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.

Protein Profiling of Aedes aegypti Treated with Streptomyces sp. KSF103 Ethyl Acetate Extract Reveals Potential Insecticidal Targets and Metabolic Pathways.

The insecticidal activity of Streptomyces sp. KSF103 ethyl acetate (EA) extract against mosquitoes is known; however, the underlying mechanism behind this activity remains elusive. In this study, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was employed to investigate changes in the protein profile of Aedes aegypti larvae and adults treated with lethal concentrations of 50 (LC50) EA extract. By comparing the treated and untreated mosquitoes, this study aimed to identify proteins or pathways that exhibit alterations, potentially serving as targets for future insecticide development. Treatment with a lethal concentration of EA extract upregulated 15 proteins in larvae, while in adults, 16 proteins were upregulated, and two proteins were downregulated. These proteins were associated with metabolism, protein regulation/degradation, energy production, cellular organization and structure, enzyme activity, and catalysis, as well as calcium ion transport and homeostasis. Notably, ATP synthase, fructose-bisphosphate aldolase (FBA), and ATP citrate synthase were significantly expressed in both groups. Gene ontology analysis indicated a focus on energy metabolic processes. Molecular docking revealed a strong interaction between dodemorph, selagine (compounds from the EA extract), and FBA, suggesting FBA as a potential protein target for insecticide development. Further studies such as Western blot and transcriptomic analyses are warranted to validate the findings.

Identification of potential vectors of Dirofilaria immitis and Brugia pahangi (Spirurida: Filariidae): First observation of infective third-stage larva of B. pahangi in Culex quinquefasciatus (Diptera: Culicidae).

Information on the mosquito species that transmit canine filariosis is scanty. Hence, an experimental study was conducted to identify the potential vectors responsible for the transmission of D. immitis Leidy and B. pahangi Buckley & Edeson. A total of 367 mosquitoes belonging to six species containing both laboratory and field strains (i.e. Aedes togoi Theobald, Aedes aegypti Linnaeus, Aedes albopictus Skuse, Culex quinquefasciatus Say, Culex vishnui Theobald and Anopheles dirus Peyton & Harrison) were used in this study. All mosquitoes were artificially fed on either D. immitis or B. pahangi microfilariae (mfs) infected blood by using the Hemotek™ membrane feeding system. Out of 367 mosquitoes, 228 (64.9%) were fully engorged. After feeding on D. immitis (20%) and B. pahangi (33%) mfs positive blood, the mortality rates for Cx. quinquefasciatus were found to be slightly lower than that of other species of mosquitoes. On the other hand, majority of An. dirus were found to be incapable to withstand the infection of mfs as the mortality rates were relatively high (D. immitis = 71.4%; B. pahangi = 100.0%). Brugia pahangi was detected in Ae. togoi and Cx. quinquefasciatus with infection rates of 50% and 25%, respectively. Aedes togoi was the only species infected with D. immitis with an infection rate of 69%. Our results showed that Ae. togoi was an excellent experimental vector for both D. immitis and B. pahangi. This study also documented the observation of B. pahangi, for the first time in the head region of Cx. quinquefasciatus under a laboratory setting.

First report of Rickettsia asembonensis in small ruminants.

Rickettsioses is a group of emerging infectious diseases in Southeast Asia caused by Gram-negative obligate intracellular bacteria in the Rickettsiae tribe. However, there is limited information regarding the vertebrate hosts of Rickettsia spp. in this region. This study aims to detect and identify Rickettsia agents present in wildlife and domesticated animals in Malaysia using Polymerase Chain Reaction (PCR) and sequencing of citrate synthase gene (gltA), followed by genotyping and phylogenetic analysis. Rickettsia sp. was detected in 2 (0.67%) of 300 wildlife and domesticated animal blood samples. The positive samples were derived from a goat (5.56% of 18) and a sheep (2.22% of 45). Both sequences demonstrated 99.64% sequence similarity to Rickettsia asembonensis, a species that is known to infect humans and macaques. This study reported for the first time the detection of R. asembonensis in sheep and goats in Malaysian farms, suggesting this species may be adapting to a wider range of animals, specifically farm animals. Therefore, this bacterium may pose a zoonotic threat to the local community particularly to the farmworkers or animal handlers. The low infection rate of this pathogen across different animals highlighted the need of continuous surveillance of emerging and reemerging pathogens among animal populations.

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.

Integration of Microscopic, Serologic and Molecular Techniques for Detection of Filarial Parasites in Dogs in Malaysia.

PURPOSE: Canine filariosis in domestic dogs caused by several species of filarids is an emerging vector-borne disease and the spread of this disease remains a global veterinary and public health concern. However, information regarding these filarids and their epidemiological patterns remains scarce in Malaysia. The present study aimed to determine the infection rate and associated risk factors of filarial parasites in dogs in Malaysia. METHODS: A total of 399 dog blood samples were collected from veterinary hospitals and animal shelters in Malaysia to determine the infection rate and associated risk factors via a combination of microscopic, serologic and molecular diagnostic techniques. RESULTS: Two species of canine filariae identified in this study were Dirofilaria immitis (6.5%) and Brugia pahangi (1.3%), and their infections were associated with cross breed, medium size and short hair (p < 0.05). CONCLUSIONS: A new pair of primers was developed to complement the recovery of the 12S rRNA gene fragment of filarial parasites. This study represents the first molecular evidence of B. pahangi in dogs in Malaysia.

A novel clade of bat-associated Bartonella detected in the bat fly Leptocyclopodia ferrari (Diptera: Nycteribiidae) parasitizing Cynopterus brachyotis (Chiroptera: Pteropodidae).

Bartonelloses are emerging infectious diseases that are common in humans and animals worldwide. Several Bartonella species associated with companion animals such as Bartonella henselae and Bartonella rochalimae are species with zoonotic implications and have become a global concern. Other Bartonella species associated with wild animals, however, remain underappreciated particularly in the developing regions of the world. To explore further on this neglected bacterial agent, Leptocyclopodia ferrari (Nycteribiidae) bat flies collected from Cynopterus brachyotis (Pteropodidae), an endemic fruit bat species in Southeast Asia, were molecularly examined for the presence of Bartonella. Both 16 S-23 S ribosomal RNA intergenic spacer region and citrate synthase gene sequences exhibited less than 95 % similarity to all previously reported Bartonella spp. Further phylogenetic analysis revealed a novel clade of this Bartonella sp. with high bootstrap support. The vectorial capacity of bat flies in transmitting this novel pathogen merits further investigation.

Sexually anomalous individuals of the black fly Simulium trangense (Diptera: Simuliidae) infected with mermithid parasites (Nematoda: Mermithidae).

Sexually anomalous individuals, typically intersexes or gynandromorphs, bear a mixture of male and female traits. Twelve sexually anomalous individuals of the black fly Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler were discovered among 49 adults reared from pupae. All 12 sexually anomalous adults were parasitized by mermithid nematodes, although five additional parasitized adults had no overt external anomalies. Sequence analysis of the 18S rRNA gene revealed that the mermithids, possibly representing a new species, are related to Mesomermis spp., with genetic distances of 5.09-6.87%. All 12 anomalous individuals had female phenotypical traits on the head, thorax, forelegs, midlegs, and claws, but male features on the left and right hind basitarsi. One individual had mixed male and female genitalia. The findings are in accord with the trend that mermithid infections are associated with sexually anomalous adult black flies.

Characterization of benzimidazole resistance in Haemonchus contortus: integration of phenotypic, genotypic and proteomic approaches.

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 ß-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.

DNA Barcoding of Simulium asakoae (Diptera: Simuliidae) From Northern Thailand.

Previous studies suggested the presence of species complex in the so-called Simulium asakoae Takaoka & Davies (Diptera: Simuliidae) in Thailand due to its high morphological variability and genetic divergence. To investigate whether the true S. asakoae is present in Thailand, we performed a detailed morphological identification of S. asakoae and compared its DNA barcodes with the morphospecies S. asakoae from Myanmar and the typical S. asakoae from Malaysia. Phylogenetic analysis revealed the Thai materials analyzed in this study were indeed genetically similar with those from Myanmar and Malaysia, though genetic distances 0-2.27% were observed. We tentatively regard this divergence as intraspecific variation, and the automatic barcode gap discovery analysis further supports them as a single species.

Genetic diversity and differentiation in the blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia.

The population genetic structures of S. vanluni, S. cheongi and S. jeffreyi were determined from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) across different states in Peninsular Malaysia. High levels of genetic diversity and genetic differentiation were observed among three species. All three species revealed an intermediate level of gene flow among the populations. Negative values of Fu's Fs and low values of Raggedness index supported the hypothesis of population expansion in S. vanluni, S. cheongi and S. jeffreyi.

An overview of rickettsiae in Southeast Asia: Vector-animal-human interface.

Rickettsioses are emerging, and re-emerging diseases caused by obligate intracellular arthropod-borne bacteria that infect humans and animals worldwide. Various rickettsiae such as Orientia, Rickettsia, Anaplasma and Ehrlichia have been circulated in companion, domesticated and wild animals through bites of infected ticks, fleas, lice or mites. This review summarizes the infections of rickettsiae, including the newly discovered regional species Rickettsia thailandii, Candidatus Rickettsia sepangensis, Candidatus Rickettsia johorensis, Candidatus Rickettsia laoensis, Candidatus Rickettsia mahosotii, Candidatus Rickettsia khammouanensis, Candidatus Anaplasma pangolinii, and other novel genotypes in vectors, humans and animals in Southeast Asia. Issues on some unidentified rickettsiae that elicit immune responses and production of antibodies that are cross-reactive with the antigens used are discussed. Knowledge gaps which required attention are also identified in this review.

Temporal variation in diversity and community structure of preimaginal blackflies (Diptera: Simuliidae) in a tropical forest reserve in Malaysia.

Blackflies (Diptera: Simuliidae) are ecologically and medically important insects but they have been understudied in Malaysia. Accordingly, a study on the temporal variation in diversity and community structure of preimaginal blackflies was conducted for the first time in Malaysia. A total of 865 preimaginal blackflies were collected in 120 samplings from five streams across three monsoon seasons from February 2018 until January 2019. Ten species were recorded and most frequently collected species were Simulium cheongi, Simulium vanluni and Simulium jeffreyi. Relatively common species were Simulium roslihashimi, Simulium tani complex and Simulium trangense. No significant changes of rainfall was observed between three monsoon seasons as well as the seasons with species and physiochemical parameters except acidity (pH) (P < 0.01). Species relative abundance was varied between seasons, as high value of Shannon index (H) was found in northeast monsoon and lowest in the southwest monsoon. Principal Component Analysis (PCA) of all stream variables revealed two PCs that accounted for 61.4% of the total variance of physicochemical characteristics. Regression analysis revealed that species richness was positively and significantly associated with wider, deeper, faster, low conductivity and larger streambed particle with less canopy cover. Forward logistic regression analysis on three frequently collected species (>20%) indicated that S. vanluni and S. jeffreyi were commonly associated with wider, deeper and fast-flowing streams with low conductivity and larger streambed particle. In contrast, S. cheongi was associated with smaller, slower and small streambed particle. This first extensive bimonthly study has uncovered the species community structure as well as the changes of stream physicochemical parameters over time although they were not greatly and significantly influenced by the monsoon seasons. Species distribution, richness and abundance, however, were highly determined by the stream width, depth and velocity, therefore, were vital in shaping diversity and community structure of preimaginal blackflies.

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%.

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.

Occurrence of zoonotic Cryptosporidium and Giardia duodenalis species/genotypes in urban rodents.

This report describes the detection of zoonotic Cryptosporidium muris, C. parvum subgenotype IIa and Giardia duodenalis genotype B in urban rodents in Malaysia. A rare occurrence of C. meleagridis was also reported suggesting a role of rodents in mechanical transmission of this pathogen. Utilization of DNA sequencing and subtyping analysis confirmed the presence of zoonotic C. parvum subtypes IIaA17G2R1 and IIaA16G3R1 for the first time in rodents.