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 F _ST 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.     

Genetic diversity of walking catfish, Clarias macrocephalus, in Thailand and evidence of genetic introgression from introduced farmed C. gariepinus

The Thai walking catfish, Clarias macrocephalus Günther, 1864, is economically important to Thailand. It occupies marshes and swamps that are severely endangered due to population expansion and natural populations are thought to be suffering from massive back-crossing with the C. macrocephalus×C. gariepinus hybrids. Therefore, a study on genetic diversity of this species is required to enable efficient conservation and management plans. In this study, 25 natural populations were collected throughout the country, 12 populations from provinces locate in the Chaophraya river basin in the center of the country, 5 from the Mekong river basin, 1 from the east and 7 from the south. One population of hatchery origin was obtained from the Department of Aquaculture, Kasetsart University in Bangkok.

Twelve isozymes and one protein system were analyzed. Among 18 loci resolved, 8 were polymorphic. The number of alleles per locus, average polymorphism and individual polymorphism were significantly higher in collections from the Chaophraya river basin than from the Mekong, east and south. The hatchery population also had relative high genetic variation. Six out of twenty-six populations differed significantly from Hardy-Weinberg equilibrium after Bonferroni correction. None of loci pairs showed significant linkage disequilibrium after Bonferroni correction. The F_st value across loci was highly significant from zero. A neighbor-joining tree reveals that populations from the south were genetically distinct from the remaining populations.

Alleles peculiar to the African catfish [C. gariepinus (Burchell, 1822)] genome were observed in 12 of the natural populations and the hatchery population. This is evidence of genetic introgression which has probably persisted for several generations, since there was no significant genotype disequilibrium between the macrocephalus and gariepinus alleles at three diagnostic loci.     

CRISPR-based platform for rapid,sensitive and field-deployable detection of scale drop disease virus in Asian sea bass (Lates calcarifer)

Scale drop disease virus (SDDV) is a major pathogen of Asian sea bass that has emerged in many countries across the Asia Pacific since 1992 and carries the potential to cause drastic economic losses to the aquaculture sector. The lack of an approved vaccine for SDDV necessitates timely prevention as the first line of defence against the disease, but current diagnostic platforms still face challenges that render them incompatible with field applications, particularly in resource-limited settings. Here, we developed a novel detection platform for SDDV based on a CRISPR-Cas12a-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA-Cas12a). Using the viral adenosine triphosphatase (SDDV-ATPase) gene as a target, we achieved the detection limit of 40 copies per reaction and high specificity for SDDV. The coupling with fluorescence and lateral flow readouts enables naked-eye visualization and straightforward data interpretation requiring minimal scientific background. Compared with semi-nested PCR in field sample evaluation, our RPA-Cas12a assay is more sensitive and capable of detecting SDDV in asymptomatic fish. Importantly, the entire workflow can be carried out at a constant temperature of 37°C within an hour from start to finish, thus removing the need for an expensive thermal cycling apparatus and long turnaround times associated with PCR-based methods. Therefore, owing to its high accuracy, rapidity and user-friendliness, the developed RPA-Cas12a platform shows the potential for diagnosis of SDDV at point of need and could be a valuable tool to help protect fish farming communities from large-scale epidemics.