Assessing the bioefficacy of a commercial temephos formulation (Temebate®) for controlling Aedes albopictus larvae in different land use localities in Malaysia.

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.

A novel molecular and chromosomal lineage of the anthropophilic Simulium (Simulium) rufibasis subgroup (Diptera: Simuliidae) in Taiwan.

The Simulium rufibasis subgroup is one of three subgroups of the Simulium (Simulium) tuberosum species-group; it is characterized by a pair of clustered stout hairs on the ventral surface of female abdominal segment 7. A member of the S. rufibasis subgroup in Taiwan was investigated morphologically and genetically using the universal cytochrome c oxidase subunit I (COI) barcoding gene and polytene chromosomal banding pattern. The Taiwanese material is morphologically similar to S. rosliramlii Takaoka & Chen from Vietnam and represents the second species of the S. rufibasis subgroup known from Taiwan. It also represents a novel molecular lineage that is distinct from three other primary lineages identified as S. doipuiense, S. doipuiense/S. rufibasis, and S. weji previously reported from Thailand. The mitochondrial evidence for a distinct lineage in Taiwan is supported by chromosomal analysis, which revealed unique sex chromosomes. For nomenclatural stability, we associate the name S. arisanum Shiraki with the Taiwanese entity. Originally described from females from Taiwan, S. arisanum until now has remained an enigmatic species.

Simulium (Gomphostilbia) merapiense sp. nov. (Diptera: Simuliidae) from Java, Indonesia.

Simulium (Gomphostilbia) merapiense sp. nov. is described based on females, males, pupae, and mature larvae from Yagyakarta, Java, Indonesia. This new species is placed in the Simulium epistum species-group, and is characterized by the pupal gill with eight short filaments all arising at the same level from a short stalk, somewhat enlarged basal fenestra, entirely bare pupal head and thoracic integument, and small and short larval postgenal cleft. These characters rarely are found in the subgenus. Taxonomic notes are given to separate this new species from related species of the S. epistum species-group.

Evaluation of Insect Growth Regulators Against Field-Collected Aedes aegypti and Aedes albopictus (Diptera: Culicidae) from Malaysia.

Susceptibility status of Aedes aegypti (L.) and Aedes albopictus Skuse larvae obtained from 12 states in Malaysia were evaluated against five insect growth regulators (IGRs), namely, pyriproxyfen, methoprene, diflubenzuron, cyromazine, and novaluron under laboratory conditions. Field populations of Ae. aegypti exhibited moderate resistance toward methoprene and low resistance toward pyriproxyfen, with resistance ratios of 12.7 and 1.4, respectively, but susceptibility to diflubenzuron, cyromazine, and novaluron. On the other hand, field populations of Ae. albopictus exhibited low resistance against diflubenzuron and novaluron, with resistance ratio of 2.1 and 1.0, respectively, but susceptibility to other tested IGRs. Our study concluded that the tested IGRs provide promising results and can be used to control field population of Ae. aegypti and Ae. albopictus, especially cyromazine. The use of IGR should be considered as an alternative when larvae develop resistance to conventional insecticides.

Revision of the Simulium (Simulium) melanopus species-group (Diptera: Simuliidae) in Sabah, Malaysia.

Species of the Simulium (Simulium) melanopus species-group in Sabah are taxonomically revised by examining type specimens of S. (S.) crassimanum S. (S.) laterale, and S. (S.) nigripilosum, all described from females by Edwards in 1933, and newly collected samples from the vicinity of Mt. Kinabalu. The females of these three species are redescribed, and their males and pupae are described for the first time based on adults reared from pupae. Simulium (S.) liewi Takaoka, 2007 and S. (S.) kinabaluense Smart & Clifford, 1969 are synonymized with S. (S.) crassimanum and S. (S.) laterale, respectively. Simulium (S.) cheedhangi Takaoka, Sofian-Azirun & Ya'cob, 2015 is newly recorded from Sabah. Two new related species, S. (S.) lardizabalae and S. (S.) timpohonense, are described from males reared from pupae. Keys to identify eight species of the S. melanopus species-group in Sabah are provided for females, males, pupae and mature larvae.

Co-occurrence of dual lineages within Simulium (Gomphostilbia) atratum De Meijere in the Indonesian Archipelago along Wallace's Line.

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.

First report on adulticide susceptibility status of Aedes albopictus, Culex quinquefasciatus, and Culex vishnui from a pig farm in Tanjung Sepat, Selangor, Malaysia.

The present study aims to investigate the susceptibility status of Aedes albopictus, Culex quinquefasciatus, and Cx. vishnui collected from a pig farm in Tanjung Sepat, Selangor, toward 11 insecticides representing the classes of organochlorines, carbamates, organophosphates, and pyrethroids. The results of a World Health Organization adult mosquito bioassay revealed that Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui exhibited different susceptibility toward various insecticides. Overall, pyrethroids were able to induce rapid knockdown for all test mosquito species. The pyrethroids lambdacyhalothrin and etofenprox were able to cause high mortality (> 80%) of all 3 species. The findings of the present study will benefit local authorities in selecting appropriate dosage of insecticides to be used in mosquito control in this area.

From bites to barcodes: uncovering the hidden diversity of black flies (Diptera: Simuliidae) in Vietnam.

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.

Insecticidal activities of Streptomyces sp. KSF103 ethyl acetate extract against medically important mosquitoes and non-target organisms.

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.

DNA barcoding of black flies (Diptera: Simuliidae) in Indonesia.

BACKGROUND: 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. METHODS: 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. RESULTS: The DNA barcodes successfully distinguished most morphologically distinct species (> 80% of sampled taxa). Nonetheless, high maximum intraspecific distances (3.32-13.94%) in 11 species suggested cryptic diversity. Notably, populations of the common taxa Simulium (Gomphostilbia) cheongi, S. (Gomphostilbia) sheilae, S. (Nevermannia) feuerborni and S. (Simulium) tani in the islands of Indonesia were genetically distinct from those on the Southeast Asian mainland (Malaysia and Thailand). Integrated morphological, cytogenetic and nuclear DNA studies are warranted to clarify the taxonomic status of these more complex taxa. CONCLUSIONS: The findings showed that COI barcoding is a promising taxonomic tool for Indonesian black flies. The DNA barcodes will aid in correct identification and genetic study of Indonesian black flies, which will be helpful in the control and management of potential vector species.

Two new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Peninsular Malaysia.

Two new species of black flies, Simulium (Comphostilbia) terengganuense sp. nov. and Simulium (Gomphostilbia) aziruni sp. nov. (Diptera: Simuliidae), are described on the basis of reared adult, pupal, and larval specimens collected from Peninsular Malaysia. Both species are placed in the batoense species-group within the subgenus Gomphostilbia, one of two dominant subgenera of the genus Simulium in Peninsular Malaysia as well as in the Oriental Region. Strikingly, three morphological characteristics that rarely occur in the subgenus Gomphostilbia are found in these two new species: the very narrow female frons and the mushroom-like pupal terminal hooks in S. (G.) terengganuense sp. nov. and the pupal gill composed of an inflated horn-like structure and eight slender filaments in S. (G.) aziruni sp. nov.

Nationwide distribution of Culex mosquitoes and associated habitat characteristics at residential areas in Malaysia.

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

Cat Owners' Perceptions toward Flea Infestation and Flea-borne Diseases: Impact for Public Health and Community Awareness.

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.

Phenotypic profile of Aedes albopictus (Skuse) exposed to pyrethroid-based mat vaporizers and underlying detoxification mechanisms: A statewide report in Selangor, Malaysia.

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.

Organochlorines and organophosphates susceptibility of Aedes albopictus Skuse larvae from agricultural and non-agricultural localities in Peninsular Malaysia.

Aedes albopictus larvae obtained from different types of agricultural and non-agricultural localities in Peninsular Malaysia were subjected to several larvicides at World Health Organization (WHO) recommended dosages. Upon 24 h of WHO larval bioassay using two organochlorines and six organophosphates, high resistance against dichlorodiphenyltrichloroethane (DDT), temephos, chlorpyrifos and bromophos were demonstrated among all larval populations. Aedes albopictus larvae from both paddy growing areas (92.33% mortality) and rubber estates (97.00% mortality) were moderately resistant to dieldrin while only Ae. albopictus larvae from dengue prone residential areas (89.00% mortality) showed high resistance against dieldrin. All Ae. albopictus larval populations also developed either incipient or high resistance to both malathion (33.67%-95.33% mortality) and fenitrothion (73.00%-92.67% mortality). Only Ae. albopictus larvae from fogging-free residential areas that were tolerant to fenthion (97.33% mortality), whereas Ae. albopictus larvae from dengue prone residential areas were highly resistant to the same organophosphate (88.33% mortality). Cross resistance between intraclass and interclass larvicides of organochlorines and organophosphates were also exhibited in this study. The present study provided baseline data on various susceptibility levels of Ae. albopictus larval populations from different types of agricultural and non-agricultural localities against organochlorines and organophosphates at WHO recommended dosages. Nevertheless, further susceptibility investigations are suggested using revised doses of larvicides established from the local reference strain of Ae. albopictus to prevent the underestimation or overestimation of insecticide resistance level among Ae. albopictus field strains of larvae.

Adulticide Resistance Status of Aedes albopictus (Diptera: Culicidae) in Sarawak State, Malaysia.

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.

Evolutionary and biogeographic history of the black fly Simulium wayani (Diptera: Simuliidae) on the island of Timor.

A recently described species of black fly, Simulium wayani Takaoka and Chen, from the island of Timor was chromosomally mapped to provide insights into its evolutionary and biogeographic history. The morphologically based species status of S. wayani is supported by a suite of fixed chromosomal rearrangements and unique sex chromosomes derived primarily from a large pool of polymorphisms in the S. ornatipes complex in Australia. The banding patterns of its polytene chromosomes indicate that S. wayani is closely related to a pair of homosequential cryptic species (S. norfolkense Dumbleton and S. ornatipes cytoform A2) in the S. ornatipes Skuse complex on mainland Australia; all three species uniquely share the same amplified band in their chromosomal complement. The low level of polymorphism and heterozygosity in S. wayani, relative to Australian populations of the S. ornatipes complex, suggests few colonization events from the larger land mass.

A new black fly species of Simulium (Nevermannia) (Diptera: Simuliidae) from Vietnam.

Simulium pumatense sp. nov. is described from Vietnam, and is placed in the Simulium feuerborni species-group of the subgenus Simulium (Nevermannia) Enderlein. Its morphological characteristics include the relatively smaller numbers of the following three numerical features: inner teeth of the female mandible (15-18), minute conical processes (16) on the female cibarium, and male upper-eye facets (in 15 vertical columns and 16 horizontal rows). Keys are constructed to distinguish this species from four species of the same group in Vietnam. Our molecular analysis of the DNA barcoding COI gene shows that this species is most closely related to cytoform A of the S. feuerborni complex from Thailand.

A new black fly species of Simulium (Gomphostilbia) (Diptera: Simuliidae) br />from Sulawesi, Indonesia.

Simulium (Gomphostilbia) marosense sp. nov. is described based on adults, pupae and mature larvae from Sulawesi, Indonesia. This new species is placed in the Simulium duolongum subgroup of the S. batoense species-group, representing the ninth member for the subgroup in Sulawesi. 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 1.9-3.0 times as long as the six other filaments. Taxonomic notes are provided to distinguish this new species from related species.