The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 β-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.
Sexually anomalous individuals, typically intersexes or gynandromorphs, bear a mixture of male and female traits. Twelve sexually anomalous individuals of the black fly Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler were discovered among 49 adults reared from pupae. All 12 sexually anomalous adults were parasitized by mermithid nematodes, although five additional parasitized adults had no overt external anomalies. Sequence analysis of the 18S rRNA gene revealed that the mermithids, possibly representing a new species, are related to Mesomermis spp., with genetic distances of 5.09-6.87%. All 12 anomalous individuals had female phenotypical traits on the head, thorax, forelegs, midlegs, and claws, but male features on the left and right hind basitarsi. One individual had mixed male and female genitalia. The findings are in accord with the trend that mermithid infections are associated with sexually anomalous adult black flies.
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.
Given the lack of molecular evidence in altered target-site insecticide resistance mechanism in Aedes albopictus (Skuse) worldwide, the present study aims to detect the presence of A302S mutation in the gene encoding the gamma aminobutyric acid receptor resistant to dieldrin (Rdl) in Ae. albopictus for the first time from its native range of South East Asia, namely Malaysia. World Health Organization (WHO) adult susceptibility bioassay indicated a relatively low level of dieldrin resistance (two-fold) in Ae. albopictus from Petaling Jaya, Selangor. However, PCR-RFLP and direct sequencing methods revealed the presence of the A302S mutation with the predomination of heterozygous genotype (40 out of 82 individuals), followed by the resistant genotype with 11 individuals. This study represents the first field evolved instance of A302S mutation in Malaysian insect species.
This paper reports total nematode anthelmintic resistance towards albendazole, fenbendazole, levamisole and ivermectin in a commercial sheep farm located in Terengganu, Malaysia. Faecal Egg Count Reduction Test (FECRT) was conducted on 25 sheep, where five sheep in each group were treated with the respective four anthelmintics based on live bodyweight. The balance of five sheep placed in the control group were not treated with any anthelmintics. At day 13 post-treatment, faecal egg count was conducted and nematode worm egg count reduction percentage was calculated to determine the resistance status towards the respective anthelmintics tested. Results showed that nematodes were resistant to all the anthelmintics tested, namely albendazole, fenbendazole, levamisole and ivermectin with reduction percentage of 87%, 46%, 94% and 68%, respectively. Subsequently, the third stage larvae of Haemonchus contortus and Trichostrongylus colubriformis recovered from post-treatment faecal cultures were subjected to allele-specific polymerase chain reaction (AS-PCR) assay to determine the presence of the benzimidazole resistance gene. This study reports the occurrence of the classical F200Y mutation in the isotype 1 βtubulin gene, for the first time in Malaysia.
This study aims to examine the efficacy of mosquito mat vaporizers on Aedes aegypti and their associated metabolic detoxication mechanisms. For this purpose, Aedes aegypti (Linnaeus) was collected from nine districts in Selangor, Malaysia and tested with mosquito vaporizing mat bioassays. The same populations were also subjected to biochemical assays to investigate activities of detoxifying enzymes, namely non-specific esterase (EST), glutathione-S-transferase (GST) and mixed function oxidase (MFO). The efficacy of Ae. aegypti on the active ingredients tested in decreasing order were d- allethrin > dimefluthrin > prallethrin with PBO > prallethrin. The results further indicated significant enhancement mean levels of EST, GST and MFO in pyrethroid-resistant populations. The mortality rate of Ae. aegypti in response to pyrethroid active ingredients was associated with MFO activity, suggesting it is an important detoxification enzyme for the populations tested. In view of the presence of resistance against household insecticide products, pyrethroid efficacy on Ae. aegypti populations needs to be monitored closely to ensure the implementation of an effective vector control program in Malaysia.
The increasing attention on Vietnam as a biodiversity hotspot prompted an investigation of the potential for cryptic diversity in black flies, a group well known elsewhere for its high frequency of isomorphic species. We analyzed the banding structure of the larval polytene chromosomes in the Simulium tuberosum species group to probe for diversity beyond the morphological level. Among 272 larvae, 88 different chromosomal rearrangements, primarily paracentric inversions, were discovered in addition to 25 already known in the basic sequences of the group in Asia. Chromosomal diversity in Vietnam far exceeds that known for the group in Thailand, with only about 5% of the rearrangements shared between the two countries. Fifteen cytoforms and nine morphoforms were revealed among six nominal species in Vietnam. Chromosomal evidence, combined with available molecular and morphological evidence, conservatively suggests that at least five of the cytoforms are valid species, two of which require formal names. The total chromosomal rearrangements and species (15) now known from the group in Vietnam far exceed those of any other area of comparable size in the world, supporting the country's status as a biodiversity hotspot. Phylogenetic inference based on uniquely shared, derived chromosomal rearrangements supports the clustering of cytoforms into two primary lineages, the Simulium tani complex and the Southeast Asian Simulium tuberosum subgroup. Some of these taxa could be threatened by habitat destruction, given their restricted geographical distributions and the expanding human population of Vietnam.
The biodiversity of black flies (Diptera: Simuliidae), which are biting insects of medical and veterinary importance, is strikingly high in Southeast Asian countries, such as Indonesia, Malaysia, Philippines and Thailand. In 2013, we began to explore the fauna of black flies in Vietnam, which has so far been poorly studied. In this monograph, the wealth of the biodiversity of black flies in Vietnam is also confirmed on the basis of the results of our recent investigations, though limited to five provinces in the country. Morphotaxonomic studies of black flies obtained from Sapa, Lao Cai Province, northern Vietnam, in 2014 and Nghe An Province, northern Vietnam, in 2015, and reexaminations of black flies collected from Tam Dao, Vinh Phuc Province, northern Vietnam, in 2013, Thua Thien Hue Province, central Vietnam, in 2014, and Lam Dong Province, southern Vietnam, in 2014, were conducted. A total of 22 species are described as new, including one in the newly recorded subgenus Montisimulium Rubtsov, and three species are recognized as new records from Vietnam. This investigation brings the number of species of black flies known in Vietnam to 70, all of which are assigned to the genus Simulium Latreille, and are placed in four subgenera (25 in Gomphostilbia Enderlein, one in Montisimulium, seven in Nevermannia Enderlein, and 37 in Simulium Latreille s. str.). The numbers of species-groups recognized include seven in Gomphostilbia, three in Nevermannia and nine in Simulium, indicating a high diversity of putative phylogenetic lineages. New species include S. (G.) sanchayense sp. nov. (= the species formerly regarded as S. (G.) brinchangense Takaoka, Sofian-Azirun & Hashim), S. (S.) lowi sp. nov. (= the species formerly regarded as S. (S.) brevipar Takaoka & Davies), S. (S.) fuscicoxae sp. nov. [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)], S. (S.) suoivangense sp. nov. [= morphoform 'b' of the S. (S.) tani Takaoka & Davies (complex)]. Newly recorded species are S. (G.) parahiyangum Takaoka & Sigit, S. (N.) maeaiense Takaoka & Srisuka, and S. (S.) doipuiense Takaoka & Choochote (complex) [= the species formerly regarded as S. (S.) rufibasis Brunetti (in part)]. The substitute name, S. (S.) huense, is given for the species that was described under the name of S. (S.) cavum from southern Vietnam. A redescription of the female, male, pupa and larva of S. (G.) asakoae Takaoka & Davies is presented, and the female and larva of S. (G.) hongthaii Takaoka, Sofian-Azirun & Ya'cob are described for the first time. Keys to 10 subgenera in the Oriental Region and all 70 species recorded from Vietnam are provided for females, males, pupae and mature larvae. As investigations extend nationwide in all the provinces in Vietnam, more new species and records are expected to be discovered. It is hoped that this monograph will be useful as a baseline taxonomic reference for future studies of black flies in Vietnam and neighbouring countries.
The bioefficacy of commercial mosquito coils containing four different active ingredients, namely metofluthrin, d-allethrin, d-trans allethrin, and prallethrin against Aedes albopictus (Skuse) (Diptera: Culicidae) from 10 states in Malaysia, was evaluated using the glass chamber method. In this study, Ae. albopictus exhibited various knockdown rates (50% knockdown time, KT50), ranging from 2.50 to 5.00 min, 2.50 to 7.00 min, 3.00 to 8.00 min, and 5.00 to 17.00 min for metofluthrin, d-trans allethrin, d-allethrin, and prallethrin, respectively. Overall, all strains of Ae. albopictus were most susceptible to metofluthrin, with mortality rates >80%. On the other hand, mortality rates ranging from 5.0 to 100% were observed from all populations exposed to d-trans allethrin, d-allethrin, and prallethrin. In addition, significant correlations between KT50 of metofluthrin and d-allethrin (r = 0.758, P = 0.011), metofluthrin and prallethrin (r = 0.676, P = 0.032), d-allethrin and d-trans allethrin (r = 0.832, P = 0.003), d-allethrin and prallethrin (r = 0.921, P = 0.000), and d-trans allethrin with prallethrin (r = 0.941, P = 0.000) were detected, suggesting some levels of cross-resistance within the pyrethroid insecticides. This study demonstrated that metofluthrin can induce high insecticidal activity in Ae. albopictus in Malaysia, followed by d-trans allethrin, d-allethrin, and prallethrin.
This study was conducted to monitor the susceptibility status of Aedes aegypti (Linnaeus) larvae in the Sunda Islands of Indonesia against various organophosphates and organochlorines. Larval bioassay was performed in accordance with the World Health Organization standard protocol. Field-collected and reference strains of Ae. aegypti larvae were tested against diagnostic doses of eight larvicides belonging to organophosphates and organochlorines, namely bromophos (0.050 mg/liter), chlopyrifos (0.002 mg/liter), fenitrothion (0.020 mg/liter), fenthion (0.025 mg/liter), malathion (0.125 mg/liter), temephos (0.012 mg/liter), DDT (0.012 mg/liter), and dieldrin (0.025 mg/liter). Mortality rates of larvae were recorded at 24-h posttreatment. This study showed that Ae. aegypti larvae from Padang, Samarinda, Manggarai Barat, and South Central Timor were susceptible to both fenitrothion and dieldrin (mortality rates ≥ 98%). About 6 out of 10 field strains of Ae. aegypti larvae were resistant (<80% mortality rates) against fenthion, whereas Ae. aegypti larvae from Kuningan, Samarinda, Sumba, and South Central Timor exhibited some degrees of resistance (mortality rates 80-98%). All field-collected Ae. aegypti larvae were resistant against diagnostic doses of chlorpyrifos, malathion, temephos, and DDT with mortality rates ranging from 0 to 74.67%. Continued insecticide susceptibility studies are essential to identify the efficacy of insecticides for an improved dengue vector control and to delay the development of insecticide resistance.
Resistance to pyrethroid insecticides is widespread in Indonesian Aedes aegypti (Linnaeus), the primary vector of dengue viruses. This study aims to investigate the mutations in the voltage-gated sodium channel (Vgsc) conferring pyrethroid resistance against Ae. aegypti populations from Indonesia. Molecular genotyping of mutations using polymerase chain reaction assay and direct DNA sequencing were performed at positions 989 and 1,016 in IIS6 region, and 1,534 in IIIS6 region of the voltage-gated sodium channel (Vgsc) in nine populations of Indonesian Ae. aegypti. The V1016G and S989P genotyping identified the RR genotype to be predominant in six out of nine populations of Ae. aegypti, whereas the SS genotype occurred only in minority. Interestingly, co-occurrence of the V1016G and S989P mutations was detected in the aforementioned six populations with high frequency. Genotyping of F1534C showed all nine populations exhibited the SS genotype, with merely two individuals from a population were heterozygous (RS). Significant correlations were demonstrated between the allele frequencies of the V1016G mutation and the survivability rates as well as resistance ratios in pyrethroid adult bioassays. This signifies the V1016G can contribute more to the insensitivity of Vgsc than the F1534C. Homozygous 1016G mosquitoes were likelier to survive pyrethroid exposure. Identification of underlying mechanisms resulting in insecticide resistance is advantageous in developing effective mosquito control programs in Indonesia.
Aedes aegypti (L.) (Diptera: Culicidae) is the primary vector of several arthropod-borne viral infectious diseases globally. Relentless vector control efforts are performed to curtail disease transmissions, insecticides remain as the first line of defense in Indonesia. With a dearth of publication on the efficacy of mosquito coil in Indonesia, this is the first report related to mosquito coil despite its common use in households. Ae. aegypti mosquitoes were sampled from nine regencies in Indonesia and tested using the glass-chamber method against three commercially available local pyrethroid-based mosquito coils containing d-allethrin, transfluthrin, and metofluthrin. The 50% knockdown time of female Ae. aegypti tested with d-allethrin, transfluthrin, and metofluthrin containing coils ranged from 0.65 to 14.32; 0.8 to 16.4; and 0.78 to 20.57 min, respectively. Mortality rates in accordance with WHO resistance indicators showed that strains from Denpasar, Mataram, Kuningan, Padang, Samarinda, and Sumba Timur were resistant (<80% mortality rate), whereas strains from Manggarai Barat, Dompu, and Pontianak were susceptible (>98% mortality rate) to the active ingredients assayed. Moreover, the knockdown rates between d-allethrin and transfluthrin, d-allethrin and metofluthrin, as well as transfluthrin and metofluthrin displayed significant associations, portraying the presence of cross-resistance within pyrethroid insecticides. The minimal insecticidal effect of mosquito coils against some Indonesian Ae. aegypti also pointed out the development of pyrethroid resistance, prompting a revamping of the vector control system.
This study was carried out to determine from bacterial profiling to the bacterial profiles of head lice among the Orang Asli communities. The head lice were collected from Orang Asli community volunteers. The surface sterilized head lice pools were subjected to genomic DNA extraction while next generation sequencing of the 16S rRNA gene was performed using the Illumina MiSeq platform. Six female and three male head lice identified as Pediculus humanus capitis were collected. A total of 111 368 number of NGS sequencing reads were recorded while another 223 bacterial taxa sequences were obtained. Symbiotic bacteria showed the highest number of reads, with Arsenophonus and Rhodococcus sequences being the most abundant genera in the female and male samples, respectively. The female head lice contained a more distinct microbial diversity. Amongst the pathogenic bacterial species sequences noted were the methicillin-resistant Staphylococcus aureus, Streptobacillus moniliformis, Haemophilus influenzae, Bordetella pertussis and Acinetobacter baumannii. The 16S rRNA genome sequencing revealed a number of rare and pathogenic bacterial species within the head lice of the Orang Asli. The socio-economic practices of the community which involved forest foraging and hunting, and their poor living conditions potentially facilitated the transmission of zoonotic bacterial pathogens, including those found within the head lice. Hence, there is the possibility that the head lice could serve as vectors for the transmission of pathogenic bacteria. This study highlighted the diverse microbial community found within the head lice's gut of the Orang Asli, with the detection of multiple rare and pathogenic bacteria capable of causing severe infections.
Susceptibility status of Aedes albopictus (Skuse) sampled from residential areas in Interior, Sandakan and Tawau divisions of Sabah, Malaysia, was evaluated based on the WHOrecommended doses of organochlorine and organophosphate larvicides. To determine susceptibility status, larval bioassays were carried out and post 24-hour mortalities based on WHO resistance classifications were adopted. The results demonstrated that Ae. albopictus larvae were resistant toward 5 out of the 8 larvicides tested. Larvae from all populations were resistant against bromophos, fenitrothion, malathion, temephos and dichlorodiphenyltrichloroethane (DDT), with mortalities ranging from 0.00 to 89.33%. Dieldrin, on the other hand, could induce 100.00% mortalities in all populations, followed by fenthion and chlorpyrifos, with mortalities ranging from 97.33 to 100.00% and 81.33 to 100.00% respectively. Despite most populations exhibiting similitude in their resistance status, larvae from Sandakan exhibited the highest resistance level whereas the lowest level was observed in Keningau. In view of the inadequacy of some larvicides in controlling Ae. albopictus in this study, integrated management such as insecticide rotation or combination of interventions is warranted.
PURPOSE: Canine filariosis in domestic dogs caused by several species of filarids is an emerging vector-borne disease and the spread of this disease remains a global veterinary and public health concern. However, information regarding these filarids and their epidemiological patterns remains scarce in Malaysia. The present study aimed to determine the infection rate and associated risk factors of filarial parasites in dogs in Malaysia.
METHODS: A total of 399 dog blood samples were collected from veterinary hospitals and animal shelters in Malaysia to determine the infection rate and associated risk factors via a combination of microscopic, serologic and molecular diagnostic techniques.
RESULTS: Two species of canine filariae identified in this study were Dirofilaria immitis (6.5%) and Brugia pahangi (1.3%), and their infections were associated with cross breed, medium size and short hair (p
This study examines the biological efficacy of four mosquito mat vaporizers each containing different active ingredients: prallethrin with PBO, dimefluthrin, prallethrin, and d-allethrin. The glass chamber assay was used to evaluate their efficacy on Aedes albopictus (Skuse) (Diptera: Culicidae) from nine districts in Selangor, Malaysia. Aedes albopictus exhibited different knockdown rates, with 50% knockdown times, KT50, varying from 1.19 to 2.00 min, 1.22 to 2.20 min, 1.39 to 5.85 min, and 1.39 to 1.92 min for prallethrin with PBO, dimefluthrin, prallethrin and d-allethrin, respectively. In general, all populations of Ae. albopictus were completely knocked down after exposure to all active ingredients except Hulu Selangor population, which showed 96.00% knockdown against d-allethrin. On the contrary, mortality rates were observed from 84.00-100.00%, 84.00-100.00%, 90.67-100.00% and 90.67-100.00% in populations tested with prallethrin with PBO, dimefluthrin, prallethrin and d-allethrin, respectively. Moreover, significant correlations between mortality rates of prallethrin with PBO vs dimefluthrin (r = 0.836, P = 0.003), prallethrin with PBO vs prallethrin (r = 0.760, P = 0.011), and prallethrin vs d-allethrin (r = 0.694, P = 0.026) were also observed, suggesting cross-resistance among pyrethroids. d-allethrin was found to be high in insecticidal activity, followed by prallethrin, prallethrin with PBO, and dimefluthrin. In consistent with mortality due to insecticide exposure, elevated levels of enzyme activities were also demonstrated in Sabak Bernam, Hulu Selangor, Gombak, Petaling, Hulu Langat and Klang populations.
The efficacy of three groups of insect growth regulators, namely juvenile hormone mimics (methoprene and pyriproxyfen), chitin synthesis inhibitors (diflubenzuron and novaluron), and molting disruptor (cyromazine) was evaluated for the first time, against Aedes albopictus Skuse (Diptera: Culicidae) larvae from 14 districts in Sabah, Malaysia. The results showed that all field populations of Ae. albopictus were susceptible towards methoprene, pyriproxyfen, diflubenzuron, novaluron, and cyromazine, with resistance ratio values ranging from 0.50-0.90, 0.60-1.00, 0.67-1.17, 0.71-1.29, and 0.74-1.07, respectively. Overall, the efficacy assessment of insect growth regulators in this study showed promising outcomes and they could be further explored as an alternative to conventional insecticides.
Two new species, Simulium (Gomphostilbia) sunapii and S. (G.) rangatense, are described based on adults, pupae, and mature larvae from Flores, in the eastern part of the Sunda Archipelago, Indonesia. Simulium (G.) sunapii sp. nov. is placed in the S. asakoae species-group, representing the easternmost geographical record for the group in this archipelago. It is characterized by a small number of male upper-eye large facets in eight or nine vertical columns and 12 horizontal rows. Simulium (G.) rangatense sp. nov. is placed in the S. ceylonicum species-group and is characterized by the pupal gill with six filaments. This new species, together with two related species of the S. ceylonicum species-group in Flores, suggests the species radiation of this species-group might have been accompanied by a reduction of the number of pupal gill filaments from eight to four through six. Taxonomic notes are provided to distinguish these two new species from related species.
Vector-borne infections are persistent public health threats worldwide. In recent years, a number of mosquito-borne viruses have emerged or re-emerged to cause major disease outbreaks. Other vector-borne pathogens, however, remain understudied and much neglected especially in the developing regions of the world including Southeast Asia. In this study, the brown dog tick Rhipicephalus sanguineus sensu lato, cat louse Felicola subrostratus, and cat fleas Ctenocephalides felis and Ctenocephalides orientis collected from free-ranging cats and dogs in Malaysia were molecularly screened for the presence of Bartonella and Rickettsia bacteria, and Dipylidium tapeworm. Our results showed the presence of Bartonella clarridgeiea, Bartonella henselae (lineage Marseille and lineage Houston-1), and Rickettsia sp. in C. felis. We also detected Rickettsia asembonensis in C. orientis and R. sanguineus s.l. Additionally, this study provides the first documentation on a potentially new species of Dipylidium infecting F. subrostratus and C. felis. Our results highlight the role of ectoparasites from free-ranging animals including cats and dogs, in harboring multiple transmissible pathogens.
Simulium (Gomphostilbia) lemborense sp. nov. is described based on adults, pupae and mature larvae from Flores, in the eastern part of the Sunda Archipelago, Indonesia. This new species is placed in the Simulium batoense species-group of the subgenus Gomphostilbia Enderlein, representing the most eastern distribution record for the group. This new species is characterized by a narrow female frons and pupal gill with eight filaments, of which two filaments of the ventral pair are three to four times as long as the six other filaments. Taxonomic notes are provided to distinguish this new species from related species.