A new species of the Anopheles barbirostris complex (Diptera: Culicidae) from Bhutan.

Anopheles sarpangensis Somboon, Namgay & Harbach is described as a new species of the Barbirostris Complex of the subgenus Anopheles Meigen. The adults differ from other species of the complex in having wings with a prehumeral pale spot on the costa, foretarsomere 1 with an apical pale band more than half as long as foretarsomere 5, midtarsomeres 1 and 2, and occasionally 3 and 4, with incomplete apical pale bands, and abdominal sterna with many white scales as in An. campestris Reid, An. saeungae Taai & Harbach, and An. wejchoochotei Taai & Harbach. The pupa and larva are indistinguishable from the pupae and larvae of other species of the complex. Phylogenetic analysis revealed that COI sequences form a clade separate from clades consisting of the sequences of other species of the complex. The immature stages were commonly found in fishery ponds in foothills at low to moderately high altitudes in the Sarpang District of Bhutan.

The female and pupa of Uranotaenia (Pseudoficalbia) bhutanensis (Diptera: Culicidae).

Uranotaenia bhutanensis Somboon, Namgay & Harbach was recently described as a new species of the Bimaculata Series of the subgenus Pseudoficalbia Theobald based on larvae collected from a log hole at high altitude in Bhutan. The adult female and pupa are described in this article based on the subsequent discovery of 2 paratype females with associated pupal exuviae.

Antennal morphology and sensilla ultrastructure of the malaria vectors, Anopheles maculatus and An. sawadwongporni (Diptera: Culicidae).

Mosquitoes rely mainly on the olfactory system to track hosts. Sensilla contain olfactory neuron receptors that perceive different kinds of odorants and transfer crucial information regarding the surrounding environment. Anopheles maculatus and An. sawadwongporni, members of the Maculatus Group, are regarded as vectors of malaria in Thailand. The fine structure of their sensilla has yet to be identified. Herein, scanning electron microscopy is used to examine the sensilla located on the antennae of adults An. maculatus and An. sawadwongporni, collected from the Thai-Myanmar border. Four major types of antennal sensilla are discovered in both species: chaetica, coeloconica, basiconica (grooved pegs) and trichodea. The antennae of female An. maculatus have longer lengths (µm, mean ± SE) in the long sharp-tipped trichodea (40.62 ± 0.35 > 38.20 ± 0.36), blunt-tipped trichodea (20.39 ± 0.62 > 18.62 ± 0.35), and basiconica (7.84 ± 0.15 > 7.41 ± 0.12) than those of An. sawadwongporni. Using light microscopy, it is found that the mean numbers of large sensilla coeloconica (lco) on both flagella in An. maculatus (left: 32.97 ± 0.48; right: 33.27 ± 0.65) are also greater when compared to An. sawadwongporni (left: 30.40 ± 0.62; right: 29.97 ± 0.49). The mean counts of lco located on flagellomeres 1-3, 6, and 9 in An. maculatus are significantly higher than those of An. sawadwongporni. The data in this study indicate that two closely related Anopheles species exhibit similar morphology of sensilla types, but show variations in length, and likewise in the number of large sensilla coeloconica between them, suggesting they might be causative factors that affect their behaviors driven by the sense of smell.

A study of Anopheles monticola of the Baileyi Complex, Culex sasai, and Lutzia vorax (Diptera: Culicidae) in Yunnan Province, China.

This article deals with the morphological and molecular assessments of 3 species of mosquitoes collected in Yunnan Province, China, i.e Anopheles monticola Somboon, Namgay & Harbach, the only species of the Baileyi Complex identified, Culex sasai Kano, Nitahara & Awaya, and Lutzia vorax Edwards. Interestingly, some adult specimens of An. monticola exhibited a variant form in having pale fringe spots, as found in An. simlensis James, another species of the Baileyi Complex. Culex sasai was recorded for the first time in Yunnan and mainland China. The prevalence of Lt. vorax was confirmed, and previous records of Lt. halifaxii in China are regarded as misidentifications of Lt. vorax.

A multiplex PCR assay for the identification of 5 sibling species of the Lindesayi Complex of the genus Anopheles (Diptera: Culicidae) in Bhutan.

Five sibling species of the Lindesayi Complex of the genus Anopheles have been discovered in Bhutan: An. druki Somboon, Namgay & Harbach, An. himalayensis Somboon, Namgay & Harbach, An. lindesayi Giles, An. lindesayi species B, and An. thimphuensis Somboon, Namgay & Harbach. The species are morphologically similar in the adult and/or immature stages. This study aimed to develop a multiplex PCR assay to identify the 5 species. Allele-specific primers were designed for specific nucleotide segments of ITS2 sequences previously reported for each species. The assay provided products of 183 bp for An. druki, 338 bp for An. himalayensis, 126 bp for An. lindesayi, 290 bp for An. lindesayi species B, and 370 bp for An. thimphuensis. The use of the assay produced consistent results. The assay is relatively inexpensive, enables the rapid identification of a large number of specimens, and will foster further studies of the Lindesayi Complex.

Aedes yunnanensis (Diptera: Culicidae): transferred from the subgenus Hulecoeteomyia to Orohylomyia subg. nov., with descriptions of the species and the new subgenus.

Aedes yunnanensis (Gaschen), currently classified in the subgenus Hulecoeteomyia Theobald, is transferred to a new monobasic subgenus, Orohylomyia Somboon & Harbach, subg. nov., based on morphological assessment of adults, male and female genitalia, larvae, and pupae, and phylogenetic analysis. The new subgenus and its type species are described in detail.

Amphotericin B resistance correlates with increased fitness in vitro and in vivo in Leishmania (Mundinia) martiniquensis.

Amphotericin B (AmpB) deoxycholate is the available first-line drug used to treat visceral leishmaniasis caused by Leishmania (Mundinia) martiniquensis, however, some cases of AmpB treatment failure have been reported in Thailand. Resistance to drugs is known to affect parasite fitness with a potential impact on parasite transmission but still little is known about the effect of resistance to drugs on L. martiniquensis. Here we aimed to gain insight into the fitness changes occurring after treatment failure or in vitro-induced resistance to AmpB. L. martiniquensis parasites isolated from a patient before (LSCM1) and after relapse (LSCM1-6) were compared for in vitro and in vivo fitness changes together with an in vitro induced AmpB-resistant parasite generated from LSCM1 parasites (AmpBRP2i). Results revealed increased metacyclogenesis of the AmpBPR2i and LSCM1-6 strains (AmpB-resistant strains) compared to the LSCM1 strain and increased fitness with respect to growth and infectivity. The LSCM1-6 and AmpBRP2i strains were present in mice for longer periods compared to the LSCM1 strain, but no clinical signs of the disease were observed. These results suggest that the AmpB-resistant parasites could be more efficiently transmitted to humans and maintained in asymptomatic hosts longer than the susceptible strain. The asymptomatic hosts therefore may represent "reservoirs" for the resistant parasites enhancing transmission. The results in this study advocate an urgent need to search and monitor for AmpB-resistant L. martiniquensis in patients with relapsing leishmaniasis and in asymptomatic patients, especially, in HIV/Leishmania coinfected patients.

Review of the Mimeticus Subgroup of Culex (Culex) (Diptera: Culicidae), With an Assessment of the Specific Status of Three Nominal Species Described From India.

The Mimeticus Subgroup of the subgenus Culex, genus Culex Linnaeus, is reviewed and revised. Mitochondrial COI sequences of species of the subgroup available in GenBank were analysed to verify the identity of voucher specimens. The specific status of three species described from India, Cx. gaugleri Suman, Cx. katezari Karlekar, Andrew & Deshpande, and Cx. kodaikanalensis Suman, Veer & Chandra, was examined and found to be unsupportable. Culex gaugleri and Cx. kodaikanalensis are synonyms of Cx. jacksoni based on the similarity of DNA sequences and dubious evidence of morphological differentiation. Culex katezari is recognized as a nomen dubium because the morphological description and molecular information provided by the authors are insufficient for its identification. A number of specimens registered in GenBank were found to be misidentified. In addition, available data indicate that the Mimeticus Subgroup may include at least three unrecognized species, referred to informally as Cx. mimeticus form B, Cx. mimulus form X, and Cx. mimulus form Y.

Insecticide Susceptibility Status of Anopheles and Aedes Mosquitoes in Malaria and Dengue Endemic Areas, Thai-Myanmar Border.

The occurrence and spread of insecticide resistance has had a negative effect on the efficacy of insecticide-based tools and is distributed worldwide, including the Greater Mekong Subregion (GMS). This study aims to determine the insecticide susceptibility of malaria and dengue vectors in malaria and dengue hotspots on the Thai-Myanmar border. Mosquito larvae and pupae were obtained from water sources from December 2019 to April 2020 in Tha Song Yang District, Tak province, western Thailand. WHO bioassay susceptibility tests were conducted with three classes of insecticides to evaluate the knockdown and mortality rates of Anopheles and Aedes aegypti female adults. V1016G and F1534C kdr mutations in the voltage-gated sodium channel of Ae. aegypti were identified using a multiplex PCR. A total of 5764 female mosquitoes were bioassayed in this study, including Anopheles spp. (92.63%) and F1 Ae. aegypti (7.37%). After 24 h of observation, An. minimus s.l. (n = 3885) and An. maculatus s.l. (n = 1138) in Suan Oi (SO) and Tala Oka (TO) were susceptible to pyrethroids, organophosphates and carbamates (except bendiocarb) with 98-100% mortality (MR). Resistance to bendiocarb was detected with a mortality rate of 88.80%, 88.77%, and 89.92% for An. minimus s.l. (n = 125, 125) and An. maculatus s.l. (n = 66), respectively. The first generation of Ae. aegypti adult females were suspected of resistance to deltamethrin (n = 225, MR = 96.89%) and confirmed resistance to permethrin (n = 200, MR = 20.00%). V1016G and F1534C mutations were detected in three genotypes, heterozygote and homozygote forms. The correlation between the kdr alleles and deltamethrin resistance was significant. In conclusion, bendiocarb resistance was found in primary malaria vectors, An. minimus s.l. and An. maculatus s.l. F1 Ae. aegypti population was pyrethroids-resistant, associated with kdr alleles. Therefore, molecular analysis should be conducted to gain insights into the mechanism of insecticide resistance. Routine malaria vector control programmes, such as fogging implementation in hotspot villages to induce Aedes resistance available in peri-domestic sites, are questionable.

Stimulation of metacyclogenesis in Leishmania (Mundinia) orientalis for mass production of metacyclic promastigotes.

Leishmania (Mundinia) orientalis is a human pathogen causing leishmaniasis and studies on the properties of metacyclic promastigotes, the parasite's infective stage, are required for a better understanding of its transmission and infection. However, information on cultivation for mass production of L. orientalis metacyclic promastigotes and factors that stimulate their metacyclogenesis is limited. Therefore, the objective of this study was to develop a suitable methodology for generating promastigote cultures containing a high proportion and number of L. orientalis metacyclic promastigotes. Various media, i.e., Schneider's insect medium, Medium 199 and Grace's insect medium, supplemented with various quantities of dithiothreitol, Basal Medium Eagle vitamins, pooled human urine, and fetal bovine serum, were optimized for metacyclogenesis. The results revealed that the optimum culture medium and conditions of those tested were Schneider's insect medium supplemented with 100 µM dithiothreitol, 1% (v/v) Basal Medium Eagle vitamins, 2% (v/v) pooled human urine, and 10% (v/v) fetal bovine serum, pH 5.0 at 26°C. We also demonstrated that L. orientalis metacyclic promastigotes could be purified and enriched by negative selection using peanut lectin. Under these culture conditions, the highest yield of metacyclic promastigotes was obtained with a significantly higher percentage of parasite survival, resistance to complement-mediated lysis, and infection index in THP-1 macrophage cells compared to parasites cultured without media supplements at neutral pH. This is the first report providing a reliable method for mass production of L. orientalis metacyclic promastigotes for in vivo infections and other experimental studies of this emerging parasite in the future.

Novel real-time PCR assay detects widespread distribution of knock down resistance (kdr) mutations associated with pyrethroid resistance in the mosquito, Culex quinquefasciatus, in Thailand.

Susceptibility to pyrethroids in the mosquito Culex quinquefasciatus, the major vector of lymphatic filariasis, is being seriously threatened worldwide. Knockdown resistance (kdr), caused by mutations in the voltage gated sodium channel (VGSC) gene, particularly the L1014F mutation, is an important resistance mechanism. Our aim was to develop a real-time PCR with melt curve analysis to evaluate the distribution of the L1014F mutation in Cx. quinquefasciatus throughout Thailand and to determine the polymorphism pattern of a VGSC gene fragment spanning the L1014F mutation. A total of 3760 females from 18 localities across five regions of Thailand were bio-assayed by exposure to 0.05% deltamethrin WHO papers, showing mortality rates ranging from 2.4% to 83.0%. Genotyping of 753 dead and surviving mosquitoes using our novel real-time PCR assay with melt curve analysis and tetra-primer allele-specific PCR revealed the mutant F1014 allele is closely associated with the deltamethrin resistance phenotype. The L1014F mutation was found at high frequency throughout Thailand, particularly in the North. However, some survivors were homozygous for wild type L1014 allele, which were further sequenced for the IIP-IIS6 region of VGSC gene. The haplotype network of phenotypically characterized individuals indicated the presence of other possible kdr alleles/resistance mechanisms at play including two novel mutations, V978E and D992E. The finding of new putative kdr alleles and widespread distribution of the F1014 allele emphasizes the significant role of kdr mutations in pyrethroid resistance in Thai Cx. quinquefasciatus populations. Monitoring kdr variations and phenotypic resistance is critical for managing resistance in Cx. quinquefasciatus.

A Simple CO2 Generating System Incorporated with CDC Light Trap for Sampling Mosquito Vectors.

Traps for capturing mosquitoes and other blood-feeding arthropods are often baited with carbon dioxide (CO2) as an attractant. Dry ice is popularly used as a CO2 source due to its high efficiency and ease of use. However, dry ice can be difficult to obtain in many rural and remote areas. The objective of this study was to develop a simple and inexpensive method that could continuously generate CO2 overnight (about 10 h) while being used with CDC light traps for sampling adult mosquitoes. In principle, CO2 was produced from the reaction between hydrochloric acid (HCl) (12% w/w) and limestone powder (mainly composed of calcium carbonate, CaCO3). In laboratory experiments, an average of 256 mL of CO2 was produced from 1 g of limestone. For continuous production of CO2, an intravenous drip infusion set, as commonly used in hospitals, was modified for dripping the acid solution (1 L in a normal saline bag) onto limestone powder (800 g in a 1.5 L bottle) at a flow rate of 30 drops/min (about 1.6 mL/min). With this procedure, an average of 55 mL of CO2 per min was obtained (approximately equivalent to the CO2 exhaled by two chickens). The performance of this CO2 generating system incorporated with CDC light traps for sampling mosquitoes was evaluated in three rural villages of Sanpatong District, Chiang Mai Province, Thailand. Three trap sets were used, i.e., Set I, light trap alone; Set II, light trap with dry ice (1 kg); and Set III, light trap with limestone and acid. In each village, mosquitoes were collected at three fixed sites, each with one of the three trap sets. They were rotated daily for three rounds (9 nights per village and 27 nights in total). A total of 1620 mosquitoes (97.7% being females) consisting of Aedes, Anopheles, Armigeres, Coquilletidia, Culex and Mansonia were captured across three different sampling sets from all villages. The predominant species collected were Culex vishnui (n = 760, 46.91%), Cx. bitaeniorhynchus (n = 504, 31.11%) and Cx. tritaeniorhynchus (n = 157, 9.69%). Light traps alone (Set I) collected very low numbers of mosquitoes (n = 12) and species (6 spp.), whereas light traps with dry ice (Set II) collected the highest numbers of mosquitoes (n = 1341) and species (14 spp.). Although the light trap with limestone and acid (Set III) collected fewer mosquitoes (n = 267) and species (9 spp.) than the trap set with dry ice (Set II), it collected all common vector species in the study areas as collected by Set II. The presence of an acid solution had no bias in the collection of mosquitoes with different physiological ages as determined by the parous rate. The present study demonstrated that this CO2 generating system is reliable, simple and inexpensive, and could be an alternative to dry ice. The system can be modified to increase the amount of CO2 generated for higher efficacy of mosquito collection. This CO2 production method can be applied to collect other blood-sucking arthropods as well.

The Anopheles lindesayi Species Complex (Diptera: Culicidae) in Bhutan.

This paper deals with species A, C, D, and E of the Lindesayi Complex of Anopheles subgenus Anopheles (Diptera: Culicidae) that were recently recorded from Bhutan. Species B is not included due to insufficient data. Species A is Anopheles lindesayi sensu stricto, and species C, D, and E are new species that are formally described and named herein as Anopheles druki Somboon, Namgay & Harbach, Anopheles himalayensis Somboon, Namgay & Harbach and Anopheles thimphuensis Somboon, Namgay & Harbach, respectively. Morphological characteristics of the adults, larval and pupal stages of each species are provided and compared with An. lindesayi from Bhutan.

Chrysomya pinguis (Walker) (Diptera: Calliphoridae), blow fly of forensic importance: A review of bionomics and forensic entomology appraisal.

Role of blow flies as the entomological evidence used in forensic investigations has risen dramatically worldwide. As the diverse habitats of Thailand suitably endowed with biodiversity of insects, blow flies of forensic importance need investigation in their bionomics, which are further applied in forensic investigations. Chrysomya pinguis (Walker, 1858) (Diptera: Calliphoridae) make up one of the most common blow fly species found associated with the human corpses and/or death scenes in several countries of the Asia continent. Given the major species as forensically important, this review is performed by the need for gathering information of C. pinguis from literature search in the future application in the regions where this species exists. This review deals with morphology, current knowledge on bionomics and forensic entomology involvement. Important morphological characteristics of egg, larva, puparium and adult were highlighted with illustration and/or micrographs. Search pertaining to molecular analysis used for fly identification and developmental rate of larvae were included. Furthermore, we outline potential issues and challenges of C. pinguis research that necessitate forensic applications in the future.

Uranotaenia (Pseudoficalbia) bhutanensis (Diptera: Culicidae), A New Species From Bhutan.

A new species of the subgenus Pseudoficalbia of the genus Uranotaenia is described from larvae found in a hole in a log at high altitude in montane forest in the Mongar District of Bhutan. Based on morphology, the larvae are sufficiently distinct from other species of the subgenus to be described and formally named here as Uranotaenia bhutanensis Somboon, Namgay & Harbach, n. sp. The species is a member of the Bimaculata Series. The chaetotaxy of the fourth-instar larva is provided.

Significance of eggshell morphology as an additional tool to distinguish species of sand flies (Diptera: Psychodidae: Phlebotominae).

Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica (Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b (Cytb) as a molecular marker. The chorionic sculpturing of Ph. stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr. indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs.

The Anopheles baileyi species complex (Diptera: Culicidae: Anophelinae) in Bhutan.

This paper deals with three species (informal species A, B and C) of the Baileyi Complex of Anopheles subgenus Anopheles (Diptera: Culicidae) that were recently reported from Bhutan. Species A is An. baileyi sensu stricto, while species B and C are new species which are formally described and named herein as An. monticola Somboon, Namgay & Harbach and An. bhutanensis Somboon, Namgay & Harbach, respectively. Morphological characters of the adults, male genitalia, larval and pupal stages of the two new species, and the chaetotaxy of the immature stages, are provided and compared with An. baileyi from Bhutan. Although the patterns of wing spots of the adults and the pupal setae of the three species are very similar, significant differences in structures of the male genitalia distinguish them. Larvae of An. baileyi and An. monticola are similar, but differ from the larva of An. bhutanensis in the development of setae 3- and 13-T. The first antennal flagellomere of females of An. baileyi and An. bhutanensis has a distinct patch of pale scales, which is absent in An. monticola.