The efficacy of a pyrethroid insecticide; alpha-cypermethrin against dengue vector Aedes albopictus adult populations from various types of residential and agricultural areas in West Malaysia was evaluated through the adult mosquito bioassays. Sugar-fed Ae. albopictus adult females aged 3-5 days old from each study area were exposed to alpha-cypermethrin 0.05% for an hour. The mortality percentages were calculated at 30 minutes of exposure and 24 h post-exposure. At 30 minutes of exposure, Ae. albopictus adult populations from dengue-risk residential areas (34.19 min) and oil palm plantations (56.53 min) exhibited the shortest and longest time to obtain the 50% mortalities of the population (LT50), respectively. At 24 h post-exposure, other than the reference strain, Ae. albopictus adult populations from rubber estates (99.67%), dengue-free residential areas (99.67%) and dengue-risk residential areas (98.33%) demonstrated susceptibility to alpha-cypermethrin 0.05%. These results revealed the worthwhile adulticidal effect of alpha-cypermethrin which could be used in the future vector control strategies at rubber estates, dengue-free residential areas and dengue-risk residential areas selected in this study.

We investigated the knowledge, attitudes, and practices (KAPs) regarding dengue and its prevention among local residents in all five divisions of Sabah, Malaysia. A cross-sectional questionnaire survey was conducted with 1,000 residents aged 18 years and above. The results revealed that less than one-tenth of the respondents scored high in knowledge (9.6%), while more than half scored high in attitude (64.6%), and less than one-fifth (15.6%) scored high in dengue prevention practices. Economic factors (monthly salary and occupation) were the final independent predictors in all KAP domains. KAP parameters were correlated with each other, but wide gaps between the scores implied that a positive attitude was not translated into either high knowledge or practice scores. Therefore, it is imperative for the authorities to employ multifaceted approaches in future dengue awareness campaigns to increase all KAP domains, especially among those with lower economic status.

The susceptibility levels of Malaysian Aedes albopictus larvae sampled from several agricultural, fogging-free residential and dengue prone residential areas against different larvicides were evaluated using revised diagnostic doses derived from the 2xLC99 values of the reference strain. Upon 24-hour recovery period of WHO larval bioassay, incipient resistance was observed among Ae. albopictus larvae from rubber estates against fenitrothion (96.67% mortality) and permethin (97.00% mortality) while Ae. albopictus larvae from rice cultivation areas were moderately resistant to fenthion (94.33% mortality). Aedes albopictus larvae from dengue prone residential areas developed moderate to high resistance against dichlorodiphenyltrichloroethane (DDT), fenitrothion, fenthion, propoxur and permethrin (79.67% - 97.33% mortality). Moderate to high resistance were also demonstrated among all populations of Ae. albopictus larvae against temephos and chlorpyrifos (63.00% - 97.67% mortality). Except for Ae. albopictus larvae from oil palm plantations, all Ae. albopictus larval populations were also highly resistant to bendiocarb (65.67% - 89.67% mortality). Cross resistance between larvicides from similar and different insecticide classes were also revealed in this study. The use of revised diagnostic doses established from the local reference strain could reduce the possibility of underestimation or overestimation of the insecticide susceptibility status of field strain populations.

Mitochondrial cytochrome c oxidase subunit I (COI) sequences were utilized to infer the population genetic structure of Simulium (Gomphostilbia) atratum De Meijere, an endemic simulid species to Indonesia. Both median joining haplotype network and maximum-likelihood tree revealed two genetic lineages (A and B) within the species, with an overlap distribution in Lombok, which is situated along Wallace's line. Genetic differentiation and gene flow with varying frequencies (FST = 0.02-0.967; Nm = 0.01-10.58) were observed between populations of S. (G.) atratum, of which population pairs of different lineages showed high genetic differentiation. Notably, the high genetic distance of up to 5.92 % observed within S. (G.) atratum in Lombok was attributed to the existence of two genetically distinct lineages. The co-occurrence of distinct lineages in Lombok indicated that Wallace's line did not act as faunistic border for S. (G.) atratum in the present study. Moreover, both lineages also exhibited unimodal distributions and negative values of neutrality tests, suggesting a pattern of population expansion. The expansion and divergence time estimation suggested that the two lineages of S. (G.) atratum diverged and expanded during the Pleistocene era in Indonesia.

A commercial bacterial product of Bacillus thuringiensis israelensis (Bti); Mosquito BTI from USA was tested for its efficacy in exterminating the local populations of Aedes albopictus larvae. Larval surveys were carried out to obtain field populations of Ae. albopictus from various land use namely dengue-risk and dengue-free housing localities as well as agrarian localities like oil palm plantations, rubber estates and paddy fields. A 24-hour larval bioassay using Mosquito BTI was performed on each Ae. albopictus population. Only a maximum of 2.00% mortality was observed by the end of the first four hours of exposure while a maximum of 25.00% mortality was displayed at 24 hours of exposure. Results showed the potential use of Bti in reducing local Ae. albopictus larval populations but more than 24 hours may be required to achieve complete mortalities among these populations upon exposure to Bti. Some limitations of this study were also discussed.

Widespread insecticide resistance among Aedes albopictus is a major public health threat as it could impede the fluidity of vector control program. Hence, profiling insecticide resistance mechanisms is pivotal to gain better insights in tailoring future control strategies. Accordingly, biochemical and molecular assays were performed on Ae. albopictus larvae and adults from 14 districts across all five divisions of Sabah, Malaysia. Field-collected Ae. albopictus demonstrated influx of activities in detoxification enzymes such as non-specific esterases (ESTs), mixed function oxidase (MFO) and acetylcholinesterase (AChE). In larval populations, ESTs were significantly associated with organophosphate resistance. On the other hand, in adult populations, MFO and AChE were respectively associated with DDT and carbamate resistance. Glutathion-S-transferase (GST) activity elevation was insignificant, and no knockdown resistance (kdr) detected in Ae. albopictus, suggesting that these two mechanisms play no role in resistance build up. In short, these findings could serve as fundamentals for authorities in strategizing better Aedes control that could incorporate efficient insecticide utilization whilst minimizing resistance dispersal.

Temephos is the World Health Organization (WHO) recommended larvicide and is still being utilized worldwide to control larvae of dengue vectors; Aedes aegypti and Aedes albopictus. The efficacy of a commercial temephos product; Temebate® to exterminate the local populations of Ae. albopictus larvae originated from different land use particularly dengue-risk and dengue-free housing localities as well as agrarian localities including oil palm plantations, rubber estates and paddy fields was assessed to verify its bioefficacy in these localities. Field populations of Ae. albopictus larvae were attained via a larval survey at each study locality. Each Ae. albopictus larval population was subjected to a 24-h larval bioassay using Temebate® at operational dosage of 1 mg/L. Almost all Ae. albopictus larval populations demonstrated mortalities between 7.00% and 100.00% by the end of the first 4 h of Temebate® exposure with the resistance ratios between 0.94 and 8.33. After 24 h of Temebate® exposure, all sixteen Ae. albopictus larval populations exhibited increased mortalities with ten of them showing 100% mortalities. These results confirmed the relevance of Temebate® to be continuously used by the residents of these localities as their control efforts against dengue vectors. Nevertheless, Temebate® application by consumers in dengue-risk localities need to be carefully monitored to prevent further development of temephos resistance among Ae. albopictus populations and substantiated with other vector control approaches.

DNA barcoding is a valuable taxonomic tool for rapid and accurate species identification and cryptic species discovery in black flies. Indonesia has 143 nominal species of black flies, but information on their biological aspects, including vectorial capacity and biting habits, remains underreported, in part because of identification problems. The current study represents the first comprehensive DNA barcoding of Indonesian black flies using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. Genomic DNA of Indonesian black fly samples were extracted and sequenced, producing 86 COI sequences in total. Two hundred four COI sequences, including 118 GenBank sequences, were analysed. Maximum likelihood (ML) and Bayesian inference (BI) trees were constructed and species delimitation analyses, including ASAP, GMYC and single PTP, were performed to determine whether the species of Indonesian black flies could be delineated. Intra- and interspecific genetic distances were also calculated and the efficacy of COI sequences for species identification was tested. The DNA barcodes successfully distinguished most morphologically distinct species (> 80

Background Prompt and precise identification of black flies (Simuliidae) is crucial, given their biting behaviour and significant impact on human and animal health. To address the challenges presented by morphology and chromosomes in black fly taxonomy, along with the limited availability of molecular data pertaining to the black fly fauna in Vietnam, this study employed DNA-based approaches. Specifically, we used mitochondrial and nuclear-encoded genes to distinguish nominal species of black flies in Vietnam. Methods In this study, 135 mitochondrial cytochrome c oxidase subunit I (COI) sequences were established for 45 species in the genus Simulium in Vietnam, encompassing three subgenera ( Gomphostilbia , Nevermannia , and Simulium ), with 64 paratypes of 27 species and 16 topotypes of six species. Of these COI sequences, 71, representing 27 species, are reported for the first time. Results Combined with GenBank sequences of specimens from Malaysia, Myanmar, Thailand, and Vietnam, a total of 234 DNA barcodes of 53 nominal species resulted in a 71% success rate for species identification. Species from the non-monophyletic Simulium asakoae, S. feuerborni , S. multistriatum , S. striatum , S. tuberosum , and S. variegatum species groups were associated with ambiguous or incorrect identifications. Pairwise distances, phylogenetics, and species delimitation analyses revealed a high level of cryptic diversity, with discovery of 15 cryptic taxa. The current study also revealed the limited utility of a fast-evolving nuclear gene, big zinc finger (BZF), in discriminating closely related, morphologically similar nominal species of the S. asakoae species group. Conclusion This study represents the first comprehensive molecular genetic analysis of the black fly fauna in Vietnam to our knowledge, providing a foundation for future research. DNA barcoding exhibits varying levels of differentiating efficiency across species groups but is valuable in the discovery of cryptic diversity. Graphical abstract

A potentially novel actinobacterium isolated from forest soil, Streptomyces sp. KSF103 was evaluated for its insecticidal effect against several mosquito species namely Aedes aegypti, Aedes albopictus, Anopheles cracens and Culex quinquefasciatus. Mosquito larvae and adults were exposed to various concentrations of the ethyl acetate (EA) extract for 24 h. Considerable mortality was evident after the EA extract treatment for all four important vector mosquitoes. Larvicidal activity of the EA extract resulted in LC50 at 0.045 mg/mL and LC90 at 0.080 mg/mL for Ae. aegypti; LC50 at 0.060 mg/mL and LC90 at 0.247 mg/mL for Ae. albopictus; LC50 at 2.141 mg/mL and LC90 at 6.345 mg/mL for An. cracens; and LC50 at 0.272 mg/mL and LC90 at 0.980 mg/mL for Cx. quinquefasciatus. In adulticidal tests, the EA extract was the most toxic to Ae. albopictus adults (LD50 = 2.445 mg/mL; LD90 = 20.004 mg/mL), followed by An. cracens (LD50 = 5.121 mg/mL; LD90 = 147.854 mg/mL) and then Ae. aegypti (LD50 = 28.873 mg/mL; LD90 = 274.823 mg/mL). Additionally, the EA extract exhibited ovicidal activity against Ae. aegypti (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), Ae. albopictus (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), and An. cracens (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), evaluated up to 168 h post-treatment. It displayed no toxicity on the freshwater microalga Chlorella sp. Beijerinck UMACC 313, marine microalga Chlorella sp. Beijerinck UMACC 258 and the ant Odontoponera denticulata. In conclusion, the EA extract showed promising larvicidal, adulticidal and ovicidal activity against Ae. aegypti, Ae. albopictus, An. cracens, and Cx. quinquefasciatus (larvae only). The results suggest that the EA extract of Streptomyces sp. KSF103 has the potential to be used as an environmental-friendly approach in mosquito control. The current study would serve as an initial step toward complementing microbe-based bioinsecticides for synthetic insecticides against medically important mosquitoes.

This study inferred the genetic diversity and dispersal pattern of Aedes albopictus , for the first time from various populations in Sarawak, the largest Malaysian state situated in northwest Borneo Island. A set of sequences of 120 Ae . albopictus from Sarawak aligned as 633 characters of the cytochrome c oxidase subunit I (COI) revealed 22 haplotypes (A1–A22), of which A1 was the most widespread haplotype. The overall haplotype and nucleotide diversities were 0.65014 and 0.00176, respectively, with no significant genetic differentiation and high gene flow ( N m = 1.07). The genetic distances among the populations ranged from 0 to 0.0227. In the global dataset, sequence alignment of Ae. albopictus from Sarawak and other regions of the world revealed 72 haplotypes. Based on uniquely shared haplotypes, Ae . albopictus in Sarawak has a close affinity with those from Southeast Asian and Oceania regions.

Insects or arthropods collected from a human deceased during crime scene investigation often revealed information related to the body of the deceased or the crime scene. Morphologically identical immature larvae or maggots have been identified using DNA identification methods. However, a big challenge to the DNA identification method is the presence of morphologically identical but genetically distinct taxa. The genetic diversity of Chrysomya megacephala flies from Malaysia will be revealed in this study. A total of 74 Chrysomya megacephala from Peninsular Malaysia were subjected to phylogenetic and haplotype analyses based on the Cytochrome b (Cyt b) gene. Chrysomya megacephala from Kuala Lumpur and Selangor are more diverse genetically compared to those from Perak, Johor and Pahang. The Cyt b gene revealed three distinct genetic clades of Chrysomya megacephala , one clade is for populations from Selangor and Kuala Lumpur, whereas the other two clades consisted of specimens from all five studied populations that is Perak, Pahang, Selangor, Kuala Lumpur and Johor. Detection of hidden lineages of Chrysomya megacephala based on the Cyt b gene may offer some clues for forensic entomological investigation in the country.

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.

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 pyrethmids. 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.

Insecticide application is one of the most important control measures for protection from mosquito bites. A descriptive, community-based cross-sectional survey involving 986 respondents was conducted to probe the knowledge, attitudes, and practices regarding household insecticide use in Selangor—the state that recorded the highest number of dengue cases in Malaysia. In this study, we classified the responses as either low (poor) or high (good) based on median cut-off points. Age, gender, ethnicity, and household income were found to be significantly associated factors relative to the respondents’ mosquito knowledge score, whereas no factors were significantly associated with attitudes toward the use of household insecticide. Practices, on the other hand, were associated with age and high attitude scores. Most respondents (71.4%) had good knowledge of mosquitoes. Conversely, 51.1% and 59.2% of respondents scored high on attitudes and practices, respectively, on the use of household insecticides. This study highlights the need to expand health promotion activities through social mobilization to raise awareness about vector-borne disease prevention and promoting positive attitudes and safe practices in the use of household insecticides.

Resistance status of Aedes albopictus (Skuse) from 13 districts in Sarawak State, Malaysia, was evaluated against four major classes of adulticides, namely organochlorine, organophosphate, carbamate, and pyrethroid. Adult bioassays were performed according to the World Health Organization (WHO) standard protocols to assess knockdown and mortality rates of Ae. albopictus. Among the tested pyrethroids, only cyfluthrin was able to exhibit complete knockdown. On the other hand, different susceptibility and resistance patterns were observed in other adulticides. As for mortality rates, the mosquitoes were susceptible to cyfluthrin and dieldrin but exhibited various susceptibilities to other tested adulticides. Cross-resistance was discovered within and between tested insecticide classes. Significant correlations were found within pyrethroid and carbamate classes (i.e., bendiocab and propoxur, P = 0.036; etofenprox and permethrin, P = 0.000; deltamethrin and lambda-cyhalothrin, P = 0.822; deltamethrin and permethrin, P = 0.042). Additionally, insecticides belonging to different groups were also found significantly correlated (i.e., malathion and deltamethin, P = 0.019; malathion and bendiocarb, P = 0.008; malathion and propoxur, P = 0.007; and bendiocarb and deltamethrin, P = 0.031). In conclusion, cyfluthrin was effective for Aedes albopictus control in Sarawak State and these data may assist local authorities to improve future vector control operations.

Susceptibility status of Aedes albopictus (Skuse) sampled from residential areas in Interior, Sandakan and Tawau divisions of Sabah, Malaysia, was evaluated based on the WHO-recommended 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.

A structured questionnaire was distributed to a total of 531 cat owners in Peninsular Malaysia to evaluate their perceptions toward fleas and flea-borne diseases (FBD) using a knowledge-attitude-practice (KAP) approach. This study showed that majority of cat owners (50.5%) had a moderate level of knowledge on fleas, while 65.3% of them had a relatively low level of knowledge on FBD. However, most of them had positive attitudes (87.9%) and good practices (94.5%) toward efforts on prevention from flea infestation and FBD. Statistical analysis showed significant associations between knowledge on fleas and FBD toward demographic and pet ownership profiles of the respondents. Educated cat owners from urban area were more likely to have positive attitudes toward prevention of fleas and FBD. Female cat owners with monthly income of RM 5,001-7,000 ($1143-1600) were more likely to have good practices. Middle-aged cat owners showed the highest odds toward both positive attitudes and good practices. Correlation analyses indicated significant linear relationships between both knowledge, which were also associated with attitudes, but not with practices.

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.

Some filarial nematodes, such as Wuchereria bancrofti, Brugia malayi, and Brugia timori, cause lymphatic diseases in humans in the tropics, whereas other filarial parasites from wild animals cause zoonotic diseases in humans worldwide. To elucidate the prevalence and diversity of filarial parasites in Malaysia, we investigated the filarial parasites from wild animals in Gemas, Negeri Sembilan. To find adult filarial parasites, we dissected 26 animals, which included five frogs, one skink, one snake, two birds, six common treeshrews, and 11 rats. Then, we examined microfilariae in the blood smears and skin snips obtained from each animal. We found two types of microfilariae in the blood smears of common treeshrews: one was very similar to Malayfilaria sofiani and the other closely resembled Brugia tupaiae. These findings indicate an additional distribution of these filarial parasites in Gemas.