Protistan parasites in order to ensure their viability and demonstrate successful progression in their life cycle need to respond towards various environmental stressors. Blastocystis sp. is known to be the most commonly found intestinal protistan parasite in any human stool surveys and has been incriminated to be responsible for diarrhea and bloating stomach. The present study demonstrates for the first time the presence of HSP70 in subtypes of Blastocystis sp. when the cultures were subjected to temperature of 39 and 41 °C where the growth of parasites was reduced to a minimum to majority being granular forms. The growth of parasites exposed to higher temperatures however doubled compared to the controls when the parasites were re-cultured back at 37 °C. Upon thermal stress at 41 °C, subtype 3 and subtype 5 isolates' growth reached up to 2.97 × 10(6) and 3.05 × 10(6) cells/ml compared to their respective controlled culture tubes at 37 °C which peaked only at 1.34 × 10(6) and 1.70 × 10(6) cells/ml respectively. The designed primer set that amplified Blastocystis sp. subtype 7 HSP70 gene in subtypes 1, 3 and 5 was against a conserved region. The gene was amplified at 318 bp. The multiple sequence alignment showed that the targeted sequence length ranges from 291-295 bp. The pair wise alignment result showed that the sequence identity among the four sequence ranges from 88% to 96%. These findings were further evidenced by the up regulation of HSP70 gene in thermal stressed isolates of subtype 3 and 5 at 41 °C. Higher number of granular forms was significantly found in thermal stressed isolates of subtype 3 and 5 which implicates that this life cycle stage has a role in responding to thermal stress.
The Malaysian giant prawn is among the most commonly cultured species of the genus Macrobrachium. Stocks of giant prawns from four rivers in Peninsular Malaysia have been used for aquaculture over the past 25 years, which has led to repeated harvesting, restocking, and transplantation between rivers. Consequently, a stock improvement program is now important to avoid the depletion of wild stocks and the loss of genetic diversity. However, the success of such an improvement program depends on our knowledge of the genetic variation of these base populations. The aim of the current study was to estimate genetic variation and differentiation of these riverine sources using novel expressed sequence tag-microsatellite (EST-SSR) markers, which not only are informative on genetic diversity but also provide information on immune and metabolic traits. Our findings indicated that the tested stocks have inbreeding depression due to a significant deficiency in heterozygotes, and FIS was estimated as 0.15538 to 0.31938. An F-statistics analysis suggested that the stocks are composed of one large panmictic population. Among the four locations, stocks from Johor, in the southern region of the peninsular, showed higher allelic and genetic diversity than the other stocks. To overcome inbreeding problems, the Johor population could be used as a base population in a stock improvement program by crossing to the other populations. The study demonstrated that EST-SSR markers can be incorporated in future marker assisted breeding to aid the proper management of the stocks by breeders and stakeholders in Malaysia.
DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5' terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5' region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.