Siti Azizah Mohd Nor

DNA Barcoding Reveals Cryptic Diversity within Commercially Exploited Indo-Malay Carangidae (Teleosteii: Perciformes)

Background DNA barcodes, typically focusing on the cytochrome oxidase I gene (COI) in many animals, have been used widely as a species-identification tool. The ability of DNA barcoding to distinguish species from a range of taxa and to reveal cryptic species has been well documented. Despite the wealth of DNA barcode data for fish from many temperate regions, there are relatively few available from the Southeast Asian region. Here, we target the marine fish Family Carangidae, one of the most commercially-important families from the Indo-Malay Archipelago (IMA), to produce an initial reference DNA barcode library. Methodology/Principal Findings Here, a 652 bp region of COI was sequenced for 723 individuals from 36 putative species of Family Carangidae distributed within IMA waters. Within the newly-generated dataset, three described species exhibited conspecific divergences up to ten times greater (4.32–4.82%) than mean estimates (0.24–0.39%), indicating a discrepancy with assigned morphological taxonomic identification, and the existence of cryptic species. Variability of the mitochondrial DNA COI region was compared within and among species to evaluate the COI regions suitability for species identification. The trend in range of mean K2P distances observed was generally in accordance with expectations based on taxonomic hierarchy: 0% to 4.82% between individuals within species, 0% to 16.4% between species within genera, and 8.64% to 25.39% between genera within families. The average Kimura 2-parameter (K2P) distance between individuals, between species within genera, and between genera within family were 0.37%, 10.53% and 16.56%, respectively. All described species formed monophyletic clusters in the Neighbour-joining phylogenetic tree, although three species representing complexes of six potential cryptic species were detected in Indo-Malay Carangidae; Atule mate, Selar crumenophthalmus and Seriolina nigrofasciata. Conclusion/Significance This study confirms that COI is an effective tool for species identification of Carangidae from the IMA. There were moderate levels of cryptic diversity among putative species within the central IMA. However, to explain the hypothesis of species richness in the IMA, it is necessary to sample the whole family across their broad geographic range. Such insights are helpful not only to document mechanisms driving diversification and recruitment in Carangidae, but also to provide a scientific framework for management strategies and conservation of commercially-important fisheries resources.

Morphological Deformities in Chironomus spp. (Diptera: Chironomidae) Larvae as a Tool for Impact Assessment of Anthropogenic and Environmental Stresses on Three Rivers in the Juru River System, Penang, Malaysia

ABSTRACT Morphological deformities in parts of the head capsule of Chironomus spp. larvae inhabiting three polluted rivers (Permatang Rawa [PRR], Pasir [PR], and Kilang Ubi [KUR]) in the Juru River Basin, northeastern peninsular Malaysia, were studied. Samples of the fourth-instar larvae at one location in each river were collected monthly from November 2007 to March 2008 and examined for deformities of the mentum, antenna, mandible, and epipharyngis. At each sample location, in situ measurements of water depth, river width, water pH, dissolved oxygen, and water temperature were made. Samples of river water and benthic sediments were also collected monthly from each larval sample location in each river and taken to the laboratory for appropriate analysis. Total suspended solids (TSSs), ammonium-N, nitrate-N, phosphate-P, chloride, sulfate, and aluminum content in water were analyzed. Total organic matter and nonresidual metals in the sediment samples were also analyzed. Among the three rivers, the highest mean deformity (47.17%) was recorded in larvae collected from KUR that received industrial discharges from surrounding garment and rubber factories, followed by PRR (33.71%) receiving primarily residues of fertilizers and pesticides from adjacent rice fields, and PR (30.34%) contaminated primarily by anthropogenic wastes from the surrounding residential areas. Among the various head capsule structures, deformity of the mentum was strongly reflective of environmental stress and amounted to 27.9, 20.87, and 30.19% in the PRR, PR, and KUR, respectively. Calculated Lenats toxic score index satisfactorily explained the influence of prevailing environmental variables on the severity of mentum deformities. Redundancy analysis and forward selection selected TSSs, sediment Zn, Mn, Cu, and Ni, and water pH, dissolved oxygen, water temperature, total organic matter, nitrate-N, chloride, phosphate-P, ammonium-N, sulfate, and aluminum as parameters that significantly affected some proportion of deformities. The total deformities correlated closely with deformities of mentum but only weakly with deformities in other parts of head. The total deformity incidence was strongly correlated with high contents of sediment Mn and Ni. The mentum and epipharyngis deformities incidence was highly correlated with an increase of TSSs, total aluminum, and ammonium-N and a decrease in pH and dissolved oxygen.

Distribution of Chironomidae (Insecta: Diptera) in polluted rivers of the Juru River Basin, Penang, Malaysia

The influence of physical and chemical parameters on the abundance and diversity of chironomids was studied in six rivers with moderate to highly polluted water in the Juru River Basin. The rivers: Ceruk Tok Kun (CTKR) as reference site, and polluted rivers of Pasir (PR), Juru (JR), Permatang Rawa (PRR), Ara (AR) and Kilang Ubi (KUR) were sampled over a period of five months (November 2007-March 2008). Nine chirnomid species: Chironimus kiiensis, C. javanus, Polypedilum trigonus, Microchironomus sp., Dicrotendipes sp., Tanytarsus formosanus, Clinotanypus sp., Tanypus punctipennis and Fittkauimyia sp. were identified. Assessment of their relationships with several environmental parameters was performed using the canonical correspondence analysis (CCA). Tanytarsus formosanus was the most dominant in the relatively clean CTKR and moderately polluted JR with mean densities of 19.66 and 25.32 m(-2), respectively while C. kiiensis was abundant in more polluted rivers. Tanytarsus formosanus, Dicrotendipes sp. and Microchironomus sp. were grouped under moderate to high water temperature, total organic matter (TOM), total suspended solids (TSS), velocity, pH, phosphates and sulphates. However, Tanypus punctipennis, Fittkauimyia sp., and Clinotanypus sp. were associated with high contents of river sediment such as TOM, Zn and Mn and water ammonium-N and nitrate-N and they were associated with higher dissolved oxygen (DO) content in the water. Chironomus kiiensis, C. javanus and P. trigonus showed positive relationships with TOM, ammonium-N and nitrate-N as well as trace metals of Zn, Cu and Mn. These three species could be considered as tolerant species since they have the ability to survive in extreme environmental conditions with low DO and high concentrations of pollutants. Based on the water parameter scores in all rivers, the highest diversity of chironomid larvae was reported in CTKR. With higher concentrations of organic and/or inorganic pollutants as reported in PPR, KUR and AR, the chironomid larval diversity decreased, and the abundance of tolerant species, mainly Chironomus spp., increased.

Genotoxicity of heavy metals to the larvae of Chironomus kiiensis Tokunaga after short-term exposure.

The genotoxic effects of increasing concentrations (below lethal concentration [LC50]) of cadmium ([Cd] 0.1, 1 and 10 mg/L), copper ([Cu] 0.2, 2 and 20 mg/L) and zinc ([Zn] 0.5, 5 and 50 mg/L) on Chironomus kiiensis were evaluated using alkaline comet assay after exposure for 24 h. Both the tail moment and the olive tail moment showed significant differences between the control and different concentrations of Cd, Cu and Zn (Kruskal–Wallis, p < 0.05). The highest concentration of Cd was associated with higher DNA damage to C. kiiensis larvae compared with Cu and Zn. The potential genotoxicity of these metals to C. kiiensis was Cd > Cu > Zn.

Dietary Prebiotics and Probiotics Influence the Growth Performance, Feed Utilisation, and Body Indices of Snakehead (Channa striata) Fingerlings

This study used a two-phase feeding trial to determine the influence of selected dietary prebiotics and probiotics on growth performance, feed utilisation, and morphological changes in snakehead (Channa striata) fingerlings as well as the duration of these effects over a post-experimental period without supplementation. Triplicate groups of fish (22.46 ±0.17 g) were raised on six different treatment diets: three prebiotics (0.2% β-glucan, 1% galacto-oligosaccharides [GOS], 0.5% mannan-oligosaccharides [MOS]), two probiotics (1% live yeast [Saccharomyces cerevisiae] and 0.01% Lactobacillus acidophilus [LBA] powder) and a control (unsupplemented) diet; there were three replicates for each treatment. All diets contained 40% crude protein and 12% crude lipid. Fish were fed to satiation three times daily. No mortalities were recorded during Phase 1; however, 14% mortality was documented in the control and prebiotic-amended fish during Phase 2. At the end of Phase 1, growth performance and feed utilisation were significantly higher (p<0.05) in the LBA-treated fish, followed by live yeast treatment, compared with all other diets tested. The performance of fish on the three prebiotic diets were not significantly different from one another but was significantly higher than the control diet. During Phase 2 (the post-feeding phase), fish growth continued until the 6th week for the probiotic-based diets but levelled off after four weeks for the fish fed the prebiotic diets. The feed conversion ratio (FCR) was higher in all treatments during the post-feeding period. The hepatosomatic index (HSI) did not differ significantly among the tested diets. The visceral somatic index (VSI) and intraperitoneal fat (IPF) were highest in the LBA-based diet and the control diet, respectively. The body indices were significantly different (p<0.05) between Phases 1 and 2. This study demonstrates that probiotic-based diets have a more positive influence on the growth, feed utilisation, and survival of C. striata fingerlings compared with supplementation with prebiotics.

A diminutive new species of cave-dwelling Wolf Snake (Colubridae: Lycodon Boie, 1826) from Peninsular Malaysia.

A newly discovered, diminutive, cave-dwelling, lowland species of the colubrid snake genus Lycodon Boie is described from a limestone cave along the Thai-Malaysian border in the state of Perlis, northwestern Peninsular Malaysia. Lycodon cavernicolus sp. nov. is most closely related to L. butleri Boulenger, an endemic, upland, forest-dwelling species from Peninsular Malaysia of the fasciatus group but is separated from L. butleri and all other species of the L. fasciatus group and the closely related L. ruhstrati group by having the combination of 245 (male) and 232 (female) ventral scales; 113 (male) and 92 (female) paired, subcaudal scales; a single precloacal plate; nine or 10 supralabials; 10 or 11 infralabials; a maximum total length of 508 mm (female); a relative tail length of 0.25-0.27; an immaculate venter in juveniles and dark-brown, posterior, ventral scale margins in adults; and dorsal and caudal bands in juveniles white. The discovery of L. cavernicolus sp. nov. adds to a rapidly growing list of newly discovered reptiles from karst regions and limestone forests of Peninsular Malaysia, underscoring the fact that these areas should be studied before they are quarried as they harbor a significant portion of the Peninsular Malaysias herpetological diversity.

Cytochrome b gene reveals panmixia among Japanese Threadfin Bream, Nemipterus japonicus (Bloch, 1791) populations along the coasts of Peninsular Malaysia and provides evidence of a cryptic species

Abstract We evaluated genetic differentiation among ten presumed Japanese threadfin bream, Nemipterus japonicus populations along the coast of Peninsular Malaysia based on the partial sequence of the mitochondrial cytochrome b gene (982 bp). Genetic divergences (Kimura-2 parameter) ranged from 0.5% to 0.8% among nine of the ten populations while these nine populations were 4.4% to 4.6% diverged from the Kuala Besar population located at the Northeast coast. The constructed Neighbour Joining (NJ) phylogenetic trees based on haplotypes showed the Kuala Besar population forming an isolated cluster. The Analysis of Molecular Variance (AMOVA) of the ten populations a priori assigned into four regions, revealed that most of the variation occurred within population with a fairly low but significant level of regional differentiation (FST = 0.07, p < 0.05, FSC = 0.00, p > 0.05 and FCT = 0.07, p < 0.05) attributed to the Kuala Besar population. p Value after Bonferroni correction revealed that only pairwise FST values involving the Kuala Besar population with the other nine populations were significant. Thus, this study revealed that the N. japonicus populations off Peninsular Malaysia were panmictic. However, the Kuala Besar population, although morphologically identical was composed of a genetically discrete taxon from the rest. These findings are important contributions in formulating sustainable fishery management policies for this important fishery in Peninsular Malaysia.

DNA Barcoding Reveals High Cryptic Diversity of the Freshwater Halfbeak Genus Hemirhamphodon from Sundaland.

DNA barcoding of the cytochrome oxidase subunit I (COI) gene was utilized to assess the species diversity of the freshwater halfbeak genus Hemirhamphodon. A total of 201 individuals from 46 locations in Peninsular Malaysia, north Borneo (Sarawak) and Sumatra were successfully amplified for 616 base pairs of the COI gene revealing 231 variable and 213 parsimony informative sites. COI gene trees showed that most recognized species form monophyletic clades with high bootstrap support. Pairwise within species comparisons exhibited a wide range of intraspecific diversity from 0.0% to 14.8%, suggesting presence of cryptic diversity. This finding was further supported by barcode gap analysis, ABGD and the constructed COI gene trees. In particular, H. pogonognathus from Kelantan (northeast Peninsular Malaysia) diverged from the other H. pogonognathus groups with distances ranging from 7.8 to 11.8%, exceeding the nearest neighbor taxon. High intraspecific diversity was also observed in H. byssus and H. kuekanthali, but of a lower magnitude. This study also provides insights into endemism and phylogeographic structuring, and limited support for the Paleo-drainage divergence hypothesis as a driver of speciation in the genus Hemirhamphodon.

High connectivity in Rastrelliger kanagurta: influence of historical signatures and migratory behaviour inferred from mtDNA cytochrome b.

Phylogeographic patterns and population structure of the pelagic Indian mackerel, Rastrelliger kanagurta were examined in 23 populations collected from the Indonesian-Malaysian Archipelago (IMA) and the West Indian Ocean (WIO). Despite the vast expanse of the IMA and neighbouring seas, no evidence for geographical structure was evident. An indication that R. kanagurta populations across this region are essentially panmictic. This study also revealed that historical isolation was insufficient for R. kanagurta to attain migration drift equilibrium. Two distinct subpopulations were detected between the WIO and the IMA (and adjacent populations); interpopulation genetic variation was high. A plausible explanation for the genetic differentiation observed between the IMA and WIO regions suggest historical isolation as a result of fluctuations in sea levels during the late Pleistocene. This occurrence resulted in the evolution of a phylogeographic break for this species to the north of the Andaman Sea.

Phenotypic differences of Haemonchus contortus from sheep and goats in the States of Perak and Kelantan, Peninsular Malaysia

The large stomach worm, Haemonchus contortus is a highly pathogenic nematode parasite of sheep (Ovis aries) and goats (Capra hircus). It sucks blood and causes acute disease, anaemia and severe economic losses due to morbidity and mortality. This paper compares characteristics of surface cuticular ridges (synlophe) and morphometrics of total body length, cervical papillae, spicules, barbs, gubernaculum and vulvar flap morphology of H. contortus recovered in the two hosts from the States of Perak and Kelantan, Peninsular Malaysia. The morphological variation in H. contortus samples recovered from the two hosts in Kelantan was found to be higher than from Perak. A discriminant function, based on three measurements of the spicule, confirmed the identity of male worms in the two hosts as belonging to the same species H. contortus.