The use of locus specific microsatellite markers for detecting genetic variation in hatchery bred probarbus jullieni.

This study is to demonstrated that microsatellites markers developed for Tor tambroides can be used to amplify microsatellite loci in other family. It is assumed that microsatellite loci are more conserved for aquatic species compared to terrestrial ones due to aquatic environments are less mutagenic than terrestrial ones. Development of microsatellites still requires investment of time and resources. Thus using loci already developed in a related species may provide a cost-effective alternative to microsatellite isolation and development in a species of interest in present study, Probarbus jullieni. In this study we investigated the possibility of the conservation of microsatellite flanking regions among different species. Nine pairs of SSR primers, five gave very strong banding profile (SYK1, SYK 2, SYK 5 SYK 8 and SYK 9) which could be used for population studies by using the nested protocol. Results showed that SYK 2 and SYK 9 flanked the same (CA)n repeats and thus are highly conserved in a different species. The products of the SYK 5, 8 and 1 primer pairs showed differences in the microsatellite regions which they flanked in Probarbus jullieni when compared to those of the source species, Tor tambroides. The mean observed heterozygosity levels for all the primers ranged 0.23-0.81. The primers are all polymorphic with the mean number of alleles from 2-5

A simple and low-cost technique of DNA extraction from edible mushrooms examined by molecular phylogenetics

The first and most important step of molecular techniques is to isolate the high quality and standard quantity of DNA. The DNA extracted using the recommended method could successfully amplify the regions of interest and demonstrated reliable results can be applied in other molecular assays. Moreover, the designed primers of ITS1-UM2 and ITS4-UM2 were perfectly matched with the species of Basidiomycetes, can be used in phylogenetic studies of other mushrooms. We here evaluated the quality and quantity of DNA using a spectrophotometer, showed reliable OD260/280, and concentration. The protocol is efficient, rapid, low-cost, and simple, needs low amount of sample, and requires minimum facilities. The standard yield in addition to the high quality of DNA will enable mycologists to establish molecular techniques easier. In the current study, the constructed phylogenetic tree based on the obtained sequences of Internal Transcribed Spacer (ITS) I and II regions distinctly classified the examined material

A simple and low-cost technique of DNA extraction from edible mushrooms examined by molecular phylogenetics

The first and most important step of molecular techniques is to isolate the high quality and standard quantity of DNA. The DNA extracted using the recommended method could successfully amplify the regions of interest and demonstrated reliable results can be applied in other molecular assays. Moreover, the designed primers of ITS1-UM2 and ITS4-UM2 were perfectly matched with the species of Basidiomycetes, can be used in phylogenetic studies of other mushrooms. We here evaluated the quality and quantity of DNA using a spectrophotometer, showed reliable OD260/280, and concentration. The protocol is efficient, rapid, low-cost, and simple, needs low amount of sample, and requires minimum facilities. The standard yield in addition to the high quality of DNA will enable mycologists to establish molecular techniques easier. In the current study, the constructed phylogenetic tree based on the obtained sequences of Internal Transcribed Spacer (ITS) I and II regions distinctly classified the examined material

Impact of urbanisation and agriculture on the diet of fruit bats

This study was funded by the Institutional Links grant 172,726,351 under the Newton - Ungku Omar Fund, through the British Council in the UK and the Malaysian Industry-Government Group for High Technology in Malaysia

Proteomic Analysis of Differentially Expressed Protein in Hemocytes of Wild Giant Freshwater Prawn Macrobrachium Rosenbergii Infected with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV)

Epizootic diseases cause huge mortality and economical loses at post larvae stages in freshwater prawn aquaculture industry. These prawns seem less susceptible to viral diseases except for infectious hypodermal and hematopoietic necrosis virus (IHHNV). During viral infection in prawns, hemocytes are the primary organ that shows immunological response within the early stages of infection. We applied proteomic approaches to understand differential expression of the proteins in hemocytes during the viral disease outbreak. To aid the goal, we collected Macrobrachium rosenbergii broodstocks from the local grow out hatchery which reported the first incidence of IHHNV viral outbreak during larvae stage. Primarily, application of the OIE primer targeting 389 bp fragments of IHHNV virus was used in identification of the infected and non-infected samples of the prawn breeding line. Analysis of two-dimensional gel electrophoresis showed specific down-regulation of Arginine kinase and Sarcoplasmic calcium-binding protein and up/down-regulation of Prophenoloxidase1 and hemocyanin isoforms. These proteins were validated using semi quantitative RT-PCR and gene transcripts at mRNA level. These identified proteins can be used as biomarkers, providing a powerful approach to better understanding of the immunity pathway of viral disease with applications in analytic and observational epidemiology diagnosis. Proteomic profiling allows deep insight into the pathogenesis of IHHNV molecular regulation and mechanism of hemocyte in freshwater prawn

Bioinformatic characterization and gene expression pattern of apoptosis inhibitor from Macrobrachium rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus

Apoptosis is genetically programmed cellular killing processes that execute unnecessary or infected cells. It plays an important role in embryogenesis, homeostasis, insect metamorphosis and immunity. Apoptosis inhibitor (MrIAP) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrIAP consisted of 1753 base pair nucleotides encoded 535 polypeptide with an estimated molecular mass of 60 kDa. MrIAP amino acid sequence contains IAP superfamily domain between 5 and 490. The deduced amino acid sequences of the MrIAP were aligned with the other IAP family members. The highest sequence similarity was observed in IAP-5 from ant Camponotus floridanus (67%) followed by IAP from body louse Pediculus humanus corporis (66%) and the lowest (62%) in IAP-5 isoform-5 from common chimpanzee Pan troglodytes and IAP-5 from Aedes aegypti. The IAP phylogenetic tree showed that MrIAP closely related to other arthropod blacklegged tick Ixodes scapularis, formed a sister group with IAP from a hemichordate acorn worm Saccoglossus kowalevskii and finally clustered together with IAPs from fish groups. The quantitative real time PCR analysis revealed that significantly (P < 0.05) highest expression was noticed in hepatopancreas and significantly (P < 0.05) lowest expression in pleopods. Based on the results of gene expression analysis, MrIAP mRNA transcription in M. rosenbergii challenged to infectious hypodermal and hematopoietic necrosis virus (IHHNV) was highly induced in hepatopancreas. The collective results of this study indicate that the MrIAP is an essential immune gene and influences the immune response against IHHNV infection in M. rosenbergii. (C) 2011 Elsevier Ltd. All rights reserved

Molecular cloning, characterization and gene expression of an antioxidant enzyme catalase (MrCat) from Macrobrachium rosenbergii

In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85-100% enzyme activity from 30 °C to 40 °C