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Microalgal lipid production by Chlorella protothecoides using sugarcane bagasse hydrolysate was investigated in this study. First, maximum glucose and reducing sugar concentrations of 15.2 and 27.0 g/L were obtained in sugarcane bagasse hydrolysate (SCBH), and the effects of different percentages of glucose and xylose on algal cultivation were investigated. Afterwards, SCBH was used as a carbon source for the cultivation of C. protothecoides and higher biomass concentration of 10.7 g/L was achieved. Additionally, a large amount of fatty acids, accounting up to 16.8% of dry weight, were accumulated in C. protothecoides in the nitrogen-limited (0.1–1 mmol/L) culture. Although SCBH inhibited fatty acid accumulation to a certain degree and the inhibition was aggravated by nitrogen starvation, SCBH favored microalgal cell growth and fatty acid production. The present study is of significance for the integration of cost-effective feedstocks production for biodiesel with low-cost SCBH as well as environmentally friendly disposal of lignocellulosic wastes.

Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

A survey of 30 collections sites in Peninsular Malaysia, representing five types of habitats, namely, hot springs, eutrophicated freshwater lake, palm oil mill effluent ponds, brackish and marine water habitats, yielded 79 environmental samples. The total number of algal taxa and genera identified were 73 and 52 respectively; with 9 genera and 11 taxa of Cyanophyta; 25 genera and 33 taxa of Bacillariophyta; 13 genera and 16 taxa of Chlorophyta and 5 genera and 13 taxa of Euglenophyta. In terms of cell numbers, the samples from the oil palm mills had the highest cell density. Of the isolates obtained from the samples collected, only six genera, namely the Cyanophyte Cyanosarcina, the Chlorophytes Chlorella, Chlamydomonas, Chlorococcum,Scenedesmus and the Euglenophyte Euglena, are discussed in terms of their growth and biochemical profiles in this paper. Chlorococcum and Euglena had the highest specific growth rate, µ, followed by Chlorella andScenedesmus. Biomass productivity at day 12 was generally higher than that at day 8 for all genera. Highest biomass productivity was from Chlorella followed by Chlamydomonas. Highest lipid productivity was from Chlorella andChlamydomonas on day 12. Chlorella had the highest carbohydrate productivity followed by Chlamydomonas.Chlorella and Chlamydomonas had the highest protein productivity. In this short survey, some interestingalgae were identified and isolated, which proved to have potential for use as feedstocks for biofuel production. Keywords:microalgae, checklist, specific growth rate, carbohydrate, protein, lipid productivity

Angiostrongylus cantonensis is a bursate nematode parasite that causes eosinophilic meningitis (or meningoencephalitis) in humans in many parts of the world. The genomic data from A. cantonensis will form a useful resource for comparative genomic and chemogenomic studies to aid the development of diagnostics and therapeutics. We have sequenced, assembled and annotated the genome of A. cantonensis. The genome size is estimated to be ∼260 Mb, with 17,280 genomic scaffolds, 91X coverage, 81.45% for complete and 93.95% for partial score based on CEGMA analysis of genome completeness. The number of predicted genes of ≥300 bp was 17,482. A total of 7737 predicted protein-coding genes of ≥50 amino acids were identified in the assembled genome. Among the proteins of known function, kinases are the most abundant followed by transferases. The draft genome contains 34 excretory–secretory proteins (ES), a minimum of 44 Nematode Astacin (NAS) metalloproteases, 12 Homeobox (HOX) genes, and 30 neurotransmitters. The assembled genome size (260 Mb) is larger than those of Pristionchus pacificus, Caenorhabditis elegans, Necator americanus, Caenorhabditis briggsae, Trichinella spiralis, Brugia malayi and Loa loa, but smaller than Haemonchus contortus and Ascaris suum. The repeat content (25%) is similar to H. contortus. The GC content (41.17%) is lower compared to P. pacificus (42.7%) and H. contortus (43.1%) but higher compared to C. briggsae (37.69%), A. suum (37.9%) and N. americanus (40.2%) while the scaffold N50 is 42,191. This draft genome will facilitate the understanding of many unresolved issues on the parasite and the disorder it causes.

Despite the large number of species discovered in Halymenia, many remain poorly known due to the scarce information available. In order to facilitate species discrimination of foliose Halymenia species in Southeast Asia, molecular analysis and morphological studies were made on Halymenia collections from Malaysia, the Philippines, and Indonesia. The rbcL phylogenetic analyses showed that there are at least six taxa of foliose Halymenia occurring in Southeast Asia. Among the six taxa, a new species, Halymenia malaysiana P.-L. Tan, P.-E. Lim, S.-M. Lin et S.-M. Phang, is proposed based on both rbcL sequence analyses and morphological observations. Halymenia malaysiana is characterized by thalli possessing oblong or suborbiculate blades with a supple cartilaginous structure and gelatinous (slimy) texture, arising from a small discoid holdfast without a stipe, abruptly expanding into a broad blade and having a smooth surface with sinusoidally undulated margins. The phylogenetic analyses also revealed that Halymenia is a polyphyletic genus, which requires further taxonomic studies.

The single cell oil (SCO) production by the mono and mixed culture of microalgae Chlorella pyrenoidosa and red yeast Rhodotorula glutinis was investigated using non-detoxified cassava bagasse hydrolysate (CBH) as carbon source. The results suggested that the two strains were able to tolerate and even degrade some byproducts presented in the CBH, and the mixed culture approach enhanced the degradation of certain byproducts. Biomass (20.37 ± 0.38 g/L) and lipid yield (10.42 ± 1.21 g/L) of the mixed culture achieved in the batch culture were significantly higher than that of the mono-cultures (p 0.05). The fed-batch culture further raised the biomass and lipid yield to 31.45 ± 4.93 g/L and 18.47 ± 3.25 g/L, respectively. The lipids mainly composed of oleic acid and palmitic acid, suggesting the potential applications such as biofuel feedstock, cosmetics, food additives and lubricant. This study provided new insights for the integration of the economical SCO production with agro-industrial waste disposal.

Many studies classifying Gracilaria species for the exploitation of agarophytes and the development of the agar industry were conducted before the prevalence of molecular tools, resulting in the description of many species based solely on their morphology. Gracilaria firma and G. changii are among the commercially important agarophytes from the western Pacific; both feature branches with basal constrictions that taper toward acute apices. In this study, we contrasted the morpho-anatomical circumscriptions of the two traditionally described species with molecular data from samples that included representatives of G. changii collected from its type locality. Concerted molecular analyses using the rbcL and cox1 gene sequences, coupled with morphological observations of the collections from the western Pacific, revealed no inherent differences to support the treatment of the two entities as distinct taxa. We propose merging G. changii (a later synonym) into G. firma and recognize G. firma based on thallus branches with abrupt basal constrictions that gradually taper toward acute (or sometimes broken) apices, cystocarps consisting of small gonimoblast cells and inconspicuous multinucleate tubular nutritive cells issuing from gonimoblasts extending into the inner pericarp at the cystocarp floor, as well as deep spermatangial conceptacles of the verrucosa-type. The validation of specimens under different names as a single genetic species is useful to allow communication and knowledge transfer among groups from different fields. This study also revealed considerably low number of haplotypes and nucleotide diversity with apparent phylogeographic patterns for G. firma in the region. Populations from the Philippines and Taiwan were divergent from each other as well as from the populations from Malaysia, Thailand, Singapore and Vietnam. Establishment of baseline data on the genetic diversity of this commercially important agarophyte is relevant in the context of cultivation, as limited genetic diversity may jeopardize the potential for its genetic improvement over time.

Through the years, several strains and color morphotypes of Kappaphycus and Eucheuma have emerged in cultivation areas as a result of environmental and anthropogenic factors. This led to confusion in nomenclature of these seaweeds. However, the advent of molecular technology has brought notable changes to seaweed research over the past two decades. New molecular techniques have proved useful, particularly in the molecular identification and cataloging of economically important carrageenophytes, i.e. Kappaphycus and Eucheuma which were commercially introduced into many countries worldwide. The following chapter discusses the application and findings of molecular studies within the context of these red seaweeds, along with potential areas for future research.

The tephritid fruit fly Zeugodacus tau (Walker) is a polyphagous fruit pest of economic importance in Asia. Studies based on genetic markers indicate that it forms a species complex. We report here (1) the complete mitogenome of Z. tau from Malaysia and comparison with that of China as well as the mitogenome of other congeners, and (2) the relationship of Z. tau taxa from different geographical regions based on sequences of cytochrome c oxidase subunit I gene. The complete mitogenome of Z. tau had a total length of 15631 bp for the Malaysian specimen (ZT3) and 15835 bp for the China specimen (ZT1), with similar gene order comprising 37 genes (13 protein-coding genes—PCGs, 2 rRNA genes, and 22 tRNA genes) and a non-coding A + T-rich control region (D-loop). Based on 13 PCGs and 15 mt-genes, Z. tau NC_027290 (China) and Z. tau ZT1 (China) formed a sister group in the lineage containing also Z. tau ZT3 (Malaysia). Phylogenetic analysis based on partial sequences of cox1 gene indicates that the taxa from China, Japan, Laos, Malaysia, Bangladesh, India, Sri Lanka, and Z. tau sp. A from Thailand belong to Z. tau sensu stricto. A complete cox1 gene (or 13 PCGs or 15 mt-genes) instead of partial sequence is more appropriate for determining phylogenetic relationship.

Unique ORFs of Gracilaria firma homologous to red algal plasmids. The G. firma ORFs showed significant similarity (e-valueâ

The emergence of type 2 diabetes mellitus (T2DM) as the pre-eminent global non-infectious disease has driven the search for new anti-diabetic strategies including utilising traditional food and herbs. In this investigation, we describe the anti-diabetic potential of six selected Malaysian seaweed species against recognised pharmaco- logical targets. Specifically, we measured their ability to inhibit α-glucosidase and dipeptidyl-peptidase-4 (DPP-4) and also their ability to stimulate incretin hormone secretion in vitro.Crudewater extractsofHalimedamacroloba,Padina sulcata, Sargassum binderi and Turbinaria conoides pos- sessed potent inhibitory activities against α-glucosidase and DPP-4. The highest inhibitory activity against α-glucosidase was found in water extracts of the green seaweed species H. macroloba with an IC50 value of 6.388 mg mL �1 . Crude water extracts of the brown seaweeds studied namely P. sulcata, S. binderi and T. conoides, exhibited potent DPP-4 inhibition compared with the green seaweed H. macroloba. The brown seaweed also stimulates secretion of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) from pGIP neo STC-1 cells in vitro. H. macroloba stimulated GLP-1 secretion but not secretion of GIP.

A multilocus sequence analysis using mitochondria-encoded cytochrome c oxidase subunit I (COI), cytochrome B (CytB), NADH dehydrogenase subunit 5 (ND5); nuclear encoded 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) genes was performed to determine the levels of genetic variation between the closely related species Haematobia irritans Linnaeus and Haematobia exigua de Meijere. Among these five genes, ND5 and CytB genes were found to be more variable and informative in resolving the interspecific relationships of both species. In contrast, the COI gene was more valuable in inferring the intraspecific relationships. The ribosomal 18S and 28S sequences of H. irritans and H. exigua were highly conserved with limited intra- and inter-specific variation. Molecular evidence presented in this study demonstrated that both flies are genetically distinct and could be differentiated based on sequence analysis of mitochondrial genes.

Both Malaysia and Indonesia have high biodiversity for a variety of organisms, including freshwater red algae. The type localities of over a dozen freshwater red algal taxa are located in Malaysia and Indonesia. These species were described prior to the advent of molecular systematics, and no molecular data were available for specimens from these two countries. Therefore, the goal of this study was to visit type and other locales in Malaysia and Indonesia to recollect freshwater red algal taxa for both morphological and molecular studies. A total of 11 previously published species were identified, with eight taxa belonging to the Batrachospermales, and one each in the Compsopogonales (Compsopogon caeruleus), Ceramiales (Caloglossa beccarii) and Thoreales (Nemalionopsis shawii). The rbcL gene provided numerous insights including two new species, Batrachospermum phangii sp. nov. from Malaysia and Kumanoa celebes sp. nov. from Indonesia. The placement of Batrachospermum cylindrocellulare in section Aristata rather than section Batrachospermum was clarified. Specimens from Malaysia identified as Sirodotia delicatula were distantly related to specimens from South America, suggesting a cryptic species in South America. Likewise, Balliopsis prieurii from Malaysia was distantly related to Balliopsis prieurii from South America. A gametophyte specimen and numerous chantransia stage specimens were conspecific with Batrachospermum macrosporum from South America, and this is a new record of this taxon in Malaysia. Chantransia stage sporophyte specimens from Indonesia had a similar sequence to Sheathia arcuata from Hawaii. The sequence placement of N. shawii from Indonesia points to the need for further systematic research on this genus. Although Kumanoa gibberosa was not recollected at the type location in Malaysia, it was found in Indonesia; likewise, the type locality in Indonesia did not yield S. delicatula but this species was present in Malaysia. Given the previously described diversity, the two new taxa proposed in this study and the insights gained from the present molecular data, we suggest that future focus on freshwater red algae from Southeast Asia will yield considerable knowledge of the flora of the region and freshwater red algal diversity in general.

The supply of agar as an important gelling and thickening agent in various industrial applications depends heavily on harvesting of natural seaweed resources and seaweed farming. To facilitate the selection of good seaweed source with higher agar yield and stronger gel strength, accurate and rapid screening method using molecular markers is necessary to replace the tedious, laborious and time-consuming conventional method which involves agar extraction and gel analysis. In this study, we characterized the expression of a number of algal transcripts and proteins from an agar producing seaweed, Gracilaria changii with the aim to identify potential markers for agar yield and gel strength. In total, 15 candidate transcripts that are directly or indirectly related to putative agar biosynthetic pathway were identified based on literature search. The transcript abundance of 4 and 11 of these candidates were found to be significantly (P

The genera Kappaphycus Doty and Eucheuma J. Agardh are amongst the most economicallyimportant seaweeds in Indonesia. Several species of Kappaphycus and Eucheuma are cultivated in many farms in Indonesia including Nusa Tenggara Barat (NTB). However the cultivated Kappaphycus is infl uenced by environmental changes. Variation in seaweed germplasm is necessary in order to produce elite lines with required quantity. More than 10 wild specimens of Eucheum/Kappaphycus species are obtained in the NTB marine waters, including E. spinosum (green and brown), E. striatum (green and brown), Eucheuma/Kappaphycus sp-1 and Eucheuma/Kappaphycus sp-2. Distribution of the species in NTB has been mapped, showing that Lombok Island has more diverse species of Eucheuma compared to Sumbawa Island. In addition, morphological and physiological characterisation of the species have been undertaken, and it is observed that there are morphological differences in the same species obtained from different coastal zones, and thus indicating morphological plasticity of the species to different environmental conditions. (Keywords: West Nusa Tenggara, E. spinosum, E. striatum, E. edulis, E. serra, natural diversity, seaweeds)

Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture-independent microbial community profiling of a commercial sugarcane variety (Q208(A) ) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208(A) ) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N2 and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture-independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth-promoting capacity, is a necessary step towards designing effective microbial inoculants.

The commercial importance of carrageenophytes Kappaphycus and Eucheuma is well known, with much interest in terms of cultivation, marketing, and research. Considering the many lucrative prospects, these red seaweeds were introduced into various parts of the world for farming, where merely a few were comprehensively documented. Despite being extensively cultivated throughout Southeast Asia, the genetic diversity of Kappaphycus and Eucheuma is poorly studied, where heavy reliance is placed on the use of local or commercial names for identifications. This study used the mitochondrial-encoded cox1 and cox2–3 spacer genetic markers to investigate the Kappaphycus and Eucheuma haplotypes, cultivated and wild, available throughout Southeast Asia. Concatenated cox1–cox2–3 spacer datasets were also analyzed. The near full-length cox1 gene is preferred at revealing the genetic diversity of Kappaphycus and Eucheuma, provided a larger reference database is available. Both molecular markers were capable of delineating common members of the genus Kappaphycus (i.e., Kappaphycus alvarezii, Kappaphycus striatus, and Kappaphycus cottonii) and Eucheuma denticulatum, and revealed interesting genotypes and new species which may be potential alternatives to the common cultivars as well as materials for research. The relative scarcity of Eucheuma species is discussed and future sites for sampling are recommended.

Five species of Pterocladiella (Gelidiales) are reported from Malaysia, identified using a combination of morphological data and partial rbcL (chloroplast) and coxI (mitochondrial) gene sequences. Two new species, Pterocladiella phangiae sp. nov. and Pterocladiella megasporangia sp. nov., are proposed based on phylogenetic analyses and distinct morphological features. Pterocladiella phangiae sp. nov. showed a sister relationship with P. caerulescens, P. beachiae, P. psammophila and P. australafricanensis. Pterocladiella megasporangia sp. nov. formed a monophyletic clade with P. bartlettii and P. melanoidea with strong support in all analyses. Two other species are reported as new records for Malaysia and Southeast Asia. Pterocladiella beachiae grouped robustly with P. beachiae from the Caribbean Sea; P. bartlettii was resolved in a clade among sequences of P. bartlettii from the Caribbean Sea and Texas, USA. The identity of P. caerulescens, which had previously been reported from Malaysia based on morphol...

A phylogenetic study of two parasites found on the common host species Gracilaria salicornia from Japan and Malaysia based on nuclear, plastid, and mitochondrial molecular markers was conducted. The Japanese and Malaysian parasites were placed in the same cluster in the nuclear phylogenies inferred from the LSU rRNA gene and ITS region, supporting the monophyly of these parasites despite the slight anatomical variations observed. In addition to the molecular analyses, shared morphological and reproductive features including the lack of rhizoids, similar coloration to hosts, and the presence of deep spermatangial conceptacles supported the inclusion of both the Japanese and Malaysian parasites in Congracilaria babae. Phylogenetic analyses based on ITS sequences indicated that C. babae most likely evolved directly from its current host species, G. salicornia. C. babae was shown to be a member of Gracilaria sensu stricto along with its host. C. babae growing on G. salicornia is characterized by having plastid rbcL and mitochondrial cox1 gene sequences identical to those of its host, whereas the nuclear LSU rRNA gene and ITS region sequences are different from those of its host.

The need to develop biomass-based domestic production of high-energy liquid fuels (biodiesel) for transportation can potentially be addressed by exploring microalgae with high lipid content. Selecting the strains with adequate oil yield and quality is of fundamental importance for a cost-efficient biofuel feedstock production based on microalgae. This work evaluated 29 strains of Chlorella isolated from Malaysia as feedstock for biodiesel based on volumetric lipid productivity and fatty acid profiles. Phylogenetic studies based on 18S rRNA gene revealed that majority of the strains belong to true Chlorella followed by Parachlorella. The strains were similarly separated into two groups based on fatty acid composition. Of the 18 true Chlorella strains, Chlorella UMACC187 had the highest palmitic acid (C16:0) content (71.3 ± 4.2 % total fatty acids, TFA) followed by UMACC84 (70.1 ± 0.7 %TFA), UMACC283 (63.8 ± 0.7 %TFA), and UMACC001 (60.3 ± 4.0 %TFA). Lipid productivity of the strains at exponential phase ranged from 34.53 to 230.38 mg L−1 day−1, with Chlorella UMACC050 attaining the highest lipid productivity. This study demonstrated that Chlorella UMACC050 is a promising candidate for biodiesel feedstock production.

A new species, Kappaphycus malesianus, is established as a new member of the genus Kappaphycus. Locally known as the “Aring-aring” variety by farmers in Malaysia and the Philippines, this variety has been commercially cultivated, often together with Kappaphycus alvarezii due to the similarities in morphology. Despite also producing kappa-carrageenan, the lower biomass of the K. malesianus when mixed with K. alvarezii ultimately affects the carrageenan yield. Morphological observations, on both wild and cultivated plants, coupled with molecular data have shown K. malesianus to be genetically distinct from its Kappaphycus congeners. The present study describes the morphology and anatomy of this new species as supported by DNA data, with additional morphological features for distinguishing between commercial Kappaphycus cultivars.

Studies on the crustose brown algae are relatively few despite a long history of studies conducted since the 1800s, with temperate species forming the bulk of these studies. There is a need for more focus on crustose brown algae particularly in the tropics as they are generally different from those in the temperate regions. Taxonomic confusion arising from morphological simplicity largely dependent on the reproductive structures and overlap in morpho-anatomical features among species necessitates the use of molecular techniques. This study is dedicated to a better understanding of the diversity of these understudied algae in the Indo–Malay region. Specimens collected from Peninsular Malaysia, Sabah (Borneo) and Lombok Island in Indonesia were identified using molecular markers from the plastid rubisco large subunit (rbcL) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in tandem with morphology and anatomy. Three Mesospora spp., two putative Diplura spp. and the cosmopolitan Neoralfsia expansa were identified in this study, including a new record of Mesospora negrosensis for Malaysia. Despite their morpho-anatomical similarities, Mesospora and Diplura occur in widely divergent clades within the brown algae, the former in the Mesosporaceae in the Ralfsiales, the latter in an unclassified clade sister to the Ishigeales. All six species occurred both in Malaysia and Lombok Island except for M. elongata and M. negrosensis, respectively. The rbcL marker performed better in the elucidation of phylogeny among the brown algal orders, whereas cox1-5′ is more suited as a barcoding marker for species level identification.

The present study explored the intraspecific genetic diversity, dispersal patterns and phylogeographic relationships of Culex quinquefasciatus Say (Diptera: Culicidae) in Malaysia using reference data available in GenBank in order to reveal this species' phylogenetic relationships. A statistical parsimony network of 70 taxa aligned as 624 characters of the cytochrome c oxidase subunit I (COI) gene and 685 characters of the cytochrome c oxidase subunit II (COII) gene revealed three haplotypes (A1-A3) and four haplotypes (B1-B4), respectively. The concatenated sequences of both COI and COII genes with a total of 1309 characters revealed seven haplotypes (AB1-AB7). Analysis using tcs indicated that haplotype AB1 was the common ancestor and the most widespread haplotype in Malaysia. The genetic distance based on concatenated sequences of both COI and COII genes ranged from 0.00076 to 0.00229. Sequence alignment of Cx. quinquefasciatus from Malaysia and other countries revealed four haplotypes (AA1-AA4) by the COI gene and nine haplotypes (BB1-BB9) by the COII gene. Phylogenetic analyses demonstrated that Malaysian Cx. quinquefasciatus share the same genetic lineage as East African and Asian Cx. quinquefasciatus. This study has inferred the genetic lineages, dispersal patterns and hypothetical ancestral genotypes of Cx. quinquefasciatus.

Background: Given that there is limited available information on the insensitive acetylcholinesterase in insect species in Malaysia, the present study aims to detect the presence of G119S mutation in the acetylcholinesterase gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. Results: The ace-1 sequence and PCR-RFLP test revealed the presence of glycine-serine ace-1 mutation in the wild populations of Cx. quinquefasciatus. Both direct sequencing and PCR-RFLP methods demonstrated similar results and revealed the presence of a heterozygous genotype at a very low frequency (18 out of 140 individuals), while a homozygous resistant genotype was not detected across any study site in Malaysia. In addition, statistical analysis also revealed that malathion resistance is associated with the frequency of ace-1R in Cx. quinquefasciatus populations. Conclusion: This study has demonstrated the first field-evolved instance of G119S mutation in Malaysian populations. Molecular identification of insensitive acetylcholinesterase provides significant insights into the evolution and adaptation of the Malaysian Cx. quinquefasciatus populations. © 2013 Society of Chemical Industry.

Poong S.-W., Lim P.-E., Phang S.-M., Gerung G.S., and Kawai H. 2013. Mesospora elongata sp. nov. (Ralfsiales, Phaeophyceae), a new crustose brown algal species from the Indo-Pacific region. Phycologia 52: 74–81. DOI: 10.2216/12-42.1 Crustose brown algae have relatively simple morphology and anatomy, and the limited characters were a major obstacle in delineating species of Mesospora (Ralfsiales, Phaeophyceae). The type species, Mesospora schmidtii, was described using material collected in Indonesia. A subsequent study, using material from Japan, was identified as M. schmidtii, but we questioned the identity. Therefore, we analyzed morphological characters as well as the plastid-encoded RuBisCO large subunit (rbcL) and mitochondrial-encoded cytochrome c oxidase subunit 1 (cox1) genes using new material obtained from the type locality (Indonesia) and new material obtained from Japan. The molecular phylogenetic analysis distinguished two clades, one representing material from the type location and the other...

For de novo transcriptome analysis, choosing a closest reference model specie in terms of evolutionary distance is a general approach for gene mapping and genome annotations. However, not every selected reference model species possesses comprehensive genome annotations and curated information, and the total number of mapped genes from the selected reference species could not be fully expected either. Due to inefficient mapped genes from the selected reference model species, the following functional pathway analysis on transcriptome datasets would be seriously affected. To solve this problem, we proposed an improved approach based on multiple reference model species selection, especially for KEGG pathway analysis on differentially expressed genes. Applying union operations on individually mapped genes from different selected species, we could significantly promote the integrity of gene mapping results in KEGG pathways and provide realistic P-values for each identified pathway. Furthermore, based on mapped genes and KGML datasets, we applied various gray-levels, colors and shapes to present gene expression conditions on each biological pathway. Taking NGS transcriptomic datasets from an unknown Antarctic green alga species as an experimental example and selecting three published known species including Chlamydomonas reinhardtii, Chlorella variabilis, and Coccomyxa subellipsoidea as candidate reference species, we compared the results of pathway enrichment analysis by adopting different selections of reference species. We found that integrating all mapped genes from various model species provided a better result compared to using any single reference species. Some missed important biological pathways could be retrieved under an identical threshold setting of P-value, such as Ribosome, Pyrimidine metabolism and ABC transporters pathways. Therefore, we believe appropriate selection of multiple reference species is necessary and significant for transcriptome analysis on de novo species.

Zeugodacus caudatus is a pest of pumpkin flowers. It has a Palearctic and Oriental distribution. We report here the complete mitochondrial genome of the Malaysian and Indonesian samples of Z. caudatus determined by next-generation sequencing of genomic DNA and determine their taxonomic status as sibling species and phylogeny with other taxa of the genus Zeugodacus. The whole mitogenome of both samples possessed 37 genes (13 protein-coding genes—PCGs, 2 rRNA and 22 tRNA genes) and a control region. The mitogenome of the Indonesian sample (15,885 bp) was longer than that of the Malaysian sample (15,866 bp). In both samples, TΨC-loop was absent in trnF and DHU-loop was absent in trnS1. Molecular phylogeny based on 13 PCGs was concordant with 15 mitochondrial genes (13 PCGs and 2 rRNA genes), with the two samples of Z. caudatus forming a sister group and the genus Zeugodacus was monophyletic. The Malaysian and Indonesian samples of Z. caudatus have a genetic distance of p = 7.8 % based on 13 PCGs and p = 7.0 % based on 15 mitochondrial genes, indicating status of sibling species. They are proposed to be accorded specific status as members of a species complex.

Bactrocera latifrons is a serious pest of solanaceous fruits and Bactrocera umbrosa is a pest of Artocarpus fruits, while Bactrocera melastomatos infests the fruit of Melastomataceae. They are members of the subgenus Bactrocera. We report here the complete mitochondrial genome of these fruit flies determined by next-generation sequencing and their phylogeny with other taxa of the subgenus Bactrocera. The whole mitogenomes of these three species possessed 37 genes namely, 13 protein-coding genes (PCGs), 2 rRNA and 22 tRNA genes. The mitogenome of B. latifrons (15,977 bp) was longer than those of B. melastomatos (15,954 bp) and B. umbrosa (15,898 bp). This difference can be attributed to the size of the intergenic spacers (283 bp in B. latifrons, 261 bp in B. melastomatos, and 211 bp in B. umbrosa). Most of the PCGs in the three species have an identical start codon, except for atp8 (adenosine triphosphate synthase protein 8), which had an ATG instead of GTG in B. umbrosa, whilst the nad3 (NADH dehydrogenase subunit 3) and nad6 (NADH dehydrogenase subunit 6) genes were characterized by an ATC instead of ATT in B. melastomatos. The three species had identical stop codon for the respective PCGs. In B. latifrons and B. melastomatos, the TΨC (thymidine-pseudouridine-cytidine)-loop was absent in trnF (phenylalanine) and DHU (dihydrouracil)-loop was absent in trnS1 (serine S1). In B. umbrosa, trnN (asparagine), trnC (cysteine) and trnF lacked the TψC-loop, while trnS1 lacked the DHU-stem. Molecular phylogeny based on 13 PCGs was in general concordant with 15 mitochondrial genes (13 PCGs and 2 rRNA genes), with B. latifrons and B. umbrosa forming a sister group basal to the other species of the subgenus Bactrocera which was monophyletic. The whole mitogenomes will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general.

Phylogenetics is the study of evolutionary relationships among groups of organisms, and systematics the study of the diversity of organisms and the relationships among them. The relationships are presented in evolutionary trees. In addition to morphological data, gene-enzyme systems were used extensively in earlier days for phylogenetic and systematic studies. Currently, molecular sequencing data are commonly used for phylogenetics and systematics. Cytogenetic data in some cases also serve as good parameters for differentiating sibling/cryptic species. In general, sound systematics and phylogenetics are important and relevant to various fields of biology. This review discusses selected taxonomic groups in which identification has proven problematic based on morhphological characters. The role of phylogenetics and systematics is illustrated by nematode parasites of the genus Angiostrongylus, stingless bees of the genus Tetragonilla, tephritid fruit flies of Bactrocera caudata complex, crab-eating frogs of Fejervarya cancrivora complex, and murid rats of the genera Hapalomys and Maxomys.

Microalgae are promising producers of many valuable compounds serving the food, feed, healthcare, and pharmaceutical industries. Microalgae grow rapidly and generally tolerate a wide range of environments. They can serve as cell factories for economical and sustainable production of diverse products. Microalgae can be transformed through genetic and metabolic engineering methods to overproduce the desired chemicals. Advancements in the “omics” technologies are generating information to allow design and creation of super algal strains for producing biofuels and other products.

Gracilaria edulis and Gracilaria tenuistipitata var liui are agarophytes with high commercial value which are currently cultivated in countries like India and Thailand. They have great potential for mariculture in Malaysia. Experiments were carried out to study carpospore germination and determine the effects of irradiance and salinity on the growth of these two species. Both species showed the Dumontia type of carpospore development. Both species showed increased daily growth rate (% day−1) with increasing irradiance and tolerance for a wide range of salinity with a preference for low salinity. G. edulis grew best at 100 μmol photons m−2 s−1 and 15 psu while G. tenuistipitata var liui grew best at 60–130 μmol photons m−2 s−1 and 15 psu. The highest growth rate obtained for G. edulis and G. tenuistipitata var liui was 13.57 and 19.7 % day−1 respectively. tenuistipitata var liui. ANOVA showed that both irradiance and salinity have significant effect on the growth of both species (P

Gracilaria is a red seaweed that has been cultivated worldwide and is commercially used for food, fertilizers, animal fodder, and phycocolloids. However, the high morphological plasticity of seaweeds often leads to the misidentification in the traditional identification of Gracilaria species. Molecular markers are important especially in the correct identification of Gracilaria species with high economic value. Microsatellite markers were developed from the expressed sequence tags of seaweeds deposited at the National Center for Biotechnology Information database and used for differentiating Gracilaria changii collected at various localities and two other Gracilaria species. Out of 33 primer pairs, only one primer pair gave significant results that can distinguish between three different Gracilaria species as well as G. changii from various localities based on the variation in repeated nucleotides. The unweighted pair group method using arithmetic mean dendrogram analysis grouped Gracilaria species into five main clades: (a) G. changii from Batu Besar (Malacca), Sandakan (Sabah), Bintulu (Sarawak), Batu Tengah (Malacca), Gua Tanah (Malacca), Middle Banks (Penang), Sungai (Sg.) Merbok (Kedah), Teluk Pelandok (Negeri Sembilan), Pantai Dickson (Negeri Sembilan), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore); (b) Gracilaria manilaensis from Cebu, Philippines; (c) G. changii from Morib (Selangor); (d) Gracilaria fisheri from Pattani, Thailand; and (e) G. changii from Pantai Dickson (Negeri Sembilan), Gua Tanah (Malacca), Sg. Merbok (Kedah), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore). This result shows that this primer pair was able to distinguish between three different species, which are G. changii from Morib (Malaysia), G. fisheri from Pattani (Thailand), and G. manilaensis from Cebu (Philippines), and also between different genotypes of G. changii. This suggested that the simple sequence repeat primer we developed was suitable for differentiating between different Gracilaria species due to the polymorphisms caused by the variability in the number of tandem repeats.

Advances in DNA-based genetic markers provide the essential tools in measurement of genetic diversity relating to the evolution, biogeography, and systematics of red algae by exploiting genetic variation in the entire genome of organisms. The understanding of genetic diversity in Gracilaria changii (Gracilariaceae, Rhodophyta) will provide valuable information for conservation, plant breeding management, and strain selection for cultivation. However, information of intraspecific genetic variation is still rudimentary. In this study, two mitochondrial encoded markers, cytochrome oxidase subunit 1 (cox1) and intergenic spacer between the cytochrome oxidase subunits 2 and 3 (cox2-3 spacer) were used to investigate genetic diversity in 40 individuals of G. changii collected from 11 different geographically distinct populations from Peninsular Malaysia. Seven distinct mitochondrial haplotypes were identified with the cox1 gene and three mitochondrial haplotypes with the cox2-3 spacer. Intraspecific nucleotide differences ranged from 0 to 6 bp for the cox1 and 0–4 bp for the cox2-3 spacer, respectively. This is the first report comparing the suitability of mitochondrial markers of the cox1 gene and the cox2-3 spacer for genetic diversity studies on G. changii. The present study showed that the cox1 gene is a potential molecular marker to infer intraspecific genetic variation in red macroalgae. The cox1 marker is more variable compared to the cox2-3 spacer and revealed genetic variation and phylogeographic structure for this ecologically and economically important species.

Sun Z., Hanyuda T., Lim P.E., Tanaka J., Gurgel C.F.D. and Kawai H. 2012. Taxonomic revision of the genus Lobophora (Dictyotales, Phaeophyceae) based on morphological evidence and analyses rbcL and cox3 gene sequences. Phycologia 51: 500–512. DOI: 10.2216/11-85.1 A taxonomic revision of Lobophora based on molecular phylogenetic analyses of rbcL and cox3 gene sequences as well as anatomical observations was carried out, mostly based on specimens collected from eastern Asia and southeastern Australia. In the molecular phylogenetic analyses, nine major clades supported by high bootstrap values were recognized. In combination with evaluation of morphological characters, four clades were concluded to be new species. The Australian species L. australis sp. nov. possessed erect thalli with sporangial sori scattered on the ventral surface and had a closer phylogenetic relationship with another Australian species, L. nigrescens, but it was distinguished from the latter in having fewer layers of cortical cells and ...

1 ABSTRACT. A standardized larval dipping method was used to determine the infestation rates of Culex and other species of mosquitoes in stagnant water at 20 residential areas. This study also examined the associations between Culex distribution and various habitat characteristics across all states in Malaysia. Identification of 7,848 specimens yielded 6 species dominated by Culex quinquefasciatus (82.74%), followed by Cx. vishui (14.39%), Cx. gelidus (2.70%), Lutzia fuscanus (0.11%), Armigeres subalbatus (0.05%), and Anopheles separatus (0.01%). The Culex larvae occurred in stagnant water with pH ranging from 6.4 to 8.2; conductivity, 139.7 to 6635.2 ms/cm; salinity, 0.07 to 3.64 ppt; total dissolved solids, 0.09 to 4.27g/liter; and dissolved oxygen, 5.11 to 8.11 mg/liter. The mean number of Culex larvae was positively correlated with pH, conductivity, salinity, and total dissolved solids. In contrast, the elevation and dissolved oxygen were found negatively correlated with mean number of Culex larvae. This study documented baseline information on the habitat characteristics of Culex species for the 1st time at different residential areas in Malaysia. The findings of this study will be a timely reminder to local authorities that effective control measures should be monitored regularly in order to reduce the nuisance of these mosquitoes and the risks of disease transmission.

NI-NI-WIN, Hanyuda T., Draisma S.G.A., Verheij E., Prud'homme van Reine W.F., Lim P.-E., Phang S.-M., and Kawai H. 2012. Morphological and molecular evidence for two new species of Padina (Dictyotales, Phaeophyceae), P. sulcata and P. calcarea, from the central Indo-Pacific. Phycologia 51: 576–585. DOI: 10.2216/11-94.1 Two new species of Padina – 1. Padina sulcata sp. nov. and 2. P. calcarea sp. nov. – from Malaysia, Indonesia and Palau were described based on morphological and molecular phylogenetic observations. Padina sulcata was a three-layered species characterised by a covering of thick fibrous hairs from the base to the middle of the inferior surface (away from the in-rolled margin) of the thallus; conspicuous equally spaced hairlines that alternated between both frond surfaces; and broad, indusiate oogonial and tetrasporangial sori that occupied nearly the entire fertile zones, and the fertile zones were separated by sterile zones of equal width. Padina calcarea was a two-layered species character...

The genera Kappaphycus Doty and Eucheuma J. Agardh are important sources of carrageenan in Malaysia, offering lucrative revenues to the carrageenan industry, economy, and the local community. The extensive range of morphotypes and the lack of distinct morphological characteristics led to the application of molecular systematics in elucidating this taxonomic confusion. Local varieties of Kappaphycus and Eucheuma, identified using putative external morphology, were analyzed using the mitochondrial cox2–3 spacer and plastid RuBisCO spacer molecular markers. Phylogenetic analysis of these and non-local specimens indicate that Kappaphycus and Eucheuma are genetically distinct. Three main genotypes of Kappaphycus alvarezii were identified, of which two are extant in Hawaii. Morphological and color variations are not supported by molecular data, indicating that most of the local names are not genetically based. Both the cox2–3 spacer and RuBisCO spacer generated phylogenetic trees with similar topology except in variation of nodal supports. The two markers showed clear separation between Kappaphycus and Eucheuma and the existence of three Malaysian Kappaphycus cultivars. Cox2–3 spacer data is more variable and provides better resolution than the RuBisCO spacer, showing that Kappaphycus is more diversified with a larger number of genotypes, strains, and species which are unique to Southeast Asia. Kappaphycus sp. “Aring-aring” appeared to be phenotypically and genotypically different from other Kappaphycus congeners, whereas Kappaphycus striatum exhibited two different genotypes. Our data indicate that Eucheuma denticulatum is the dominant species in Malaysian waters and also suggested paraphyly in Eucheuma which will require further studies. The application of molecular taxonomy on Malaysian Kappaphycus and Eucheuma proves useful, offering valuable insights into the taxonomy and distribution of these commercially important Rhodophytes.

Of four species of Sargassaceae, representing the genera Carpophyllum, Cystoseira, Landsburgia, and Sargassum, the intergenic spacer of the ribosomal cistron was amplified using a forward primer annealing at the 3′-end of the large subunit (LSU) of the ribosomal cistron and a reverse primer annealing at the 5S rRNA gene. The PCR products were cloned and the DNA sequences of multiple clones were determined. Almost each clone showed a unique DNA sequence. Intra-individual variation of this LSU-5S intergenic spacer was extremely high and was characterized by great length variation and a high number of short tandem repeats. Sequences were unalignable and therefore it was concluded that the LSU-5S intergenic spacer is unsuitable for phylogenetic and phylogeographic studies of sargassacean taxa.

Summary Length-weight relationships of three sympatric species of stingrays from a coastal mudflat, Malaysia were estimated. A total of 290 individuals (150 Himantura walga, 78 Dasyatis bennetti, and 57 Dasyatis zugei) were sampled using barrier net, gill net and beam trawl. The length-weight relationship based on disc length and width generally showed positive allometric growth (b > 3) for all species. This study reports the first findings regarding the length-weight relationships of these stingray species in Malaysian waters.

The seaweed industry has been identified for priority development in Malaysia. The seaweeds commercially farmed for carrageenan belong to the genera Kappaphycus Doty and Eucheuma J. Agardh. At least six varieties of Kappaphycus and Eucheuma are found in the farms around Sabah, east Malaysia. These seaweeds produce carrageenan with gel strength ranging from 250 to 550 g.cm-2. This paper provides a simple description of gross morphological features and illustrations that may be used in the field for differentiating the main seaweed varieties cultivated in Sabah.

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades – Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5–400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.

Mitochondrial cytochrome c oxidase subunit I (cox1) was employed to investigate the intraspecific genetic diversity of Gracilaria changii collected from various localities distributed along the west coast of Peninsular Malaysia. Gracilaria changii is an agarophyte with potential for commercialization in Malaysia as it has high yields of good quality agar with high gel strength for the production of food grade agar and agarose. The phylogeographic aspect of G. changii has not been studied despite its abundance and potential commercialization. In this study, six mitochondrial haplotypes (C1–C6) were revealed from 62 specimens varying by 0–3 bp over 923 bp. Results indicate that haplotype C1 is the common ancestor and the most widespread haplotype due to its prevalence in Morib, Gua Tanah, Middle Banks, Batu Besar, Batu Tengah, Sungai Pulai, and Kuala Sungai Merbok. In this study, Morib was suggested as contributing the highest intra-population diversity with the identification of three haplotypes. The mitochondrial marker cox1 is a highly divergent mitochondrial marker and is applicable for studies on species identification and assessment of genetic diversity of G. changii.

In 2008, a scientific expedition to Bachok (the new Marine Station), Kelantan and the vicinity of Terengganu, was organised by the Institute of Ocean and Earth Sciences (IOES), University of Malaya. The objective of the expedition was to collect and document the natural resources of Bachok and its vicinity. Our team collected and identified the marine algae resources of the area. A total of 133 taxa of marine algae and four taxa of seagrasses were compiled from the results as well as previous records for Kelantan and Terengganu. The marine algae comprise 3 families of Cyanophyta with 4 genera and 4 taxa; 10 families of Chlorophyta with 13 genera and 35 taxa; 16 families of Rhodophyta with 38 genera and 63 taxa; 4 families of Phaeophyta with 8 genera and 31 taxa. Only 35 taxa were collected during this present survey, the most from the Rhodophyta (14 taxa) followed by the Phaeophyta (12 taxa), the Chlorophyta (8 taxa) and the Cyanophyta (4 taxa). Of these, the Cyanophyta Lyngbya, Oscillatoria, Microcoleus and Brachytrichia; the Chlorophyta Ulva compressa, Ulva flexuosa, Ulva intestinalis, Caulerpa serrulata var. boryana; the Rhodophyta Gelidiella acerosa, Gelidiu pusillum, Actinotrichia fragilis, Acanthophora spicifera; and the Phaeophyta Dictyota ciliolata, Lobophora variegata, Padina antillarum, Hormophysa cuneiformis and Turbinaria conoides are new records for Kelantan and Terengganu. The Rhodophyta,Pterocladiella is a new record for Malaysia. Two families of seagrasses with 3 genera and 4 taxa were collected and included in this checklist. A new species of Halophila will be studied further.

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males.

P.-E. Lim, M. Sakaguchi, T. Hanyuda, K. Kogame, S.-M. Phang and H. Kawai. 2007. Molecular phylogeny of crustose brown algae (Ralfsiales, Phaeophyceae) inferred from rbcL sequences resulting in the ...

The following nine elongate unpatterned muraenid species of the subfamily Muraeninae, including one new species, are recognized from Taiwan and adjacent waters: Gymnothorax albimarginatus (TemminckSchlegel), G. dorsalis Seale, G. melanosomatus Loh, ShaoChen, G. phasmatodes (Smith), G. prolatus SasakiAmaoka, G. sagmacephalus Böhlke, Pseudechidna brummeri (Bleeker), Strophidon sathete (Hamilton) and G. pseudomelanosomatus new species, described from two specimens. This new moray eel is distinguished from its similar species, G. melanosomatus, by the following features: grey brown body (vs. black), snout length 20.5% (vs. 17.8%) of head length, smaller eye diameter 8.2% (vs. 10.0%) of head length; preanal length 49.5% (vs. 58.5%) total length, and preanal vertebrae 89-89 (vs. 105-109). Phylogenetic relationships of the nine species were examined using nucleotide sequence data from partial sequences of mitochondrial ND5 gene (600 bp), and seven species form COI (600 bp). The genetic analyses suggest that G. pseudomelanosomatus is distinct from G. melanosomatus and the other six species of Gymnothorax. Morphological features and mitogenetic affinities strongly suggest that "G." dorsalis should be placed in Strophidon rather than in Gymnothorax. The results also suggest that employment of ND5 and COI gene sequences are rather useful for identification of species and for obtaining reasonable insights into the phylogeny of the muraenid species.

Angiostrongylus malaysiensis is a nematode parasite of various rat species. When first documented in Malaysia, it was referred to as A. cantonensis. Unlike A. cantonensis, the complete mitochondrial genome of A. malaysiensis has not been documented. We report here its complete mitogenome, its differentiation from A. cantonensis, and the phylogenetic relationships with its congeners and other Metastrongyloid taxa. The whole mitogenome of A. malaysiensis had a total length of 13,516 bp, comprising 36 genes (12 PCGs, 2 rRNA and 22 tRNA genes) and a control region. It is longer than that of A. cantonensis (13,509 bp). Its control region had a long poly T-stretch of 12 bp which was not present in A. cantonensis. A. malaysiensis and A. cantonensis had identical start codon for the 12 PCGs, but four PCGs (atp6, cob, nad2, nad6) had different stop codon. The cloverleaf structure for the 22 tRNAs was similar in A. malaysiensis and A. cantonensis except the TΨC-arm was absent in trnV for A. malaysiensis but present in A. cantonensis. The Angiostrongylus genus was monophyletic, with A. malaysiensis and A. cantonensis forming a distinct lineage from that of A. costaricensis and A. vasorum. The genetic distance between A. malaysiensis and A. cantonensis was p = 11.9% based on 12 PCGs, p = 9.5% based on 2 rRNA genes, and p = 11.6% based on 14 mt-genes. The mitogenome will prove useful for studies on phylogenetics and systematics of Angiostrongylus lungworms and other Metastrongyloid nematodes.