Morphological and genetic analyses of Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka (Diptera: Simuliidae) from the Nansei Islands, Japan: redescription and transfer from the S. ceylonicum species-group to the S. asakoae species-group

@#Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka, both from the Nansei Islands, Japan, were morphologically reexamined and genetically analysed by using the COI gene sequences. The female, male, pupa and mature larva of the two species are redescribed. Morphological reexamination shows that both species are more similar to species in the S. asakoae species-group than to those in the S. ceylonicum species-group, by having a medium-long female sensory vesicle, yellow tuft hairs (S. (G.) okinawense) or yellow tuft hairs mixed with a few to several dark hairs (S. (G.) tokarense) at the base of the radial vein in the female and male, and medium-long larval postgenal cleft. However, the body of the male ventral plate (viewed ventrally) is parallel-sided (S. (G.) okinawense) or parallelsided or slightly narrowed (S. (G.) tokarense) and not emarginated basally, differing from those of most species in the S. asakoae species-group. Our genetic analysis shows that S. (G.) tokarense is in the S. asakoae species-group, and S. (G.) okinawense formed a separate sister clade with other members of the S. asakoae species-group with high bootstrap support. From the results of morphological and genetic analysis combined, S. (G.) okinawense and S. (G.) tokarense are transferred from the S. ceylonicum species-group to the S. asakoae species-group.

Morphological redescription of Simulium takahasii (Rubtsov), the first species of the subgenus Wilhelmia Enderlein (Diptera: Simuliidae) recognized in East Asia

@#Simulium takahasii (Rubtsov), which was originally described from Japan, and recorded from Korea and China, is the first among the 19 species of the subgenus Wilhelmia Enderlein recorded from East Asia. It is striking in mating, blood-feeding and ovipositing in captivity and in experimentally transmitting Dirofilaria immitis (Leidy) and Brugia pahangi (Buckley & Edeson), and it is a severe biter of cattle and horses, rarely of humans. Nevertheless, updated information about its morphological characteristics was lacking, making comparisons with related species described from China difficult, since species of the subgenus Wilhelmia are almost indistinguishable from one another, in particular, in their female terminalia, male genitalia and most of larval features. In this study, as many morphological characteristics as possible of S. takahasii based on specimens from Japan are redescribed. New information about many features of this species including the length of the female sensory vesicle against the third palpal segment, number of male upper-eye (large) facets, arrangement of the eight pupal gill filaments, presence or absence of tiny dark setae on the dorsum of the larval abdomen and the number of rows and hooklets of the larval posterior circlet will be useful in evaluating the species status of several Wilhelmia species in China including the species regarded as S. takahasii.

First observation of Afromorgus chinensis (boheman, 1858) (Coleoptera: Trogidae) on a rabbit (Oryctolagus cuniculus L., 1758) carcass and its implications in forensic entomology

@#Beetles (Coleoptera) are known to constitute forensic evidence in medico-legal investigations as their presence can be used to date human remains in almost all decomposition stages. Many forensic studies focus on the successional colonization pattern of flies (Diptera); however, beetles have not so far been studied extensively for this aspect. A beetle of the genus Afromorgus Scholtz, 1986, A. chinensis (Boheman, 1858) (Scarabaeoidea: Trogidae), was found beneath a late decaying rabbit carcass at Paya Indah Wetland, Dengkil, Malaysia, for the first time. Both genus and species are already known to occur in Malaysia from literature.

Morphological revision of Simulium (Gomphostilbia) ogatai (Rubtsov) in the S. ceylonicum species-group (Diptera: Simuliidae) from Japan

@#Simulium (Gomphostilbia) ogatai (Rubtsov) from Japan, the only Palaearctic species in the S. ceylonicum species-group, is morphologically revised. Its female is described for the first time and its male and pupa are redescribed based on specimens from Gifu, Honshu. It is characterized in the female and male by the base of the radius with a tuft of yellow hairs, in the female by the head slightly wider than the thorax, 5.89 times as wide as the greatest width of the frons, and short sensory vesicle 0.21–0.24 times as long as the third palpal segment; in the male by the number of upper-eye (large) facets in 15 vertical columns and 16 horizontal rows, and hind basitarsus spindle-shaped, enlarged, 3.79 times as long as wide, and 0.83 and 1.00 times as wide as the greatest width of the hind tibia and femur, respectively; and in the pupa by the terminal hooks simple and cone-like, and the cocoon with an anterodorsal projection. Taxonomic notes for this species are given relevant to several related species in the Oriental Region, and its assignment to the S. ceylonicum species-group is confirmed.

Lactic acid bacteria waste infusion as a source of attraction and oviposition stimulation of gravid female Aedes albopictus mosquitoes

@#The Plasmodium knowlesi secreted protein with an altered thrombospondin repeat (PkSPATR) is an important protein that helps in the parasite’s invasion into the host cell. This protein has been regarded as one of the potential vaccine candidates against P. knowlesi infection. This study investigates the genetic diversity and natural selection of PkSPATR gene of P. knowlesi clinical isolates from Malaysia. PCR amplification of the full length PkSPATR gene was performed on 60 blood samples of infected P. knowlesi patients from Peninsular Malaysia and Malaysian Borneo. The amplified PCR products were cloned and sequenced. Sequence analysis of PkSPATR from Malaysia showed higher nucleotide diversity (CDS p: 0.01462) than previously reported Plasmodium vivax PvSPATR (p = 0.0003). PkSPATR from Peninsular Malaysia was observed to have slightly higher diversity (CDS p: 0.01307) than those from Malaysian Borneo (CDS p: 0.01212). Natural selection analysis on PkSPATR indicated significant purifying selection. Multiple amino acid sequence alignment revealed 69 polymorphic sites. The phylogenetic tree and haplotype network did not show any distinct clustering of PkSPATR. The low genetic diversity level, natural selection and absence of clustering implied functional constrains of the PkSPATR protein.

Assessment of the susceptibility status of Aedes albopictus (Diptera: Culicidae) from Interior, Sandakan and Tawau divisions of Sabah, Malaysia based on WHO diagnostic doses of larvicides

@# Susceptibility status of Aedes albopictus (Skuse) sampled from residential areas in Interior, Sandakan and Tawau divisions of Sabah, Malaysia, was evaluated based on the WHOrecommended doses of organochlorine and organophosphate larvicides. To determine susceptibility status, larval bioassays were carried out and post 24-hour mortalities based on WHO resistance classifications were adopted. The results demonstrated that Ae. albopictus larvae were resistant toward 5 out of the 8 larvicides tested. Larvae from all populations were resistant against bromophos, fenitrothion, malathion, temephos and dichlorodiphenyltrichloroethane (DDT), with mortalities ranging from 0.00 to 89.33%. Dieldrin, on the other hand, could induce 100.00% mortalities in all populations, followed by fenthion and chlorpyrifos, with mortalities ranging from 97.33 to 100.00% and 81.33 to 100.00% respectively. Despite most populations exhibiting similitude in their resistance status, larvae from Sandakan exhibited the highest resistance level whereas the lowest level was observed in Keningau. In view of the inadequacy of some larvicides in controlling Ae. albopictus in this study, integrated management such as insecticide rotation or combination of interventions is warranted.

Bioefficacy of mosquito mat vaporizers and associated metabolic detoxication mechanisms in Aedes aegypti (Linnaeus) in Selangor, Malaysia: A statewide assessment

@#This study aims to examine the efficacy of mosquito mat vaporizers on Aedes aegypti and their associated metabolic detoxication mechanisms. For this purpose, Aedes aegypti (Linnaeus) was collected from nine districts in Selangor, Malaysia and tested with mosquito vaporizing mat bioassays. The same populations were also subjected to biochemical assays to investigate activities of detoxifying enzymes, namely non-specific esterase (EST), glutathione-S-transferase (GST) and mixed function oxidase (MFO). The efficacy of Ae. aegypti on the active ingredients tested in decreasing order were d- allethrin > dimefluthrin > prallethrin with PBO > prallethrin. The results further indicated significant enhancement mean levels of EST, GST and MFO in pyrethroid-resistant populations. The mortality rate of Ae. aegypti in response to pyrethroid active ingredients was associated with MFO activity, suggesting it is an important detoxification enzyme for the populations tested. In view of the presence of resistance against household insecticide products, pyrethroid efficacy on Ae. aegypti populations needs to be monitored closely to ensure the implementation of an effective vector control program in Malaysia.

Two new black fly species of the Simulium (Simulium) rufibasis subgroup (Diptera: Simuliidae) from Taiwan

@#Simulium (Simulium) rufibasis Brunetti originally described from India was once considered a geographic generalist widely distributed in the Oriental Region. In this study, the species previously regarded as S. (S.) rufibasis in Taiwan was morphologically re-evaluated and found to be distinguished in the male and pupa from true S. (S.) rufibasis from India. This new species is described as S. (S.) hehuanense sp. nov. based on a female, a male and their pupal exuviae in Taiwan. This new species is placed in the S. rufibasis subgroup of the S. tuberosum species-group, and is similar to S. (S.) yamatoense Takaoka, Adler & Fukuda from Japan and Korea, but it is barely distinguished by the slenderer forebasitarsi of the female and male. Another new, related species, S. (S.) xiulinense sp. nov., is described based on a male and its pupal exuviae. This new species is distinguished from S. (S.) hehuanense sp. nov. by the number of male upper-eye (large) facets in 20 or 21 vertical columns and 22 horizontal rows (19 vertical columns and 19 horizontal rows in the latter species), and presence of a pair of dorsolateral shiny spots on male abdominal segment 5 (absence in the latter species). Both new species inhabit streams at high elevations (ca, 2,600 m). They differ from S. (S.) sp. (probably S. (S.) arisanum Shiraki) from Taiwan, of the same subgroup, which breeds in streams at low elevations (433–685 m), by the pupal head and thorax covered with tubercles (bare in the latter species).

Serological survey of canine vector-borne diseases in two animal shelters in central Peninsular Malaysia

@#Canine vector-borne diseases (CVBDs) are increasingly becoming a cause for global concern because of their high morbidity and mortality rates in dogs. However, information on their occurrence in Malaysia is still scanty. In this study, a total of 103 dog blood samples were collected from two animal shelters in central Peninsular Malaysia and tested for the antibodies against Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi, and the antigen of Dirofilaria immitis. Of the 103 tested dogs, 44.7% (46) were found to be seropositive for Ehrlichia spp., 30.1% (31) for Anaplasma spp. and 13.6% (14) for D. immitis. Co-infections of Anaplasma spp. + Ehrlichia spp. (18.5%, 19) were most prevalent, followed by Anaplasma spp. + D. immitis (1.9%; two) and D. immitis + Ehrlichia spp. (1.0%; one). Furthermore, three dogs (2.9%) were also found to have triple infection, testing seropositive for Ehrlichia spp., Anaplasma spp. and D. immitis. The dogs which were found to be seropositive with at least one pathogen were 66.7% (32/51) at shelter A, and 55.8% (29/52) at shelter B. Serological evidence showed that the exposure of major vector-borne diseases in dogs in shelters was relatively high in the surveyed areas. Routine detection and control of vector-borne diseases are of paramount importance for reducing the risk of CVBDs transmission in dogs and humans.

Bacterial profiling of head lice isolated from the Orang Asli: A first report in Malaysia

@#This study was carried out to determine from bacterial profiling to the bacterial profiles of head lice among the Orang Asli communities. The head lice were collected from Orang Asli community volunteers. The surface sterilized head lice pools were subjected to genomic DNA extraction while next generation sequencing of the 16S rRNA gene was performed using the Illumina MiSeq platform. Six female and three male head lice identified as Pediculus humanus capitis were collected. A total of 111 368 number of NGS sequencing reads were recorded while another 223 bacterial taxa sequences were obtained. Symbiotic bacteria showed the highest number of reads, with Arsenophonus and Rhodococcus sequences being the most abundant genera in the female and male samples, respectively. The female head lice contained a more distinct microbial diversity. Amongst the pathogenic bacterial species sequences noted were the methicillin-resistant Staphylococcus aureus, Streptobacillus moniliformis, Haemophilus influenzae, Bordetella pertussis and Acinetobacter baumannii. The 16S rRNA genome sequencing revealed a number of rare and pathogenic bacterial species within the head lice of the Orang Asli. The socio-economic practices of the community which involved forest foraging and hunting, and their poor living conditions potentially facilitated the transmission of zoonotic bacterial pathogens, including those found within the head lice. Hence, there is the possibility that the head lice could serve as vectors for the transmission of pathogenic bacteria. This study highlighted the diverse microbial community found within the head lice’s gut of the Orang Asli, with the detection of multiple rare and pathogenic bacteria capable of causing severe infections.

Descriptions of the female, male and mature larva of Simulium contractum Takaoka (Diptera: Simuliidae) from Sulawesi, Indonesia

@#Simulium (Simulium) contractum Takaoka from Sulawesi, Indonesia was known only as the pupa. Its female, male and mature larva are described for the first time. The tentative assignment of this species in the Simulium dumogaense species-group is confirmed by the adult characters including the female and male genitalia. The female and male of this species are similar to those of Simulium (Simulium) tumpaense Takaoka & Roberts but are distinguished by the yellowish femora.

Risk factors associated with flea infestation on cats

@#Fleas are the common cause of skin disorders in cats. They are well-known for transmitting various pathogens to both cats and humans. Accordingly, this study was conducted to gain insights on the risk factors associated with flea infestation on cats. Flea combing conducted on 426 cats from four distinct regions in Peninsular Malaysia revealed a relatively high rate of flea infestation on 306 cats (71.83%). A total of 651 fleas were collected, all of them were identified as Ctenocephalides felis with the total intensity of 2.13 and abundance of 1.53. The sex ratio of fleas was female-biased at 2.5:1 (♀=464, ♂=187). Statistical analysis of the data revealed that flea infestation was significantly (P<0.05) associated with several risk factors including region, age, weight, status (stray, sheltered, pet), body condition, and hair length. Higher flea prevalence was also observed in female cats (77.99%), big-sized cats (91.76%), stray cats (84.94%), cats with clean body condition (73.35%), and cats with long hairs (78.38%) as compared to their contemporaries within the same comparison variables. The high infestation of fleas in this study is indicative of cats as a flea reservoir particularly C. felis. Thus the findings of this study and the knowledge gained on the risk factors can be used to develop and improve control measures and management of flea infestations.

Preliminary dengue vector surveillance in the Sunda Islands, Indonesia: Interchange of breeding habitat preferences of Aedes aegypti and Aedes albopictus

@#Ovitrap surveillance was conducted to determine the infestation patterns of dengue vectors in fourteen study sites across eight provinces located in the Sunda Islands, Indonesia. High ovitrap indices up to 70% and 90% were obtained from indoor and outdoor areas, respectively. Mean numbers of Ae. aegypti and Ae. albopictus larvae ranged from 0.13 to 14.50 and 0.10 to 18.60, respectively. Mixed infestation (<10%) and interchange of breeding habitat preferences of Ae. albopictus and Ae. aegypti were also observed in the present study.

A new species of the Simulium (Simulium) crocinum species-group (Diptera: Simuliidae) from Vietnam

@#A new black fly species, Simulium yukawai, is described on the basis of one female and its pupal exuviae from Vietnam. This new species is placed in the S. crocinum species-group of Simulium (Simulium) (Diptera: Simuliidae). It is characterized in the female by the paraproct covered with 48–50 distinct hairs on its ventral and lateral surfaces, and in the pupa by the frons and most of the thorax bare, gill with six dark filaments in three pairs closely arising from the base, of which the two outer filaments of the dorsal and middle pairs are much longer than other filaments, abdomen with distinct spine-combs on the dorsal surface of segments 7–9, and cocoon wall-pocket shaped, with a large anterolateral window on each side. Taxonomic notes are given to separate this new species from related species. This species is the fourth member of the S. crocinum species-group known from Vietnam.

Observations of scrotal mass, liver mass, haemolytic jaundice, and central vestibular disorder in Brugia pahangi-infected dogs

@#Brugia pahangi is known to infect humans and dogs. Its associated symptoms and complications, however, have not been fully understood in dogs. Herein, we reported the observations of B. pahangi infections in dogs with scrotal mass, liver mass, haemolytic jaundice, and central vestibular disorder.

Three new species of black flies (Diptera: Simuliidae) from the Lesser Sunda Archipelago, Indonesia

@#Simulium (Gomphostilbia) dhangi sp. nov., S. (G.) sumbaense sp. nov. and S. (Nevermannia) wayani sp. nov. are described from the Lesser Sunda Archipelago, Indonesia. Simulium (G.) sumbaense sp. nov. is placed in the S. varicorne species-group and is characterized by the pupal gill with eight filaments arranged as (1+1+1+1+2)+2 from dorsal to ventral, while S. (G.) dhangi sp. nov., unplaced to group, is characterized by the pupal gill composed of two inflated trunks and four slender filaments all arising basally, and the short larval antenna as long as the stem of the labral fan. Simulium (N.) wayani sp. nov. belongs to the S. ruficorne species-group and is characterized by the female spermatheca with an unsclerotized neck, and pupal gill with four inflated filaments. The number of species of black flies from the archipelago increases from 19 to 22.

Nematode control failure due to anthelmintic resistance in a sheep farm in Malaysia: First identification of the F200Y mutation in the isotype 1 β-tubulin gene

@#This paper reports total nematode anthelmintic resistance towards albendazole, fenbendazole, levamisole and ivermectin in a commercial sheep farm located in Terengganu, Malaysia. Faecal Egg Count Reduction Test (FECRT) was conducted on 25 sheep, where five sheep in each group were treated with the respective four anthelmintics based on live bodyweight. The balance of five sheep placed in the control group were not treated with any anthelmintics. At day 13 post-treatment, faecal egg count was conducted and nematode worm egg count reduction percentage was calculated to determine the resistance status towards the respective anthelmintics tested. Results showed that nematodes were resistant to all the anthelmintics tested, namely albendazole, fenbendazole, levamisole and ivermectin with reduction percentage of 87%, 46%, 94% and 68%, respectively. Subsequently, the third stage larvae of Haemonchus contortus and Trichostrongylus colubriformis recovered from post-treatment faecal cultures were subjected to allele-specific polymerase chain reaction (AS-PCR) assay to determine the presence of the benzimidazole resistance gene. This study reports the occurrence of the classical F200Y mutation in the isotype 1 β- tubulin gene, for the first time in Malaysia.

Distribution and abundance of black flies (Diptera: Simuliidae) in recreation parks in Selangor State, Peninsular Malaysia

A preliminary survey of larvae and pupae of black flies (Diptera: Simuliidae) was conducted in three recreation parks [Templer Recreation Park (TRP), Congkak River Forest Reserve (CRFR) and Ampang Forest Reserve (AFR)] located in Selangor State, which is located 18 to 35 km from Kuala Lumpur city center, Malaysia. This study was initiated to determine the distribution and abundance of filarial vectors, Simulium spp. A total of 12 species of black flies belonging to three subgenera (Gomphostilbia, Simulium and Nevermannia) were collected. Simulium (Simulium) nobile was incriminated as the most dominant species in all recreation parks. This study is a first report on the distribution and abundance of black flies obtained from recreation parks in Malaysia.

First molecular genotyping of A302S mutation in the gamma aminobutyric acid (GABA) receptor in Aedes albopictus from Malaysia

Given the lack of molecular evidence in altered target-site insecticide resistance mechanism in Aedes albopictus (Skuse) worldwide, the present study aims to detect the presence of A302S mutation in the gene encoding the gamma aminobutyric acid receptor resistant to dieldrin (Rdl) in Ae. albopictus for the first time from its native range of South East Asia, namely Malaysia. World Health Organization (WHO) adult susceptibility bioassay indicated a relatively low level of dieldrin resistance (two-fold) in Ae. albopictus from Petaling Jaya, Selangor. However, PCR-RFLP and direct sequencing methods revealed the presence of the A302S mutation with the predomination of heterozygous genotype (40 out of 82 individuals), followed by the resistant genotype with 11 individuals. This study represents the first fieldevolved instance of A302S mutation in Malaysian insect species.