This study aims to improve the larvicidal activity of crude Chisocheton erythrocarpus extract (CCEE) against Aedes aegypti larvae via nano-emulsion formulation method. The fruits of C. erythrocarpus possesses limonoids with good larvicidal activity against Aedes mosquitoes. Using ternary phase diagrams, four selected formulations were prepared using non-ionic surfactants, methylester and water. The optimized nano-emulsion (S5) was discovered to possess submicron particle size (36.32 nm), low polydispersity index (0.348) and higher zeta potential value (-18.2 mV). After 48-hours, the CCEE and S5 demonstrated respective LC50 values of 139.44 ppm and 75.83 ppm against Ae. aegypti larvae. Higher larvicidal activity of S5 signified improved cuticular penetration of the active ingredient. Moreover, morphological deformities of treated larvae indicated possible interference with the larvae's breathing mechanism, contributing to their death. Thus, the nano-emulsion formulation of C. erythrocarpus extract has the potential to be used as a vector control agent to prevent the spread of dengue infections.

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.

Black flies play a prominent role in public health and the epidemiology of parasitic diseases of humans, domesticated and wild animals. Correct identification and a comprehensive survey are required to identify vector and pest species and thus understand their biological attributes which play a vital role in the monitoring program. DNA barcoding is an established molecular tool that provides rapid and accurate species identification. Our study strengthens the molecular database for black flies in Malaysia by adding 59 cytochrome c oxidase I sequences for 22 species, of which 14 are included for the first time. These sequences, combined with those in public databases, represent a total of 338 sequences for 52 Malaysian species, nearly 50% of which were collected from type localities. At the subgeneric level, barcode gap analysis most accurately identified species in the subgenus Nevermannia (92%), followed by Simulium s. l. (91%), and Gomphostilbia (81%). The remaining sequences were ambiguous and could not be distinguished from those of nearest neighbour species due to an overlap in genetic divergence and low genetic diversity, especially between insular species. Tree analyses indicate that certain species had incomplete lineage sorting and low mitochondrial signals. Possible cryptic species were indicated in the Simulium (Gomphostilbia) batoense and S. (G.) epistum species groups. Species delimitations were consistent with morphological identifications except in large species groups such as the S. (G.) asakoae, S. (G.) batoense, S. (G.) epistum, and S. (Simulium) melanopus groups. The use of type specimens or specimens collected from type localities (topotypes) in barcoding is strongly recommended for reference sequences to increase the reliability of the molecular database.

Mermithids are the most common parasites of black flies and are associated with host feminization and sterili-zation in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mer-mithids in the present study, with two requiring further investigation as they may represent intraspecific vari-ation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host - parasite interactions in black fly mermithids.

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.

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

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

Species of the genus Pelecitus Railliet & Henry, 1910 the most widely distributed avian filariae in Africa and South America. Zoonotic cases in humans were reported in South America. While investigating the filarial fauna of wild animals in Malaysia, we discovered an undescribed filaria from the swollen footpad of the left leg of Copsychus malabaricus (Scopoli) in Pahang, Peninsular Malaysia. Adults of both sexes have a corkscrew-shaped body. Based on comparison of their morphological characteristics (i.e. pre-oesophageal cuticular ring distinct, oesophagus divided, vulva protuberant and situated at the level of anterior half of oesophagus, spicules strongly sclerotized and left spicule with broad blade) with other Pelecitus species, they are here described as Pelecitus copsychi Uni, Mat Udin & Martin n. sp. Multi-locus sequence analyses based on seven genes (12S rDNA, cox1, 18S rDNA, 28S rDNA, MyoHC, rbp1 and hsp70) were performed to determine the phylogenetic position of the new species. The calculated p-distance between the cox1 gene sequences for P. copsychi n. sp. and Pelecitus fulicaeatrae (Diesing, 1861) was 14.1%. Intraspecific genetic variation between two individuals of the new species was 0.4%. In both the Bayesian inference and maximum-likelihood trees, P. copsychi n. sp. was positioned in the second clade of ONC5, containing three genera of the subfamily Dirofilariinae (Foleyella Seurat, 1917, Pelecitus and Loa Stiles, 1905). Immunostaining and molecular analyses remained negative for the presence of Wolbachia endosymbionts. Our findings corroborate the division of the subfamily Dirofilariinae into ONC3 with Dirofilaria Railliet & Henry, 1911 and ONC5 with Pelecitus.

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.

Biocontrol has been proposed as an effective approach in controlling mosquito population. In this study, three Odonata (dragonfly) nymphs (Neurothemis fluctuans, Orthetrum chrysis and O. sabina) were investigated for their feasibility as biocontrol agents against dengue virus vectors Aedes aegypti, Ae. albopictus and Culex quinquefasciatus. Each Odonata nymph species was separately fed each of the mosquito species IV instar larvae maintained at a fixed level by replenishing every three hours for 24 hours under controlled laboratory conditions and 12-hour light-dark period. N. fluctuans and O. sabina nymphs preferred Ae. aegypti as their prey, while O. chrysis favored Cx. quinquefasciatus. Amount of larval consumption is significantly higher during light compared to dark period (p -value < 0.05). However, overall there are no significant differences in consumption rates of the three dragonfly nymph species for the test mosquito larvae. Thus, Odonata nymphs are potential biocontrol agents against mosquito vectors of dengue disease.

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.

Although the microbiome of blood-feeding insects serves an integral role in host physiology, both beneficial and pathogenic, little is known of the microbial community of black flies. An investigation, therefore, was undertaken to identify culturable bacteria from one of Malaysia's most common black flies, Simulium tani Takaoka and Davies, using 16S rDNA sequencing, and then evaluate the isolates for antibiotic resistance and virulence genes. A total of 20 isolates representing 11 bacterial species in four genera were found. Five isolates showed β-hemolysis on Columbia agar, and virulence genes were found in three of these isolates. Some degree of resistance to six of the 12 tested antibiotics was found among the isolates. The baseline data from this study suggest rich opportunities for comparative studies exploring the diversity and roles of the microbiome of S. tani and other Southeast Asian black flies.

The phylogenetic relationships among species of the genus Eurema from Peninsular Malaysia were reconstructed using nucleotide sequences of mitochondrial CO1 (307 bp) and nuclear ribosomal 28S DNA (471 bp). A total of twenty-eight sequences generated through PCR amplification for each gene region were used to construct the Maximum Likelihood (ML) and Maximum Parsimony (MP) phylogenetic trees. The separate taxonomic grouping of the genus Eurema and the genus Gandaca, and their close association is tested here together with sequences of other pierid butterflies obtained from GenBank. All trees reveal a strongly supported monophyletic group of Eurema conspecifics and well-resolved interspecific genetic distances, indicating the usefulness of the genetic markers in local species identification. The combined phylogenetic analyses of CO1 and 28S genes strongly supports a close relationship of E. hecabe with E. blanda, while E. andersonii is recovered as a sister taxon to E. ada.

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.