Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges.

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3'UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis.

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.

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.

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.

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.

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.

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.

Culex (Culex) longitubus, A New Species of the Mimeticus Subgroup (Diptera: Culicidae) From Bhutan.

Culex longitubus Somboon, Namgay & Harbach is described as a new species of the Mimeticus Subgroup of the subgenus Culex. The larva is most similar to the larva of Cx. tianpingensis Chen from China, but is distinguished by the length of the siphon and the anal papillae, the form of the comb scales and pecten spines, and the development of setae 7-P, 13-T, 1-X, and 4-X. The adults have wing markings and male genitalia similar to those of species of the Mimeticus Complex. Phylogenetic analysis of COI sequences revealed that the new species is closely related to Cx. murrelli Lien of the Mimulus Complex. The immature stages of the new species were found in stagnant pools and marshes at high altitudes in several districts of Bhutan.

Culex bhutanensis, a new species of the Mimeticus Subgroup of the nominotypical subgenus of the genus Culex Linnaeus (Diptera: Culicidae) from Bhutan.

Culex bhutanensis Somboon, Namgay & Harbach is described as a new species of the Mimeticus Subgroup of the subgenus Culex Linnaeus. Based on morphological distinctions and phylogenetic analysis of COI sequences, the new species is a member of the Mimulus Complex. The adults have wing markings similar to those of Cx. mimulus Edwards and Cx. murrelli Lien, but are distinguished by the presence of a line of pale scales or scattered pale scales along the posterior margin of the proximal portion of the costa. The larva is most similar to the larva of Cx. orientalis Edwards from Japan and South Korea, but is distinguished by the form of the comb scales, pecten spines and seta 2-X. The pupa differs from the pupae of other species of the subgroup in having seta 8-C with more branches. The immature stages of the new species were found in rice paddies, stagnant pools and marshes at high altitudes in several districts of Bhutan.

Reinertia, a New Subgenus of the Genus Aedes Meigen and Its Type Species Aedes (Reinertia) suffusus (Diptera: Culicidae), Newly Recorded From Bhutan.

A new subgenus, Reinertia Somboon, Namgay & Harbach, of the genus Aedes Meigen and its type species, Ae. suffusus Edwards, are described from specimens reared from larvae and pupae found in a tree hole in Bhutan. The scutum of the adults is mostly covered with narrow pale falcate scales. The proboscis, maxillary palpus, tibiae, and tarsi are dark-scaled. The gonocoxite of the male genitalia bears a unique setose basomesal sclerite. The larva closely resembles larvae of the subgenus Downsiomyia Vargus in having setae 4-6-C with numerous branches and inserted more or less on level with seta 7-C, abdominal seta 12-I is present and the comb is composed of 6-10 spine-like scales arranged in an irregular row. Surprisingly, Reinertia shares features of the adult habitus, male genitalia, and larva with the Palearctic subgenus Dahliana Reinert, Harbach & Kitching. However, in phylogenetic analyses of the mitochondrial COI gene of species representing 38 subgenera of Aedes and six other genera of the tribe Aedini Neveu-Lemaire, Reinertia was not associated with Dahliana or Downsiomyia. In both maximum likelihood and Bayesian analyses of the data, Ae. suffusus was recovered as the weakly supported sister of a clade composed of five species of the subgenus Protomacleaya Theobald. In the absence of strong support, and because Protomacleaya is an unnatural group of species that resemble each other phenetically by virtue of what they lack, Ae. suffusus cannot be placed in the subgenus Protomacleaya. Thus, the morphological and molecular data attest the uniqueness of Ae. suffusus and its recognition as a monobasic subgeneric lineage.

Molecular and morphological evidence of sibling species in Anopheles baileyi Edwards (Diptera: Culicidae) in Bhutan and Thailand.

This paper reports the results of a molecular and morphological study of Anopheles baileyi in Bhutan and Thailand. Phylogenetic analyses of ribosomal (ITS2) and mitochondrial DNA (COI) sequences revealed the presence of four genetically distinct clades, three in Bhutan (Clades I, II and III) and one in Thailand (Clade IV). Most of the larvae in the Bhutanese clades differed from those in the Thai clade in having seta 4-C branched, whereas it is single in the latter. The adults of each clade showed variation of wing markings and overlapping characters. The combination of characteristics of thoracic setae 1,2-P and abdominal seta 3-I was found to be useful for distinguishing the larvae. Pupae were inseparable. We provisionally recognize mosquitoes of Clades I, II, III and IV as members of a sibling species complex, the Baileyi Complex, denoted as species A, B, C and D, respectively. Species A is most likely the type form of An. baileyi s.s. because it was found adjacent to the type locality (Yatung, Tibet), and the others are unrecognized species.

Molecular and morphological evidence for sibling species within Anopheles (Anopheles) lindesayi Giles (Diptera: Culicidae) in Bhutan.

This paper reports the results of a comparative molecular and morphological study of An. lindesayi collected from various districts of Bhutan and An. l. cameronensis from Thailand, compared with GenBank accessions and publications for An. l. japonicus from Japan, South Korea and China, An. l. pleccau from Taiwan, and An. lindesayi from India. Phylogenetic analyses based on ribosomal (ITS2) and mitochondrial (COI) DNA sequences using the Maximum Likelihood method revealed five genetically distinct clades (A, B, C, D and E) in Bhutan. Specimens in Clade A correspond to the original description of An. lindesayi, particularly in wing markings, the pattern of basal pale scales on the hindfemur and the single seta 4-C of larvae, and their COI sequences were closely related to one Indian sequence. Larvae of Clades B, C, D and E are similar in having seta 4-C branched rather than single. The adults of Clades C, D and E (B not available) are distinguishable from those of Clade A and other subspecies. Specimens of Clade C are unique in having a long pale spot on wing vein R and the subcosta, scattered pale scales on several veins and a dark spot at the tip of vein R2. The adults of Clades D and E are similar in having a dark spot at the tip of vein R2 and no scattered pale scales on all other veins. We provisionally recognize mosquitoes of Clades A, B, C, D and E as species A, B, C, D and E, respectively, of the Lindesayi Complex. Species A is An. lindesayi sensu stricto and the others are unnamed species. Concomitantly, the previous concept of the "Lindesayi Complex", which included An. lindesayi, An. menglangensis, An. nilgiricus and An. wellingtonianus, is now recognized as the Lindesayi Subgroup of the Lindesayi Group (Anopheles Series, subgenus Anopheles) with the five sibling species of An. lindesayi comprising a more apposite Lindesayi Complex within the subgroup.

Description of Aedes (Hulecoeteomyia) bhutanensis n. sp. (Diptera: Culicidae) from Bhutan.

A new species of the subgenus Hulecoeteomyia of the genus Aedes reared from pupae found with two larvae in a small pool on a log in mountainous forest of Bhutan is diagnosed and formally named Aedes bhutanensis Somboon & Harbach, n. sp. The specific status of the species is supported by differential morphological characters of females, larvae and pupae, and sequences of the mitochondrial COI gene. Dichotomous keys are provided for identification of the adult females and larvae of the species of the subgenus.

Morphological and molecular evidence for a new species of Lutzia (Diptera: Culicidae: Culicini) from Thailand.

We found a species of LutziaTheobald (1903) (Diptera: Culicidae) in Chiang Mai Province and other provinces in northern Thailand which bears morphological and DNA sequence differences from the three species of Lutzia, subgenus Metalutzia Tanaka, previously recorded from Thailand, namely Lt. fuscana (Wiedemann), Lt. halifaxii (Theobald) and Lt. vorax Edwards. The adults of the Chiang Mai form (CM form) have abdominal banding patterns similar to those of Lt. vorax from Japan (which includes the type locality of this species), but differ in having the mediocubital crossvein usually positioned before rather than beyond the radiomedial crossvein. The thoracic and abdominal integument of Lt. vorax larvae is covered with relatively short pointed spicules whereas it is covered by denser, longer and more acutely pointed spicules in the CM form. Some differences are also found in the development of thoracic seta 1-M, and abdominal setae 8-II and 8-III. The pupa of the CM form clearly differs from the pupa of Lt. vorax in having setae 1 and 5 of abdominal segments V and VI branched rather than single. The characters of the wing, larva and pupa of the CM form are similar to those of Lt. fuscana and Lt. halifaxii. However, whereas the phallosome of males of the CM form is similar to males of Lt. vorax and Lt. halifaxii, it is distinct from males of Lt. fuscana. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunits I and II sequences revealed that the CM form falls outside a clade comprised of specimens of Lt. vorax from Japan, Korea, Thailand and Bhutan (Kimura 2-parameter, K2P, genetic distances 3.9-5.6, and 5.1-6.6, respectively). However, the two gene sequences of the CM form are not clearly distinct from clades comprised of sequences from specimens of Lt. fuscana and Lt. halifaxii (K2P 0.2-2.4%). However, based on the combination of morphological and molecular data, the current study provides evidence that the CM form is a previously unrecognized species of the genus Lutzia.

Culex (Culiciomyia) sasai (Diptera: Culicidae), senior synonym of Cx. spiculothorax and a new country record for Bhutan.

Culex (Culiciomyia) spiculothorax was described from Thailand based on the presence of spiculation on the thorax of larvae. Adult females are characterized but are indistinguishable from those of related species, such as Cx. pallidothorax. Phylogenetic analysis of mitochondrial oxidase subunit I (COI) sequences revealed that specimens identified as Cx. spiculothorax from Thailand, Japan and Bhutan form a single clade with Cx. sasai from Japan (Kimura 2-parameter genetic distances 0-0.9%) that is clearly distinct from clades comprised of other species of subgenus Culiciomyia. Attempts to collect Cx. sasai from several locations in Japan were unsuccessful - only larvae with thoracic vesicular-like spicules identified as Cx. spiculothorax were collected. Careful examination of specimens collected near the type locality of Cx. sasai revealed the presence of spicules on the thorax. Based on these findings, Cx. spiculothorax is formally synonymized with Cx. sasai, which replaces the former as the species present in Thailand and is a new country record for Bhutan.