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.

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.

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.

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.

Occurrence of five distinct clades of mermithid nematodes (Nematoda: Mermithidae) infecting black fly larvae (Diptera: Simuliidae) in tropical streams in Malaysia.

Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization 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 mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation 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.

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.

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.

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.

Culturable bacteria in adults of a Southeast Asian black fly, Simulium tani (Diptera:Simuliidae).

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 ß-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.

Flea-borne pathogens in the cat flea Ctenocephalides felis and their association with mtDNA diversity of the flea host.

Flea-borne pathogens were screened from 100 individual cat fleas using a PCR approach, of which 38 % were infected with at least one bacterium. Overall, 28 % of the flea samples were positive for Bartonella as inferred from ITS DNA region. Of these, 25 % (7/28) were identified as Bartonella clarridgeiae, 42.9 % (12/28) as Bartonella henselae consisted of two different strains, and 32.1 % (9/28) as Bartonella koehlerae, which was detected for the first time in Malaysia. Sequencing of gltA amplicons detected Rickettsia DNA in 14 % of cat flea samples, all of them identified as Rickettsia asembonensis (100 %). None of the flea samples were positive for Mycoplasma DNA in 16S rRNA gene detection. Four fleas were co-infected with Bartonella and Rickettsia DNAs. Statistical analyses reveal no significant association between bacterial infection and mtDNA diversity of the cat flea. Nevertheless, in all types of pathogen infections, infected populations demonstrated lower nucleotide and haplotype diversities compared to uninfected populations. Moreover, lower haplotype numbers were observed in infected 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.

New insights into the haplotype diversity of the cosmopolitan cat flea Ctenocephalides felis (Siphonaptera: Pulicidae).

The present study investigated the genetic profile of the cosmopolitan cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) from Malaysia and the reference data available in the National Center for Biotechnology Information (NCBI) GenBank. A set of sequences of 100 Malaysian samples aligned as 550 characters of the cytochrome c oxidase subunit I (cox1) and 706 characters of the II (cox2) genes revealed ten haplotypes (A1-A10) and eight haplotypes (B1-B8), respectively. The concatenated sequences of cox1 and cox2 genes with a total of 1256 characters revealed 15 haplotypes (AB1-AB15). Analyses indicated that haplotype AB1 was the most frequent and the most widespread haplotype in Malaysia. Overall haplotype and nucleotide diversities of the concatenated sequences were 0.52909 and 0.00424, respectively, with moderate genetic differentiation (FST = 0.17522) and high gene flow (Nm = 1.18). The western population presented the highest genetic diversity (Hd = 0.78333, Pi = 0.01269, Nh = 9), whereas the southern population demonstrated the lowest diversity (Hd = 0.15667, Pi = 0.00019, Nh = 3). The concatenated sequences showed genetic distances ranged from 0.08 % to 4.39 %. There were three aberrant haplotypes in cox2 sequences that highly divergent, suggesting the presence of cryptic species or occurrence of introgression. In the global point of view, the aligned sequences of C. felis revealed 65 haplotypes (AA1-AA65) by the cox1 gene (n = 586), and 27 haplotypes (BB1-BB27) by the cox2 gene (n = 204). Mapping of the haplotype network showed that Malaysian C. felis possesses seven unique haplotypes in both genes with the common haplotypes demonstrated genetic affinity with C. felis from Southeast Asia for cox1 and South America for cox2. The topologies of cox1 and cox2 phylogenetic trees were concordant with relevant grouping pattern of haplotypes in the network but revealed two major lineages by which Malaysian haplotypes were closely related with haplotypes from the tropical region.

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.

V1016G Point Mutation: The Key Mutation in the Voltage-Gated Sodium Channel (Vgsc) Gene of Pyrethroid-Resistant Aedes aegypti (Diptera: Culicidae) in Indonesia.

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.

Description of the female of Simulium (Gomphostilbia) aziruni (Diptera: Simuliidae) and its genetic relationships with members of the Simulium gombakense species-group.

Simulium (Gomphostilbia) aziruni Takaoka, Hashim & Chen was described initially based only on a pupa and a mature larva collected from Peninsular Malaysia. Herein, we describe the morphological characters of the female of S. aziruni for the first time. It resembles those of the other members of the Simulium gombakense species-group by the genital fork with a distinct projection directed medioposteriorly from each arm and claw with a large basal tooth. Cytochrome c oxidase I (COI) barcoding analysis indicates that S. aziruni is the sister species of S. maleewongae, but both are distantly separated by a genetic distance of 4.9%.

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.

Pyrethroids Use: Threats on Metabolic-Mediated Resistance Mechanisms in the Primary Dengue Vector Aedes aegypti (Diptera: Culicidae).

The emergence of pyrethroid resistance in Aedes aegypti (L.) has limited the success of vector control. Early detection of resistance could assist authorities in deciding well-suited control strategies to minimize operational failures of Ae. aegypti control. Herein, biochemical analysis was performed to investigate the mechanisms involved in pyrethroid resistance in nine populations of Indonesian Ae. aegypti. Enzymes of adult Ae. aegypti such as esterases (ESTs), glutathione-S-transferases (GSTs), and mixed-function oxidases (MFOs) were characterized. Elevated MFO activity was correlated with resistance phenotype, indicating the role of this enzyme in contributing to pyrethroid resistance. No significant correlations were shown between pyrethroid resistance phenotype and α-ESTs, suggesting that marginally exceeded enzyme levels relative to the reference strain in some pyrethroid-susceptible populations were causative factor for insecticide resistance in other groups of insecticides. However, significant correlation was demonstrated between ß-ESTs and pyrethroid resistance phenotype. The lowest enzyme levels in GSTs indicated that this enzyme was not predominant in causing pyrethroid resistance, despite the presence of significant correlations. Because metabolic detoxification fails to comprehensively explain the pyrethroid resistance in some Indonesian Ae. aegypti, additional mechanisms such as altered target sites in voltage-gated sodium channel may also contribute to the high pyrethroid resistance in Ae. aegypti.

A New Black Fly Species of the Simulium (Gomphostilbia) duolongum Subgroup (Diptera: Simuliidae) From Vietnam, and Molecular Comparisons With Related Species Using the COI Barcoding Gene.

Simulium (Gomphostilbia) yvonneae sp. nov. is described based on adults, pupae, and mature larvae from Vietnam. This new species belongs to the Simulium duolongum subgroup in the S. batoense species-group of the subgenus Gomphostilbia Enderlein. It is distinguished by having a relatively larger number of male upper-eye facets in 16 vertical columns and 16 horizontal rows and a pupal gill with eight filaments arranged as 3+(1+2)+2 from dorsal to ventral, of which two filaments of the ventral pair are 1.8 times as long as the longest filament of the middle and dorsal triplets. Morphological comparisons are made to distinguish this new species from all 22 related species. The genetic distinctiveness of this new species in the S. duolongum subgroup is also presented based on the DNA barcoding COI gene.

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.