The southern lesser pomfret (Pampus minor) is an economically important fish, and its numbers are declining because of overfishing and environmental pollution. In addition, owing to the similarities of its external morphological characteristics to other species in the genus Pampus, it is often mistaken for grey pomfret (P. cinereus) or silver pomfret (P. argenteus) juveniles. In this study, the genetic diversity and structure of 264 P. minor individuals from 11 populations in China and Malaysia coastal waters were evaluated for the first time, to the best of our knowledge, using mitochondrial cytochrome b fragments. The results showed that P. minor had moderate haplotype diversity and low nucleotide diversity. Furthermore, two divergent lineages were detected within the populations, but the phylogenetic structure corresponded imperfectly with geographical location; thus, the populations may have diverged in different glacial refugia during the Pleistocene low sea levels. Analysis of molecular variation (AMOVA) showed that genetic variation originated primarily from individuals within the population. Pairwise FST results showed significant differentiation between the Chinese and Malaysian populations. Except for the Xiamen population, which was classified as a marginal population, the genetic differentiation among the other Chinese populations was not significant. During the Late Pleistocene, P. minor experienced a population expansion event starting from the South China Sea refugium that expanded outward, and derivative populations quickly occupied and adapted to the new habitat. The results of this study will provide genetic information for the scientific conservation and management of P. minor resources.
Determination of feline meat in food products is an important issue for social, health, economic and religious concern. Hence this paper documented the application of species specific polymerase chain reaction-restriction fragment length polymorphism (SP-PCR-RFLP) assay targeting a short-fragments (69 bp) of mitochondrial cytochrome b (cytb) gene to screen feline meat in commercial meat products using lab-on-a-chip. The SP-PCR assay proved its specificity theoretically and experimentally while testing with different common animal, aquatic and plant species of DNA. The feline specific (69 bp, 43- and 26-bp) characteristic molecular DNA pattern was observed by SP-PCR and RFLP analysis. For assay performance, it was tested in three different types of commercial dummy meat products such as frankfurters, nuggets and meatballs and digested with AluI-restriction enzyme. The highest sensitivity of the assay using lab-on-a-chip was as low as 0.1 pg or 0.01 % (w/w) in commercial dummy meat products. We have also applied this assay to screen three important commercial meat products of six different brand from six supermarket chains located at three different states of Malaysia. Thus total 378 samples were tested to validate the specificity, sensitivity, stability of the assay and utilization of it for commercial meat product screening.
Malaria parasites in the phylum Apicomplexa (Order: Haemosporida) infect diverse vertebrates and invertebrate hosts. At least seven genera of haemosporidian parasites have been described to exclusively infect bats. Most of these parasites remain enigmatic with a poorly known host range. Here, we investigated 271 bats belonging to 21 species and seven families from six provinces of Thailand. Overall, 124 out of 271 bats (45.8%) were positive for haemosporidian parasites, while none had Plasmodium, based on microscopic examination of blood smears and PCR amplification. We obtained 19 distinct cytochrome b (cytb) nucleotide haplotypes of Hepatocystis from seven bat species (families: Craseonycteridae, Hipposideridae, Pteropodidae, and Rhinolophidae). Nycteria was found in four bat species (Craseonycteridae, Emballonuridae, Megadermatidae, and Pteropodidae) and Polychromophilus in two species (Emballonuridae, Vespertilionidae). Phylogenetic analysis inferred from cytb sequences placed Hepatocystis into 2 different clades. Most Hepatocystis infections were found in insectivorous bats and clustered together with a sequence from Hipposideros larvatus in Cambodia (in subclade 1a). A single sequence of Hepatocystis obtained from a frugivorous bat, Cynopterus brachyotis, was placed in the same clade with Hepatocystis from the same bat species previously reported in Malaysia (clade 2). Nycteria in these Thai bats were clearly separated from the African isolates previously reported in bats in the family Rhinolophidae. Polychromophilus murinus from Myotis siligorensis was placed in a distinct clade (clade 2) from Polychromophilus melanipherus isolated from Taphozous melanopogon (clade 1). These results confirmed that at least two distinct species of Polychromophilus are found in Thailand. Collectively, Hepatocystis presented no host specificity. Although Megaderma spasma seemed to be infected by only Nycteria, its respective parasite does not show specificity to only a single bat host. Polychromophilus murinus and P. melanipherus seem to infect a narrower host range or are somehow restricted to bats in the families Vespertilionidae and Emballonuridae, respectively.
Oriental giant squirrels are tree squirrels classified under family Ratufinae. In India, there are three species of genus Ratufa, i.e. Ratufa bicolor, Ratufa macroura and Ratufa indica. They are also distributed in South and Southeast Asia. However Ratufa indica is endemic to India. The fourth species Ratufa affinis is restricted to Maritime Southeast Asia (East Malaysia, Indonesia, Brunei and Thailand) and probably in Singapore. The species is near threatened .The species R.macroura is endemic to South Asia. Forests of South and Southeast Asia are hotspots of squirrel diversity but at the same time they are at a high risk of extinction because of high deforestation rate and habitat fragmentation. The present molecular study is the first study of the species of Ratufa for their identification. In this study old taxidermy samples were used for amplification of 12SrRNA and Cytochrome b genes. Maximum likelihood and neighbor-joining methods were used to delineate the species by using MEGA 6 and also for molecular analysis for variable, conserved, parsimony and singleton sites. Similarities between species through BLAST indicated 92.21-89.57% between R.macroura vs. R. bicolour; 93.22-90% Ratufa macroura vs. Ratufa indica; 96-92% Ratufa indica vs. Ratufa bicolour, 93.88% between Ratufa affinis vs. Ratufa indica, 93.5% R. affinis vs. R. bicolor, 90.5%. R. affinis vs. R. macroura. Ratufa bicolor is noted to be genetically closer to R. indica as inferred by using both markers. The BLAST result indicated that the obtained sequences matched 99-100% with their respective species. It was also noted that R. bicolor of Jalpaiguri, West Bengal are genetically closer to that of Bhutan. The study also revealed the evolution of R. indica and R. macroura from a single population.
This study was conducted to investigate the sensitivity and detection of porcine DNA in raw materials, ingredients and finished bakery products by polymerase chain reaction (PCR) - southern hybridization on chip analysis. A total of 20 samples (n=20* 3) with three replicates for each samples were obtained from a bakery factory located in Bangi, Selangor from January to December 2012. The sensitivity level of PCR-southern hybridization on chip was 0.001 ng. The species-specific oligonucleotide primers used in PCR-southern hybridization were targeted on the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene sequence, namely cty b biotin-labeled oligonucleotide primers. The amplicon from PCR amplification was 276 bp in size. None of the raw materials, ingredients and finished bakery product samples was positive towards porcine DNA, except for the positive control. The results in the present study demonstrated that the PCR- southern-hybridization technique on the gene chip (OliproTM Porcine gene chip) is a sensitive tool for monitoring the porcine component in highly processed ingredients and finished bakery products.
A Mo -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo (10 mM), the bacterium reduced Mo to form molybdenum blue. Approximately 27% of Mo added to the medium was reduced after 28 h of cultivation. The reduction of Mo with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo reduction. NADH and N,N,N',N' -tetramethyl-p-phenylenediamine served as electron donors for Mo reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo reduction. Both ferric and stannous ions strongly enhanced the activity of Mo reduction by NADH.
The need to detect genetic variation has fueled the development of novel marker systems in fisheries biology. In this study, a simple, fast and cost effective method was used to differentiate between species of freshwater fishes focusing on Malaysian freshwater fishes by employing
Restriction Fragment Length Polymorphisms (RFLPs) analysis of a 470-bp cytochrome b mtDNA segment. RFLP analysis using six restriction enzymes (AluI, BamHI, BsuRI, Csp61, HpaII and SalI) found variations in the digestion profile among most of the fish samples analyzed. Diagnostic digestion profiles were observed among the Hampala fishes, especially between H. macrolepidota and the other Hampala species/forms (using BsuRI and Csp61). Diagnostic digestion profiles were also detected between H.
bimaculata Type A and Type B (using AluI, BamHI, BsuRI and SalI), supporting their status as distinct species. Additionally, unique digestion profiles were observed in other species such as Leptobarbus hosii (Csp61), Osteocheilus hasseltii (Csp61), Osteocheilus sp. (Csp61), Puntioplites bulu (Csp61), Puntius bramoides (AluI), P. sealei (AluI) and Helostoma temmincki (AluI and Csp61), which can be used as genetic markers for discriminating these species. Overall, the RFLP analysis of the cytochrome
b mtDNA segment has proven to be a considerably effective, fast and non-expensive technique to discriminate among several freshwater fish species in Malaysia.
A technique to isolate DNA from ghee was developed for the authentication of beef fat product. The method was based on pre-mixed ghee with phosphate buffer solution (PBS) prior to DNA extraction using Epicentre extraction method. The recovery of beef DNA was then analysed by polymerase chain reaction (PCR) using beef species-specific oligonucleotide primers which targeted the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene. The amplicon was 274 bp in size. The developed ghee extraction method offers a high yield of DNA providing 100 ng per μl and useful for validating beef fat product.
This study was conducted to detect the presence of chicken and porcine DNA in meatballs using mitochondria DNA (mtDNA) of cytochrome b (cyt b) and nuclear DNA (nDNA) short interspersed nuclear element (SINE) species-specific primers, respectively. While, the mtDNA primers targeted transfer RNA-ATP8 (tRNA-ATP8) gene was used for 1 and 5% (w/w) chicken meatball spiked with commercial porcine blood plasm. Chicken meatballs spiked with 1% and 5% (v/w) fresh and commercial porcine blood plasma, respectively were prepared and heat-treated using five (n = 5) cooking methods: boiling, pan-frying, roasting, microwaving and autoclaving. Two pairs of mtDNA and nDNA primers used, produced 129 and 161 bp amplicons, respectively. Whereas, tRNA-ATP8 primers produced 212 bp of amplicon. Electrophoresis analysis showed positive results for porcine DNA at 1% and 5% (w/w or v/v) for all of the different cooking techniques, either for fresh or commercial blood plasma using SINE primers but not for tRNA-ATP8 primers. The present study has highlighted the useful of species-specific primers of SINE primers in PCR analysis for detecting porcine DNA blood plasma in heat-treated chicken meatballs.
Haemosporidians infect a wide diversity of bat genera and species, yet little is known about their transmission cycles or epidemiology. Though several recent studies have focused on the genus Hepatocystis, an Old World parasite primarily infecting bats, monkeys, and squirrels, this group is still understudied with little known about its transmission and molecular ecology. These parasites lack an asexual erythrocytic stage, making them unique from the Plasmodium vertebrate life cycle. In this study, we detected a prevalence of 31% of Hepatocystis in short-nosed fruit bats (Cynopterus brachyotis) in Singapore. Phylogenetic reconstruction with a partial cytochrome b sequence revealed a monophyletic group of Hepatocystis from C. brachyotis in Malaysia, Singapore, and Thailand. There was no relationship with infection and bat age, sex, location, body condition or monsoon season. The absence of this parasite in the five other bat species sampled in Singapore indicates this Hepatocystis species may be host restricted.
Phylogenetic relationships among Malaysia's long-tailed macaques have yet to be established, despite abundant genetic studies of the species worldwide. The aims of this study are to examine the phylogenetic relationships of Macaca fascicularis in Malaysia and to test its classification as a morphological subspecies. A total of 25 genetic samples of M. fascicularis yielding 383 bp of Cytochrome b (Cyt b) sequences were used in phylogenetic analysis along with one sample each of M. nemestrina and M. arctoides used as outgroups. Sequence character analysis reveals that Cyt b locus is a highly conserved region with only 23% parsimony informative character detected among ingroups. Further analysis indicates a clear separation between populations originating from different regions; the Malay Peninsula versus Borneo Insular, the East Coast versus West Coast of the Malay Peninsula, and the island versus mainland Malay Peninsula populations. Phylogenetic trees (NJ, MP and Bayesian) portray a consistent clustering paradigm as Borneo's population was distinguished from Peninsula's population (99% and 100% bootstrap value in NJ and MP respectively and 1.00 posterior probability in Bayesian trees). The East coast population was separated from other Peninsula populations (64% in NJ, 66% in MP and 0.53 posterior probability in Bayesian). West coast populations were divided into 2 clades: the North-South (47%/54% in NJ, 26/26% in MP and 1.00/0.80 posterior probability in Bayesian) and Island-Mainland (93% in NJ, 90% in MP and 1.00 posterior probability in Bayesian). The results confirm the previous morphological assignment of 2 subspecies, M. f. fascicularis and M. f. argentimembris, in the Malay Peninsula. These populations should be treated as separate genetic entities in order to conserve the genetic diversity of Malaysia's M. fascicularis. These findings are crucial in aiding the conservation management and translocation process of M. fascicularis populations in Malaysia.
A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy(®) Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.
Blood meal analysis (BMA) from ticks allows for the identification of natural hosts of ticks (Acari: Ixodidae). The aim of this study is to identify the blood meal sources of field collected on-host ticks using PCR analysis. DNA of four genera of ticks was isolated and their cytochrome b (Cyt b) gene was amplified to identify host blood meals. A phylogenetic tree was constructed based on data of Cyt b sequences using Neighbor Joining (NJ) and Maximum Parsimony (MP) analysis using MEGA 5.05 for the clustering of hosts of tick species. Twenty out of 27 samples showed maximum similarity (99%) with GenBank sequences through a Basic Local Alignment Search Tool (BLAST) while 7 samples only showed a similarity range of between 91-98%. The phylogenetic trees showed that the blood meal samples were derived from small rodents (Leopoldamyssabanus, Rattustiomanicus and Sundamysmuelleri), shrews (Tupaiaglis) and mammals (Tapirusindicus and Prionailurusbengalensis), supported by 82-88% bootstrap values. In this study, Cyt b gene as a molecular target produced reliable results and was very significant for the effective identification of ticks' blood meal. The assay can be used as a tool for identifying unknown blood meals of field collected on-host ticks.
A phylogenetic tree and median-joining network based on cytochrome b sequence data revealed clades consistent with morphological differences and geographical distribution of Clarias batrachus (Linnaeus, 1758) in Southeast Asia. AMOVA analysis for variation was significant among populations (P<0.05) and was in agreement with morphological differences. Pairwise differences were significant between Java and Brunei/Borneo, Brunei/Borneo and west Malaysia, and Java and west Malaysia samples (P < 0.05). Closest relationships were found between samples from Brunei/Borneo and Java, and between west Malaysia and Laos-Sumatra. Nine haplotypes were unique to geographical regions. The Java species had high haplotype (1.000 ± 0.126) but low nucleotide (0.017) diversities, suggesting a population bottleneck followed by expansion. However, SSD and Hri (P=0.5) did not support demographic expansion. Instead, purifying selection where mutations occur and accumulate at silent sites is a more acceptable explanation.
Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.
Food fraud is a global problem raising increased concerns during the past decades and food authenticity is now a burning issue. Beef, buffalo, chicken, duck, goat, sheep, and pork are heavily consumed meats bearing nutritional, economic and cultural/religious importance and are often found to be adulterated in raw and processed states. To authenticate these species, we developed and validated a highly specific multiplex (heptaplex) PCR assay targeting short length amplicons (73-263 bp) using seven pairs of species-specific primer sets targeting mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes. Specificity checking (in silico and in vitro) against 25 non-target species revealed no cross-species amplification. The developed multiplex assay was validated with various adulterated and heat-treated (boiled, microwaved and autoclaved) meatball products and were found to show high sensitivity and stability under all processing conditions. The assay was sensitive enough to detect 0.01-0.005 ng of DNA from raw meat and 0.5% (w/w) adulterated meat in mixed matrices. A market survey revealed mislabelling of 95% beef and 15% chicken products while pork products were found pure. Given some advantageous features including short sizes of amplicons, exceptional stability and superior sensitivity, the developed assay could be conveniently used for discriminatory detection of target species with a variety of raw meat as well as processed meat products undergoing extreme processing treatments.
Three species of flying fox (Pteropus hypomelanus, P. vampyrus, and P. lylei) from Malaysia and Vietnam were screened for apicomplexan parasites by thin blood smears and polymerase chain reaction. Only 1 of 16 bats sampled from 3 localities in southeast Asia was found to be infected (P. hypomelanus from Pulau Pangkor, Malaysia). We observed micro- and macrogametocytes, with morphology consistent with Hepatocystis sp. parasites, using light microscopy. Phylogenetic analysis of the cytochrome b gene showed that the parasite from P. hypomelanus groups with 2 published sequences from Hepatocystis spp., including one from Cynopterus brachyotis, another fruit bat in the Pteropodidae.
Plasmodium ovale is widely distributed in tropical countries, whereas it has not been reported in the Americas. It is not a problem globally because it is rarely detected by microscopy owing to low parasite density, which is a feature of clinical ovale malaria. P.o. curtisi and P.o. wallikeri are widespread in both Africa and Asia, and were known to be sympatric in many African countries and in southeast Asian countries. Small subunit ribosomal RNA (SSUrRNA) gene, cytochrome b (cytb) gene, and merozoite surface protein-1 (msp-1) gene were initially studied for molecular discrimination of P.o. curtisi and P.o. wallikeri using polymerase chain reaction (PCR) and DNA sequencing. DNA sequences of other genes from P. ovale in Southeast Asia and the southwestern Pacific regions were also targeted to differentiate the two sympatric types. In terms of clinical manifestations, P.o. wallikeri tended to produce higher parasitemia levels and more severe symptoms. To date, there have been a few studies that used the quantitative PCR method for discrimination of the two distinct P. ovale types. Conventional PCR with consequent DNA sequencing is the common method used to differentiate these two types. It is necessary to identify these two types because relapse periodicity, drug susceptibility, and mosquito species preference need to be studied to reduce ovale malaria. In this article, an easier method of molecular-level discrimination of P.o. curtisi and P.o. wallikeri is proposed.
Because of variable inconvenient living conditions in some places around the world, it is difficult to collect reliable physiological data for ostriches. Therefore, this study aims to provide a comprehensive in silico insight for the nature of polymorphism of important genetic loci that are related to physiological and reproductive traits. Sixty-nine mature ostriches ranging over half of Iraq were screened. Six exonic genetic loci, including cytochrome c oxidase I (COX1), cytochrome b (CYTB), secretogranin V (SCG5), feather keratin 2-like (FK2), prolactin (PRL) and placenta growth factor (PGF) were genotyped by PCR-single stranded conformation polymorphism (SSCP). Thirty-six novel SNPs, including seventeen nonsynonymous (ns) SNPs, were observed. Several computational software programs were utilized to assess the extent of the nsSNPs on their corresponding proteins structure, function and stability. The results showed several deleterious functional and stability changes in almost all the proteins studied. The total severity of each missense mutation was evaluated and compared with other nsSNPs accumulatively. It is evident from the extensive cumulative in silico computation that both p.E34D and p.E60K in PGF have the highest deleterious effect. The cumulative predictions from the present study are an impressive guide for the genotypes of African ostriches, which bypassed the expensive protocols for wet laboratory screening, to identify the effects of variants. To the best of our knowledge, this is the first investigation of its kind on the analyses and prediction outcome of missense mutations in African ostrich populations. The highly deleterious nsSNPs in the placenta growth factor are possible adaptive mutations which might be associated with adaptation in extreme and new environments. The flow and protocol of the computational predictions can be extended for various wild animals to identify the molecular nature of adaptations.
Genomic DNA of 13 fish (n=13) species consist of four freshwater which were catfish (Clarias gariepinus), shark catfish (Pangasius larnaudii), tilapia (Oreochromis mossambicus), perch (Lates calcarifer) and nine marine species which were black pomfret (Parastromateus niger), anchovy (Stolephorus commersonii), mabong (Rastrelliger kanagurta), red snapper (Lutjanus erythropterus), herring (Chirocentrus dorab), ray fish (Himantura gerrardii), sardine (Decapterus macrosoma), mackerel (Euthynnus affinis) and tuna (Thunnus tuna) were differentiated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Seven endonucleases of AluI, BsaJI, HaeIII, HindIII, HinfI, MboI and MboII were examined for the ability to digest cyt b amplicon from each species. Genomic DNA of pork (Sus scrofa domestica) were differentiated from fishes by comparing the digestion patterns produced by similar amplified region and enzymes used. In the present study, it was demonstrated that fishes and pork DNA genome were successfully differentiated using all endonucleases except for HindIII. Thus, PCR-RFLP analysis was found useful for future pork DNA detection in fish products.