Perspicuous assessments of taxonomic boundaries and discovery of cryptic taxa are of paramount importance in interpreting ecological and evolutionary phenomena among black flies (Simuliidae) and combating associated vector-borne diseases. Simulium tani Takaoka & Davies is the largest and perhaps the most taxonomically challenging species complex of black flies in the Oriental Region. We use a DNA sequence-based method to delineate currently recognized chromosomal and morphological taxa in the S. tani complex on the Southeast Asian mainland and Taiwan, while elucidating their phylogenetic relationships. A molecular approach using multiple genes, coupled with morphological and chromosomal data, supported recognition of cytoform K and morphoform 'b' as valid species; indicated that S. xuandei, cytoform L, and morphoform 'a' contain possible cryptic species; and suggested that cytoform B is in the early stages of reproductive isolation whereas lineage sorting is incomplete in cytoforms A, C, and G.
To examine the impact of regional and seasonal variations on the incidence and severity of sheath rot, a major seed-borne disease of rice caused by Sarocladium oryzae, data on incidence and severity were collected from 27 selected fields in the Gazipur, Rangpur, Bogra, Chittagong, Comilla, Gopalgonj, Jessore, Manikgonj, and Bhola districts of Bangladesh in rain-fed and irrigated conditions. Cultural variability of 29 pathogen isolates obtained from 8 different locations was studied on potato dextrose agar (PDA) and genetic variability was determined by DNA fingerprinting using variable number tandem repeat-polymerase chain reaction markers. Overall, disease incidence and severity were higher in irrigated rice. Disease incidence and severity were highest in the Bhola district in rain-fed rice and lowest in irrigated rice. Mycelial growth of 29 representative isolates was found to vary on PDA and the isolates were divided into 6 groups. The range of the overall size of conidia of the selected isolates was 2.40-7.20 x 1.20-2.40 μm. Analysis of the DNA fingerprint types of the 29 isolates of S. oryzae, obtained from the amplification reactions, revealed 10 fingerprinting types (FPTs) that were 80% similar. FPT-1 was the largest group and included 13 isolates (44.8%), while FPT-2 was the third largest group and included 3 isolates. Each of FPT-3, 4, 5, and 6 included only 1 isolate. We observed no relationship between cultural and genetic groupings.
The coastal mosquito Aedes togoi occurs more or less continuously from subarctic to subtropic zones along the coasts of the Japanese islands and the East Asian mainland. It occurs also in tropical Southeast Asia and the North American Pacific coast, and the populations there are thought to have been introduced from Japan by ship. To test this hypothesis, the genetic divergence among geographic populations of A. togoi was studied using one mitochondrial and three nuclear gene sequences. We detected 71 mitochondrial haplotypes forming four lineages, with high nucleotide diversity around temperate Japan and declining towards peripheral ranges. The major lineage (L1) comprised 57 haplotypes from temperate and subarctic zones in Japan and Southeast Asia including southern China and Taiwan. Two other lineages were found from subtropical islands (L3) and a subarctic area (L4) of Japan. The Canadian population showed one unique haplotype (L2) diverged from the other lineages. In the combined nuclear gene tree, individuals with mitochondrial L4 haplotypes diverged from those with the other mitochondrial haplotypes L1-L3; although individuals with L1-L3 haplotypes showed shallow divergences in the nuclear gene sequences, individuals from Southeast Asia and Canada each formed a monophyletic group. Overall, the genetic composition of the Southeast Asian populations was closely related to that of temperate Japanese populations, suggesting recent gene flow between these regions. The Canadian population might have originated from anthropogenic introduction from somewhere in Asia, but the possibility that it could have spread across the Beringian land bridge cannot be ruled out.
The major histocompatibility complex (MHC) plays a crucial role in the immune system, and in some species, it is a target by which individuals choose mates to optimize the fitness of their offspring, potentially mediated by olfactory cues. Under the genetic compatibility hypothesis, individuals are predicted to choose mates with compatible MHC alleles, to increase the fitness of their offspring. Studies of MHC-based mate choice in wild mammals are under-represented currently, and few investigate more than one class of MHC genes. We investigated mate choice based on the compatibility of MHC class I and II genes in a wild population of European badgers (Meles meles). We also investigated mate choice based on microsatellite-derived pairwise relatedness, to attempt to distinguish MHC-specific effects from genomewide effects. We found MHC-assortative mating, based on MHC class II, but not class I genes. Parent pairs had smaller MHC class II DRB amino acid distances and smaller functional distances than expected from random pairings. When we separated the analyses into within-group and neighbouring-group parent pairs, only neighbouring-group pairs showed MHC-assortative mating, due to similarity at MHC class II loci. Our randomizations showed no evidence of genomewide-based inbreeding, based on 35 microsatellite loci; MHC class II similarity was therefore the apparent target of mate choice. We propose that MHC-assortative mate choice may be a local adaptation to endemic pathogens, and this assortative mate choice may have contributed to the low MHC genetic diversity in this population.
Wolbachia are maternally transmitted bacteria found in most arthropods and nematodes, but little is known about their distribution and reproductive dynamics in the Malaysian dengue vector Aedes albopictus. In this study, polymerase chain reaction (PCR) was used to determine the presence of Wolbachia from field collected Ae. albopictus from various parts of the country using wsp specific primers. Ae. albopictus had Wolbachia infection ranging from 60 to 100%. No sequence diversity of wsp gene was found within all wAlbA and wAlbB sequences. Our findings suggest that Wolbachia infection amongst the Malaysian Ae. albopictus were not homogenously distributed in all districts in Malaysia. The presence of Wolbachia in different organs of Ae. albopictus was also determined. Wolbachia were only found in the ovaries and midguts of the mosquitoes, while absent in the salivary glands. The effects of Wolbachia on Ae. albopictus fecundity, longevity and egg viability were studied using infected and uninfected colonies. The removal of Wolbachia from Ae. albopictus resulted in reduced fecundity, longevity and egg viability, thus. Wolbachia seem to play a vital role in Ae. albopictus reproductive system.
Angiostrongylus cantonensis is an important emerging zoonotic parasite causing human eosinophilic meningitis (or meningoencephalitis) in many parts of the world. To-date there is only a single study using mitochondrial cytochrome b (CYTB) gene to determine its genetic structure in eight geographical localities in Thailand. The present study examined the molecular phylogeography of this rat lungworm and its phylogenetic relationship with congeners using CYTB gene marker. A total of 15 CYTB haplotypes was found in 37 sequences from 14 geographical localities (covering north, west, east, central and south regions) in Thailand. These CYTB haplotypes were distinct from those of A. cantonensis for China and Hawaii. In Thailand, some CYTB haplotypes appeared to be confined to specific geographical localities. The partial CYTB DNA nucleotide sequences separated unequivocally the A. cantonensis isolates of Thailand, China and Hawaii as well as the congeners Angiostrongylus malaysiensis, A. costaricensis and Angiostrongylus vasorum, with A. malaysiensis grouped with A. cantonensis and A. costaricensis grouped with A. vasorum. Likewise the congeners of Metastrongylus and Onchocerca genera could also be clearly differentiated. The present study added two new definitive hosts (Bandicota savilei and Rattus losea) and three new localities (Mae Hong Son in the north, Tak in the west, and Phang Nga in the south) for A. malaysiensis in Thailand, indicating its wide occurrence in the country. Three CYTB haplotypes were found in the Thailand samples of A. malaysiensis. In addition to differentiation of congeners, CYTB gene marker could be used for determining the genetic diversity of a given population/taxon.
Brucellosis is an important zoonotic disease that affects both humans and animals. We sequenced the full genome and characterised the genetic diversity of two Brucella melitensis isolates from Malaysia and the Philippines. In addition, we performed a comparative whole-genome single nucleotide polymorphism (SNP) analysis of B. melitensis strains collected from around the world, to investigate the potential origin and the history of the global spread of B. melitensis.
Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10(-33)), we observed one SNP showing significant association to POAG (CDC7-TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10(-8)). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis.
Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (phiCT = 0.015, phiSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype-mitotype associations and tree inconsistency. Widespread distribution and random associations (chi2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.
To elucidate the evolution of one of the most species-rich ant-plant symbiotic systems, the association between Crematogaster (Myrmicinae) and Macaranga (Euphorbiaceae) in South-East Asia, we conducted a phylogenetic analysis of the ant partners. For the phylogenetic analysis partial mitochondrial cytochrome oxidase I and II were sequenced and Maximum Parsimony analysis was performed. The analyzed Crematogaster of the subgenus Decacrema fell into three distinct clades which are also characterized by specific morphological and ecological traits (queen morphology, host-plants, and colony structure). Our results supported the validity of our currently used morphospecies concept for Peninsula Malaysia. However, on a wider geographic range (including North and North-East Borneo) some morphospecies turned out to be species complexes with genetically quite distinct taxa. Our phylogenetic analysis and host association studies do not indicate strict cocladogenesis between the subgenus Decacrema and their Macaranga host-plants because multiple ant taxa occur on quite distinct host-plants belonging to different clades within in the genus Macaranga. These results support the view that host-shifting or host-expansion is common in the ants colonizing Macaranga. Additionally, the considerable geographic substructuring found in the phylogenetic trees of the ants suggests that allopatric speciation has also played a role in the diversification and the current distribution of the Decacrema ants.
Kunjin (KUN) is a flavivirus in the Japanese encephalitis antigenic complex that was first isolated from Culex annulirostris mosquitoes captured in northern Australia in 1960. It is the etiological agent of a human disease characterized by febrile illness with rash or mild encephalitis and, occasionally, of a neurological disease in horses. KUN virus shares a similar epidemiology and ecology with the closely related Murray Valley encephalitis (MVE) virus, the major causative agent of arboviral encephalitis in Australia. Based on traditional antigenic methods, KUN was initially found to be similar to, but distinct from, reference strains of West Nile (WN) virus and designated as a new species. However, more recent phylogenic analyses have revealed that some strains of WN virus, including the isolates from New York, are more similar to KUN virus and form a separate lineage to other WN viruses. An unusual KUN isolate from Malaysia and the African virus Koutango appear to form additional lineages within the WN group of viruses. While these findings are in agreement with the Seventh Report of the International Committee for the Taxonomy of Viruses that designates KUN as a subtype of West Nile, they also suggest that the species should be further subdivided into additional subtypes.
Subtyping of Salmonella Paratyphi A isolates from India, Pakistan, Indonesia and Malaysia was carried out by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these isolates from different endemic countries.
A panel of monoclonal antibodies specific to Hong Kong Chinese nasopharyngeal carcinoma (NPC)-associated Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) variants has been generated. These monoclonal antibodies not only differentiate the Hong Kong Chinese NPC-associated LMP1 variants from the prototype B95-8 LMP1, derived from Caucasian infectious mononucleosis, but also differentiate the 2 highly homologous LMP1 deletion variants commonly found in Hong Kong primary NPC. The predominant deletion type variant, DV-Asp335, is characterized by an aspartic acid at residue 335 located in the cytoplasmic C-terminal region, whereas the other minor deletion variant, DV-Gly335, has a glycine in the same residue position. 335D is hitherto found predominantly in LMP1 of the China 1 strain in association with NPC in the Chinese populations located in southern China and Malaysia. These antibodies, which are applicable in ELISA, immunofluorescence, immunoprecipitation, immunoblotting and immunohistochemistry on paraffin sections, are the first variant-specific anti-LMP1 monoclonal antibodies produced, and will be useful in investigating the functional significance of 335D in NPC.
A total of 20 isolates of Blastocystis were characterized using a single set of polymerase chain reaction (PCR) primers. The amplification product revealed five types of pattern. All four isolates from Singapore yielded PCR products quite different from those of the local isolates. However, most of the local isolates showed a major product at either 280 or 500 bp, or both. We also suspected that the amplification product detected at 280 bp might be an indicator of the pathogenicity of this parasite. One isolate (M12) obtained from a monkey showed patterns similar to those of human isolates (10203 and KP1) and probably belongs to the same strain. The results indicate that the intraspecific or interstrain variations in these 20 Blastocystis isolates belong to 5 different patterns. The differences among isolates of the same strain revealed by the presence or absence of certain amplification products showed further intrastrain variations in this parasite.
We performed genome size estimation of 17 recent human isolates of Salmonella typhi from geographically diverse regions using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with restriction endonucleases XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3') and SpeI (5'-ACTAGT-3'), and summation of the sizes of restriction fragments obtained. All 17 isolates had circular chromosomes, and genome sizes differed by as much as 959 kb, ranging from 3,964 to 4,923 kb (mean genome size = 4,528 kb). The data obtained confirm the usefulness of PFGE in studies of bacterial genome size and are in agreement with recent results indicating considerable genetic diversity and genomic plasticity of S. typhi. The variation in genome sizes noted may be relevant to the observed biological properties of this important human pathogen, including its virulence.
Malaysia had 10,219 confirmed cases of COVID-19 as of September 20, 2020. About 33% were associated with a Tablighi Jamaat religious mass gathering held in Kuala Lumpur between February 27 and March 3, 2020, which drove community transmission during Malaysia's second wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to better understand the molecular epidemiology and spread. We obtained 58 SARS-CoV-2 whole genome sequences from patients in Kuala Lumpur and performed phylogenetic analyses on these and a further 57 Malaysian sequences available in the GISAID database. Nine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the Tablighi mass gathering and became predominant (65.2%) despite being relatively rare (1.4%) globally. Direct epidemiological links between lineage B.6 viruses and the mass gathering were identified. Increases in reported total cases, Tablighi-associated cases, and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly travel-associated and showed limited onward transmission. There were also temporally correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. Over 95% of global B.6 sequences originated from Asia Pacific. We also report a nsp3-C6310A substitution found in 47.3% of global B.6 sequences which was associated with reduced sensitivity using a commercial diagnostic real-time PCR assay. Lineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the Asia-Pacific. Mass gatherings can be significant causes of local and global spread of COVID-19. Shared genomic surveillance can be used to identify SARS-CoV-2 transmission chains to aid prevention and control, and to monitor diagnostic molecular assays. Clinical Trial Registration: COVID-19 paper.
The Sumatran rhinoceros (Dicerorhinus sumatrensis), once widespread across Southeast Asia, now consists of as few as 30 individuals within Sumatra and Borneo. To aid in conservation planning, we sequenced 218 bp of control region mitochondrial (mt) DNA, identifying 17 distinct mitochondrial haplotypes across modern (N = 13) and museum (N = 26) samples. Museum specimens from Laos and Myanmar had divergent mtDNA, consistent with the placement of western mainland rhinos into the distinct subspecies D. s. lasiotis (presumed extinct). Haplotypes from Bornean rhinos were highly diverse, but dissimilar from those of other regions, supporting the distinctiveness of the subspecies D. s. harrissoni. Rhinos from Sumatra and Peninsular Malaysia shared mtDNA haplotypes, consistent with their traditional placement into a single subspecies D. s sumatrensis. Modern samples of D. s. sumatrensis were genotyped at 18 microsatellite loci. Rhinos within Sumatra formed 2 sub-populations, likely separated by the Barisan Mountains, though with only modest genetic differentiation between them. There are so few remaining Sumatran rhinoceros that separate management strategies for subspecies or subpopulations may not be viable, while each surviving rhino pedigree is likely to retain alleles found in no other individuals. Given the low population size and low reproductive potential of Sumatran rhinos, rapid genetic erosion is inevitable, though an under-appreciated concern is the potential for fixation of harmful genetic variants. Both concerns underscore 2 overriding priorities for the species: 1) translocation of wild rhinos to ex situ facilities, and 2) collection and storage of gametes and cell lines from every surviving captive and wild individual.