (98% similarity on average). These alignments allowed the development and analyses of consensus barcode sequences for each species and comparison with limited sequences in public databases (GenBank and Barcode of Life Data Systems). Validation tests carried out in blinded studies and with commercially purchased catfish samples (both frozen and fresh) revealed the reliability of DNA barcoding for differentiating between these catfish species. The developed protocols and consensus barcodes are valuable resources as increasing market and governmental scrutiny is placed on catfish and other fisheries and aquaculture products labeling in the United States.

A preliminary genetic map of walking catfish (Clarias macrocephalus)

We have constructed the first linkage map for walking catfish, Clarias macrocephalus, using amplified fragment length polymorphism (AFLP) markers. Forty-seven primer combinations were used to amplify DNA samples of a female C. macrocephalus and 79 haploid embryos. The AFLP map consists of 134 loci placed into 31 linkage groups, with 12 loci remaining unlinked. The number of markers per linkage group varies from 3 to 14. The map spans an estimated 2037.4 Kosambi cM, with an average spacing of 17.07 cM. This linkage map will provide the basis for a high-resolution map for walking catfish.

A study on phylogeny and biogeography of mahseer species (Pisces: Cyprinidae) using sequences of three mitochondrial DNA gene regions

The cyprinid fishes of the three genera Tor, Neolissochilus and Naziritor, often referred to as mahseer, are an important group of fish. Mahseer are endemic to Asia with natural distribution encompassing the trans-Himalayan region in the northwest to Sumatra and Borneo islands in the southeast, across a number of countries such as Nepal, Pakistan, India, Sri Lanka, Myanmar, China, Thailand, Laos, Cambodia, Vietnam, Indonesia and Malaysia. These species are large-scaled barbels that live in upstream, clear, running waters (Shrestha, 1990; Ng, 2004). They are attractive as sport fish (Ng, 2004), and some species are of great economical value and of conservation concern (Nguyen et al., 2006), as well as of aquaculture potential (Haque et al., 1995; Ingram et al., 2005). Currently 46 mahseer species are recognized, of which 23 species are of genus Tor Gray, 22 species belong to genus Neolissochilus Rainboth and one species of genus Naziritor Mirza (Eschmeyer et al., 2004). However, there is still much confusion with regard to taxonomy and systematics, and uniformity in diagnosis of this group. Fishes of the genus Tor are considered as ‘‘true mahseer” with the presence of the median lobe, as opposed to Neolissochilus and Naziritor, where the median lobe is not present. The shape, size and length of the median lobe, the features that have often been used to distinguish species of Tor (Zhou and Cui, 1996), are highly variable (Roberts, 1999) and are also being influenced by environmental factors, leading

Study of Genetic Diversity of Orange-Spotted Grouper, Epinephelus coioides , from Thailand and Indonesia Using Microsatellite Markers

Genetic diversity of Epinephelus coioides (Hamilton, 1822), which inhabits coastal reefs from the western Indian Ocean to the western Pacific Ocean, was studied based on four polymorphic microsatellite loci. Two hundred and fifty individuals were collected from two locations in Thailand (Nakornsrithammarat-N and Trang-T) and four in Indonesia (Sibolga-S, Lampung-L, Jepara-J, and Flores-F). The genetic variation of E. coioides was relatively low; the observed heterozygosities (Ho) ranged between 0.36 (F) and 0.55 (N). The average number of alleles/locus was between 3.57 (L) and 5.09 (J). Genotypic distribution for most population pairs was significantly different after Bonferroni correction (P < 0.0024) except for J and F. Population structuring was significant (FST = 0.074). The genetic distances between populations ranged between 0.016 (L and N) to 0.086 (F and S). Mantels test showed no correlation between genetic distance and geographical distance. The NJ tree clearly separated N from the others which comprised two subgroups, T-S and L-J-F.

Preliminary study on performance of triploid Thai silver barb, Puntius gonionotus

Abstract The triploid chromosome condition was induced in Thai silver barb ( Puntius gonionotus ) by application of cold shock (2°C) to eggs at time intervals after activation of 0.5 min with a duration of 10 min which resulted in mean triploidy yield of 72.5% at 9 months of age. Growth rate of the 2–9-month-old, cold shock group (0.1–6.2 g/month) did not differ from that of the control (0.1–5.7 g/month). Gonadal somatic indices (GSI) of presumed triploid males and females were lower than that of control (GSI values of the presumed triploids were 35.0–60.2% and 28.7–75.9% of control males and females, respectively). Spermatogenesis and oogenesis were retarded in triploids. However, all stages of spermatogenic cells were observed in triploid males, including few spermatozoa. Oocytes of triploid females did not undergo vitellogenesis while normal oogenesis was observed in diploids. Nuclear volume of red blood cells (RBCs) of triploid fish was 1.63 times larger than that of diploids.

Pubertal effects of 17α-methyltestosterone on GH-IGF-related genes of the hypothalamic-pituitary-liver-gonadal axis and other biological parameters in male, female and sex-reversed Nile tilapia.

The influence of 17α-methyltestosterone (MT) on growth responses, biological parameters and the expression of genes involved in the GH-IGF pathway of the hypothalamic-pituitary-liver-gonadal axis were investigated in female, male, and sex-reversed Nile tilapia to evaluate the relationship between sex and MT-induced changes in these parameters. Female fish had a lower growth rate than male and sex-reversed fish, and MT increased growth performance and duodenal villi in females. Most but not all biological parameters of sex-reversed fish were similar to those of male fish. Male fish had higher red blood cell counts and hemoglobin levels than female and sex-reversed fish, suggesting that these hematological indices reflect a higher metabolic rate in male fish. Greater blood triglyceride levels indicated the vitellogenin process in female fish. MT increased the alternative complement activity in female fish (P<0.05). Sex and MT had no significant effects on the hypothalamic mRNAs of GHRH and PACAP. Although not statistically significant, females tended to have higher GH mRNA levels than male and sex-reversed fish. Additionally, MT tended to decrease and increase GH mRNA levels in female and male fish, respectively. There were significant differences among sexes in the expression of GHR, and IGF mRNAs at the peripheral level in the liver and gonads. Females had lower hepatic GHRs and higher ovarian GHRs than male and sex-reversed fish. While the mRNA levels of IGF-1 were lower in the ovary, the levels of IGF-2 were higher compared with those in testes. A significant correlation between GHRs and IGFs was demonstrated in the liver and gonad (except for IGF-1). Multiple regression analysis showed a significant relationship between GH mRNA and both GHRs and IGFs in the liver and gonad. MT exerted androgenic and, to some extent, estrogenic effects on several physiological parameters and GH-IGF action.

Suitable conditions for induction of gynogenesis in the catfish, Clarias macrocephalus, using sperm of Pangasius sutchi

Abstract Gynogenesis was induced in Clarias macrocephalus using UV-irradiated sperm of Pangasius sutchi . Sperm was diluted 1:100 in Ringers solution and irradiated for 2 min with a 30-W UV-lamp placed 30 cm over the sperm. Optimum shock duration was determined by applying cold shock (10°C) to eggs activated with the irradiated sperm, 4.5 min after activation, with durations of 3–12 and 8–17 min (at 1-min intervals). The highest percentage of diploid gynogens was obtained from the groups cold-shocked for 12, 13 or 14 min. To determine the optimum time for starting cold shock, a cold shock at 10°C with duration of 14 min was applied to groups of eggs 2.5–8 min after activation (at 0.5-min intervals). Results revealed that the optimum time to start cold shock was 3.5, 4 or 4.5 min after activation. Cold shock at 6–11°C applied for 14 min, 4 min after activation, was equally effective in inducing diploidization of gynogenetic fry. However, the highest mean percentage of diploid gynogens was observed in the 7°C-treated group.

Genetic diversity of wild mekong giant catfish Pangasianodon gigas collected from Thailand and Cambodia

The Mekong giant catfish Pangasianodon gigas is endemic to the Mekong River and is a critically endangered species. The genotypes of the microsatellite DNA (msDNA) and mitochondrial DNA (mtDNA) markers (right domain of the control region) were detected to evaluate the present status of genetic divergence of this species from the Mekong River in Thailand and Cambodia. The observed and expected heterozygosity values of Mekong giant catfish in Thailand and Cambodia were relatively low in comparison with those of other nonendangered freshwater fish species. These two populations from Thailand and Cambodia showed similar levels of genetic diversity, as evaluated by the 384 nucleotides of the mtDNA control region with 13 haplotypes. The pairwise FST value between the two populations based on the genotype frequencies of msDNA and mtDNA markers suggested a close genetic relationship between the populations in Thailand and Cambodia. The results of this study support the conclusion that the Mekong giant catfish is critically endangered. Care should be taken to sustain the genetic diversity of this species, as the level of genetic variability has already decreased in the wild population. This species is a target species for an ongoing stock enhancement program in the Mekong River in Thailand. It is proposed to apply these markers for proper broodstock management, such as for minimal kinship selective breeding in the hatchery.

Evaluation of genetic diversity of eight grouper species Epinephelus spp. based on microsatellite variations

Genetic diversity among eight species of grouper, Epinephelus bleekeri, E. coioides, E. malabaricus, E. ongus, E. akaara, E. maculatus, E. merra and E. fuscoguttatus, was studied using six microsatellite loci, Em-01⋆, Em-03⋆, Em-07⋆, Em-08⋆, Em-10⋆, and CA-07⋆, with the aim of exploring the feasibility of using microsatellite data for species identification. The results showed high levels of genetic differentiation among species (Fst=0.4403 and Rst=0.4954). Species identification based on fixed allelic differences was possible between E. coioides, E. ongus, and E. fuscoguttatus at Em-01⋆ and between E. fuscoguttatus and E. ongus at Em-08⋆. Private alleles were found in all species, except for E. ongus. Pairwise Fst ranged 0.238–0.578 (P<0.008 Bonferroni correction), and Nei’s genetic distance ranged 0.433–2.710. Size homoplasy was observed at Em-03⋆157 allele, which was characterized by a T-C transition at the 119th nucleotide site of PCR products. The genetic assignment test unambiguously assigned each individual to the correct species. Thus, this test can be used for species identification of unknown individuals when the multilocus genotypes of the six microsatellite loci are available. The phylogenetic (neighbor-joining) tree, which was constructed based on the genetic distance matrix, separated the eight grouper species into two main groups.

Spatial and temporal genetic variation of green mussel, Perna viridis in the Gulf of Thailand and implication for aquaculture.

The culture of green mussel (Perna viridis) in the Gulf of Thailand depends on natural spat which are believed to come from spawning grounds adjacent to major river mouths. In the present paper, genetic diversity of spatial and temporal populations of green mussel in the Gulf of Thailand was investigated using five microsatellite loci. The results showed moderate genetic variation of all 11 populations (averaged number of alleles per locus, A = 10.4–12.2; effective number of alleles per locus, Ae = 5.36–6.59; mean allelic richness, Ar = 10.23–12.06; observed heterozygosity, Ho = 0.52–0.63, and expected heterozygosity, He = 0.66–0.73) without significant differences among populations. No sign of bottleneck or genetic disequilibrium was observed. Genetic differentiation among spatial populations was low (FST = 0.0046, CI0.95 = 0.0020–0.0083 for the samples collected in January, 2007, and FST = 0.0088, CI0.95 = 0.0010–0.0162 for the samples collected in July, 2007) while temporal variation was significant as revealed by the analysis of molecular variance. Multidimensional scaling separated temporal population groups with minor exception. The assignment test revealed that most of the recruits were from other populations.