Natural Plasmodium vivax infections in Anopheles mosquitoes in a malaria endemic area of northeastern Thailand.

There was recently an outbreak of malaria in Ubon Ratchathani Province, northeastern Thailand. In the absence of information on malaria vector transmission dynamics, this study aimed to identify the anopheline vectors and their role in malaria transmission. Adult female Anopheles mosquitoes were collected monthly by human-landing catch in Na Chaluai District of Ubon Ratchathani Province during January 2014-December 2015. Field-captured mosquitoes were identified to species using morphology-based keys and molecular assays (allele-specific polymerase chain reaction, AS-PCR), and analysed for the presence of Plasmodium falciparum and Plasmodium vivax using an enzyme-linked immunosorbent assay (ELISA) for the detection of circumsporozoite proteins (CSP). A total of 1,229 Anopheles females belonging to 13 species were collected. Four anopheline taxa were most abundant: Members of the Anopheles barbirostris complex, comprising 38% of the specimens, species of the Anopheles hyrcanus group (18%), Anopheles nivipes (17%) and Anopheles philippinensis (12%). The other nine species comprised 15% of the collections. Plasmodium infections were detected in two of 668 pooled samples of heads/thoraces, Anopheles dirus (1/29) and An. philippinensis (1/97). The An. dirus pool had a mixed infection of P. vivax-210 and P. vivax-247, whereas the An. philippinensis pool was positive only for the latter protein variant. Both positive ELISA samples were confirmed by nested PCR. This study is the first to incriminate An. dirus and An. philippinensis as natural malaria vectors in the area where the outbreak occurred. This information can assist in designing and implementing a more effective malaria control programme in the province.

Entomological aspects and the role of human behaviour in malaria transmission in a highland region of the Republic of Yemen.

BACKGROUND: The Republic of Yemen has the highest incidence of malaria in the Arabian Peninsula, yet little is known of its vectors or transmission dynamics. METHODS: A 24-month study of the vectors and related epidemiological aspects of malaria transmission was conducted in two villages in the Taiz region in 2004-2005. RESULTS: Cross-sectional blood film surveys recorded an overall malaria infection rate of 15.3 % (250/1638), with highest rates exceeding 30 % in one village in May and December 2005. With one exception, Plasmodium malariae, all infections were P. falciparum. Seven Anopheles species were identified among 3407 anophelines collected indoors using light traps (LT) and pyrethrum knockdown catches (PKD): Anopheles arabiensis (86.9 %), An. sergentii (9 %), An. azaniae, An. dthali, An. pretoriensis, An. coustani and An. algeriensis. Sequences for the standard barcode region of the mitochondrial COI gene confirmed the presence of two morphological forms of An. azaniae, the typical form and a previously unrecognized form not immediately identifiable as An. azaniae. ELISA detected Plasmodium sporozoites in 0.9 % of 2921 An. arabiensis (23 P. falciparum, two P. vivax) confirming this species as the primary malaria vector in Yemen. Plasmodium falciparum sporozoites were detected in An. sergentii (2/295) and a single female of An. algeriensis, incriminating both species as malaria vectors for the first time in Yemen. A vector in both wet and dry seasons, An. arabiensis was predominantly anthropophilic (human blood index = 0.86) with an entomological inoculation rate of 1.58 infective bites/person/year. Anopheles sergentii fed on cattle (67.3 %) and humans (48.3; 20.7 % mixed both species), but only 14.7 % were found in PKDs, indicating predominantly exophilic behaviour. A GIS analysis of geographic and socio-economic parameters revealed that An. arabiensis were significantly higher (P < 0.001) in houses with televisions, most likely due to the popular evening habit of viewing television collectively in houses with open doors and windows. CONCLUSIONS: The predominantly indoor human biting vectors recorded in this study could be targeted effectively with LLINs, indoor residual spraying and/or insecticide-treated window/door curtains reinforced by education to instil a perception that effective and affordable malaria prevention is achievable.

Hemoglobin-derived porphyrins preserved in a Middle Eocene blood-engorged mosquito.

Although hematophagy is found in ~14,000 species of extant insects, the fossil record of blood-feeding insects is extremely poor and largely confined to specimens identified as hematophagic based on their taxonomic affinities with extant hematophagic insects; direct evidence of hematophagy is limited to four insect fossils in which trypanosomes and the malarial protozoan Plasmodium have been found. Here, we describe a blood-engorged mosquito from the Middle Eocene Kishenehn Formation in Montana. This unique specimen provided the opportunity to ask whether or not hemoglobin, or biomolecules derived from hemoglobin, were preserved in the fossilized blood meal. The abdomen of the fossil mosquito was shown to contain very high levels of iron, and mass spectrometry data provided a convincing identification of porphyrin molecules derived from the oxygen-carrying heme moiety of hemoglobin. These data confirm the existence of taphonomic conditions conducive to the preservation of biomolecules through deep time and support previous reports of the existence of heme-derived porphyrins in terrestrial fossils.

A new subgenus and species of Topomyia (Diptera: Culicidae: Sabethini) based on a remarkable male mosquito from Sabah, Malaysia.

Miyagiella Harbach, subgen. nov., is introduced as a new subgenus of Topomyia Leicester for a remarkable male mosquito, Topomyia discors Harbach, sp. nov., from Sabah, Malaysia. A diagnosis of the subgenus is provided that features unique anatomical characters of the genitalia of the holotype male. Miyagiella is very distinct from the two previously recognised subgenera of Topomyia, but is perhaps more closely related to the nominotypical subgenus than to subgenus Suaymyia Thurman. Salient differences that distinguish the three subgenera are contrasted; the holotype male of To. discors is described and its unique genitalia are illustrated.

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.

ELISA incrimination of Anopheles superpictus and Anopheles hyrcanus (Diptera: Culicidae) as vectors of Plasmodium vivax (Haemosporida: Plasmodiidae) in Tajikistan.

Entomological surveys were conducted in the malaria-endemic southwestern region of Tajikistan to establish which species of Anopheles may be responsible for malaria transmission. The head-thorax portions of 2,213 wild-caught Anopheles females-Anopheles superpictus Grassi (n = 1,292), Anopheles pulcherrimus Theobald (n = 376), Anopheles hyrcanus (Pallas) (n = 544), and Anopheles claviger (Meigen) (Diptera: Culicidae) (n = 1)-were tested for the presence of Plasmodium sporozoites using enzyme-linked immunosorbent assays. Four females of An. superpictus (three captured when landing on humans and one resting indoors) and one female of An. hyrcanus (captured when landing on a human indoors) were positive for Plasmodium vivax (Haemosporida: Plasmodiidae) VK-210. The infected females of An. superpictus were captured in the Hamadoni and Yovon districts of the Khatlon province, and the single infected female of An. hyrcanus was captured in the Jilikul district of the Khatlon province.

Nyx pholeocola, a new genus and cavernicolous species of tribe Aedini (Diptera: Culicidae) from southern Thailand based on morphological and molecular data.

Nyx Harbach & Linton, gen. nov., is introduced as a new mosquito genus of tribe Aedini for a previously unknown cave-dwelling species, Nyx pholeocola Linton & Harbach, sp. nov., from southern Thailand. A diagnosis of the genus is provided that features unique anatomical characters of the adult, pupal and larval stages of the type species. The affinities of Nyx are discussed in terms of its position in the phylogeny of Aedini. Nyx is more closely related to Borichinda and Isoaedes than to other genera of tribe Aedini. Salient differences that distinguish these three genera are contrasted. The male and female genitalia, pupa and fourth-instar larva of the new species are illustrated. DNA sequence for the second nuclear internal spacer region (ITS2) and the 658-bp barcode fragment of the mitochondrial cytochrome c oxidase I (COI) gene reveal very low similarity with published sequences, supporting the unique status of the new species.

The insupportable validity of mosquito subspecies (Diptera: Culicidae) and their exclusion from culicid classification.

Beginning about 80 years ago, the recognition of morphological varieties of mosquitoes was gradually replaced by the recognition of subspecies. As an examination of revisionary and detailed taxonomic studies of mosquitoes clearly shows, subspecies are untenable concepts which have been synonymized with nominotypical forms or recognized as distinct species. Thus, from our perspective, subspecies is not a functional or practical taxonomic rank. Consequently, in this study we critically assessed the taxonomic status of the 120 nominal taxa distinguished as subspecies before now to determine whether they should be recognized as separate species or synonymous names. As a result, 96 subspecies are formally elevated to specific rank, 22 are relegated to synonymy with nominotypical forms, one is considered a nomen dubium, one a species inquirenda and the names of four nominal species regarded as synonyms are revalidated. The subspecies and their new status are listed in a conspectus. The revalidated species include Anopheles argentinus (Brèthes, 1912), from synonymy with An. pseudopunctipennis Theobald, 1901c; An. peruvianus Tamayo, 1907, from synonymy with An. pseudopunctipennis as nomen dubium; Culex major Edwards, 1935, from synonymy with Cx. annulioris consimilis Newstead, 1907; and Trichoprosopon trichorryes (Dyar & Knab, 1907), from synonymy with Tr. compressum Lutz, 1905. Additionally, the type locality of Anopheles sergentii Theobald, 1907 is restricted to El Outaya, Biskra Province, Algeria. A complete list of species to be retained, added to or removed from the Encyclopedia of Life, with a few corrections, is provided.

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 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.

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 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.

Molecular evolution of the insect-specific flaviviruses.

There has been an explosion in the discovery of 'insect-specific' flaviviruses and/or their related sequences in natural mosquito populations. Herein we review all 'insect-specific' flavivirus sequences currently available and conduct phylogenetic analyses of both the 'insect-specific' flaviviruses and available sequences of the entire genus Flavivirus. We show that there is no statistical support for virus-mosquito co-divergence, suggesting that the 'insect-specific' flaviviruses may have undergone multiple introductions with frequent host switching. We discuss potential implications for the evolution of vectoring within the family Flaviviridae. We also provide preliminary evidence for potential recombination events in the history of cell fusing agent virus. Finally, we consider priorities and guidelines for future research on 'insect-specific' flaviviruses, including the vast potential that exists for the study of biodiversity within a range of potential hosts and vectors, and its effect on the emergence and maintenance of the flaviviruses.

Culex pipiens: species versus species complex taxonomic history and perspective.

The taxonomic history of Culex pipiens (1758-present) is reviewed. The central question is whether Cx. pipiens is a single polytypic species or a complex of sibling species? The taxon traditionally known as the Cx. pipiens complex is referred to as the Pipiens Assemblage to avoid difficulties associated with the meaning of the word "complex". Neotype specimens have been designated to fix the morphological identities of Cx. pipiens, Cx. molestus, and Cx. quinquefasciatus, and Cx. pallens is represented by a holotype, but whether these nominal forms represent one or more biological species remains controversial. Despite extensive morphological and physiological/behavioral variation, there is no indication of subspecific or racial differences in geographically separated populations of Cx. pipiens. Introgression occurs where populations of Cx. pipiens and Cx. quinquefasciatus overlap, but the retention of parental epiphenotypes outside the zone of introgression provides evidence of independent species cohesion. The main conclusions reached are: Cx. pipiens and Cx. quinquefasciatus are separate species which evolved in Africa and hybridize in non-indigenous areas where they were unintentionally introduced by humans; Cx. molestus is nothing more than a phenotypic and physiological variant of Cx. pipiens; and Cx. pallens has no taxonomic status under the provisions of the International Code of Zoological Nomenclature. Based on morphological similarity, the Pipiens Assemblage includes Cx. pipiens, Cx. quinquefasciatus, and possibly Cx. australicus. There is no evidence to suggest that the Pipiens Assemblage includes any other species.

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.

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.

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 chrysothorax (Newstead amp; Thomas, 1910) (Diptera: Culicidae), preoccupied by Cx. chrysothorax (Peryass, 1908) and recognized as a subjective synonym of Cx. trigeminatus Clastrier, 1970.

Culex chrysothorax (Newstead Thomas, 1910) (Diptera: Culicidae) is recognized as the junior secondary homonym of Culex chrysothorax (Peryass, 1908). Both nominal species are currently regarded as nomina dubia (Pecor et al. 1991; Harbach 2018; Wilkerson et al. 2021). Based on critical examination of the lectotype female, the former nominal species, which was treated as species inquirenda by Forattini Sallum (1989), is no longer regarded as a doubtful species, and is found to be conspecific with Cx. trigeminatus Clastrier, 1970. Because it is preoccupied by Cx. chrysothorax (Peryass, 1908), it is placed in synonymy with Cx. trigeminatus, which is transferred from the Educator to the Atratus Group of the subgenus Melanoconion based on morphological features of the female. Culex chrysothorax (Peryass, 1908) is retained in the Educator Group as a nomen dubium. Because the original description of Cx. chrysothorax (Newstead Thomas) likely included misidentified specimens of Cx. theobaldi (Lutz, 1904), a more precise description of the lectotype female is provided.

Mosquito (Diptera: Culicidae) Diversity and Community Structure in Doi Inthanon National Park, Northern Thailand.

Urbanization and human activities create new suitable aquatic habitats for the immature stages of mosquitoes in many countries. This also applies to Doi Inthanon National Park in northern Thailand, which is named for the highest mountain in the country. Despite its popularity, there is no information regarding mosquito diversity and community structure in the different ecosystems of the park. Monthly collections of immature stages from various habitats were conducted from August 2004 to December 2005 using dipping and sucking methods. The specimens collected from each habitat were reared to adults and identified based on their morphology. Diversity parameters and community structure were statistically analyzed. A total of 140 species (3795 specimens) belonging to 15 genera were identified. Among these, four genera (Culex, Aedes, Anopheles, and Uranotaenia) had high species richness, each represented by 48, 27, 19, and 15 species, respectively. Aedes albopictus was the most relatively abundant species, representing 6.7% of the total number of captured specimens, followed by Tripteroides aranoides (5.6%) and Cx. mimulus (5%). Species richness in natural habitats was significantly higher than in artificial containers. Species richness and abundance were highest in the rainy season. In comparison to agricultural areas and villages, mosquito diversity was found to be higher in forest areas. Ground pools, stream pools, rock pools, bamboo stumps, bamboo internode, and rice fields were the most preferred natural habitats. The results indicate that Doi Inthanon National Park has a high mosquito diversity. Each species exhibits differences in abundance and distribution in different habitats, which is useful information for planning conservation measures and vector control in the park.