The mosquitoes of Finland: updated distributions and bionomics.

Mosquitoes (Diptera: Culicidae) were collected in Finland between 2012 and 2018 to determine the species present and their distributions. In total, 52 466 specimens from 1031 collections formed the basis for the preparation of distribution maps for each of the 40 species that were collected. Anopheles maculipennis s.s., An. claviger, Aedes geminus and Ochlerotatus sticticus are confirmed on mainland Finland after previous records were uncertain or absent. Coquillettidia richiardii, Culiseta morsitans, Cs. ochroptera, Culex territans, Cx. torrentium, Oc. leucomelas, Oc. nigrinus, Oc. pullatus and Oc. punctodes occur more widely than previously reported. Three species, Ae. rossicus, Cs. subochrea and Oc. cyprius were not collected, although Ae. rossicus was subsequently found in Lapland by another researcher. No invasive species were collected. Ochlerotatus communis, an aggressive biter, was the most commonly encountered species. Larval collection data suggest that several species may have up to three generations per year in Finland, with Cx. torrentium and Cx. pipiens having at least two, and Oc. communis and Oc. punctor regularly found as larvae across the summer. These data, especially when coupled with historical records, are vital for monitoring species which have significant vector potential, particularly when faced with a warming climate.

Anopheles daciae, a new country record for Finland.

Mosquitoes (Diptera: Culicidae) were collected throughout Finland between 2013 and 2018 to determine species distributions. During the course of molecular identifications of specimens belonging to the Anopheles maculipennis complex, ribosomal internal transcribed spacer 2 sequences and link-reared specimens revealed the presence of Anopheles daciae Linton, Nicolescu & Harbach (n = 37), a new country record, as well as Anopheles messeae Falleroni (n = 19) in the collections. Although the sample size is low, distinctions are apparent in the distributions of these two species, with An. daciae present in south-eastern and central Finland, including the regions of Kanta-Häme, Pirkanmaa, Pohjois-Pohjanmaa, Päijät-Häme and Satakunta, and An. messeae present in the southern and south-eastern regions of Åland (Ahvenanmaa), Etelä-Savo, Kanta-Häme, Kymenlaakso, Päijät-Häme, Satakunta, Uusimaa and Varsinais-Suomi. All reports of An. messeae in Finland prior to 2018 should therefore be recognized as potentially being either An. messeae or An. daciae. Because these species are potential vectors of malarial protozoa, it is important to have full knowledge of their distributions across Europe, particularly in the face of climate warming.

A method for distinguishing the important malaria vectors Anopheles dirus and An. cracens (Diptera: Culicidae) based on antennal sensilla of adult females.

Some species of the Anopheles dirus species complex are considered to be highly competent malaria vectors in Southeast Asia. Anopheles dirus is the primary vector of Plasmodium falciparum and P. vivax while An. cracens is the main vector of P. knowlesi. However, these two species are difficult to distinguish and identify based on morphological characters. Hence, the aim of this study was to investigate the potential use of antennal sensilla to distinguish them. Large sensilla coeloconica borne on the antennae of adult females were counted under a compound light microscope and the different types of antennal sensilla were examined in a scanning electron microscope. The antennae of both species bear five types of sensilla: ampullacea, basiconica, chaetica, coeloconica and trichodea. Observations revealed that the mean numbers of large sensilla coeloconica on antennal flagellomeres 2, 3, 7, 10 and 12 on both antennae of both species were significantly different. This study is the first to describe the types of antennal sensilla and to discover the usefulness of the large coeloconic sensilla for distinguishing the two species. The discovery provides a simple, reliable and inexpensive method for distinguishing them.

A method for distinguishing the important malaria vectors Anopheles dirus and An. cracens (Diptera: Culicidae) based on antennal sensilla of adult females

@#Some species of the Anopheles dirus species complex are considered to be highly competent malaria vectors in Southeast Asia. Anopheles dirus is the primary vector of Plasmodium falciparum and P. vivax while An. cracens is the main vector of P. knowlesi. However, these two species are difficult to distinguish and identify based on morphological characters. Hence, the aim of this study was to investigate the potential use of antennal sensilla to distinguish them. Large sensilla coeloconica borne on the antennae of adult females were counted under a compound light microscope and the different types of antennal sensilla were examined in a scanning electron microscope. The antennae of both species bear five types of sensilla: ampullacea, basiconica, chaetica, coeloconica and trichodea. Observations revealed that the mean numbers of large sensilla coeloconica on antennal flagellomeres 2, 3, 7, 10 and 12 on both antennae of both species were significantly different. This study is the first to describe the types of antennal sensilla and to discover the usefulness of the large coeloconic sensilla for distinguishing the two species. The discovery provides a simple, reliable and inexpensive method for distinguishing them.

The mosquito Anopheles (Cellia) oreios sp. n., formerly species 6 of the Australasian Anopheles farauti complex, and a critical review of its biology and relation to disease.

Species 6 of the Australasian Anopheles farauti sibling species complex (Diptera: Culicidae) is described and formally named Anopheles oreios Bangs & Harbach, sp. n. Adult, pupal and fourth-instar larval specimens collected in the Baliem Valley, Papua Province, Indonesia, are characterized and compared with those of Anopheles farauti, Anopheles hinesorum, Anopheles irenicus and Anopheles torresiensis (formerly informally denoted as species 1, 2, 7 and 3, respectively). The variable wings of adult females, the male genitalia, the pupa and the fourth-instar larva of An. oreios are illustrated and DNA sequence data are included for regions coding for sections of the mitochondrial COI and COII genes. The biology of An. oreios and its relation to malaria transmission are discussed in detail and contrasted with the biology and disease relations of some members of the An. farauti and Anopheles punctulatus sibling species complexes.

Molecular taxonomy of members of the Anopheles hyrcanus group from Thailand and Indonesia.

During studies of malaria vectors in Indonesia and Thailand, several specimens identified by field staff as members of the Anopheles barbirostris group (Diptera: Culicidae) were found to belong to the Anopheles hyrcanus group, as shown by marked differences in the size of the nuclear rDNA second internal transcribed spacer (ITS2) between the barbirostris (~1500 bp) and hyrcanus (~600 bp) groups. Identification of the species concerned required a more detailed study of ITS2 sequences and subunit I of the mitochondrial DNA cytochrome oxidase gene (COI). A phylogenetic analysis, based on Bayesian methods, revealed that the hyrcanus group specimens comprised five distinct clades, two of which corresponded with known species, Anopheles peditaeniatus and Anopheles sinensis. The remaining specimens formed three additional clades, for which there are no similar sequences in GenBank and which cannot be linked to previously described species. The misidentification of hyrcanus group species has important implications for malaria vector control; more comprehensive studies employing gene sequences are required to clarify the number of species in the group, their distribution and vector status.

Mitochondrial DNA variation in the malaria vector Anopheles minimus across China, Thailand and Vietnam: evolutionary hypothesis, population structure and population history.

The effects of Pleistocene environmental fluctuations on the distribution and diversity of organisms in Southeast Asia are much less well known than in Europe and North America. In these regions, the combination of palaeoenvironmental reconstruction and inferences about population history from genetic data has been very powerful. In Southeast Asia, mosquitoes are good candidates for the genetic approach, with the added benefit that understanding the relative contributions of historical and current processes to population structure can inform management of vector species. Genetic variation among populations of Anopheles minimus was examined using 144 mtDNA COII sequences from 23 sites in China, Thailand and Vietnam. Haplotype diversity was high, with two distinct lineages that have a sequence divergence of over 2% and exhibit different geographical distributions. We compare alternative hypotheses concerning the origin of this pattern. The observed data deviate from the expectations based on a single-panmictic population with or without growth, or a stable but spatially structured population. However, they can be readily accommodated by a model of past fragmentation into eastern and western refugia, followed by growth and range expansion. This is consistent with the palaeoenvironmental reconstructions currently available for the region.

Distribution and ecology of mosquitoes in a focus of dirofilariasis in northwestern Iran, with the first finding of filarial larvae in naturally infected local mosquitoes.

A study of the distribution and ecology of mosquitoes (Diptera: Culicidae) in Ardebil Province of northwestern Iran, with special reference to the known dirofilariasis focus in Meshkinshahr County, was carried out in July-August 2005 and April-October 2006. In total, 17,533 adult mosquitoes and 3090 third- and fourth-instar larvae were collected on 14 occasions and identified using morphological characters and DNA sequence data. Twenty species belonging to seven genera were found: Anopheles claviger (Meigen), An. hyrcanus (Pallas), An. maculipennis Meigen, An. pseudopictus Grassi*, An. sacharovi Favre, An. superpictus Grassi, Aedes vexans (Meigen)*, Coquillettidia richiardii (Ficalbi)*, Culex hortensis Ficalbi, Cx. modestus Ficalbi, Cx. pipiens Linnaeus, Cx. theileri Theobald, Cx. torrentium Martini*, Cx. tritaeniorhynchus Giles, Culiseta longiareolata (Macquart), Cs. annulata (Schrank)*, Cs. subochrea (Edwards), Ochlerotatus caspius (Pallas) s.l.* (= Aedes caspius sensu auctorum), Oc. geniculatus (Olivier)* (= Aedes geniculatus sensu auctorum) and Uranotaenia unguiculata Edwards (asterisks indicate new occurrence records for the province). The most prevalent species in adult catches were An. maculipennis (52%), Cx. theileri (45%) and Cx. hortensis (1%); the most prevalent species caught as larvae were Cx. theileri (27%), Cx. hortensis (21%) and An. maculipennis (19%). Anopheles maculipennis, Cx. pipiens and Cx. theileri were most widely distributed in the province. The occurrence of Cx. torrentium in Iran is verified based on differential characters of fourth-instar larvae. Anopheles maculipennis and An. sacharovi of the Maculipennis Group were identified from their diagnostic ITS2 sequences. For the first time, cytochrome c oxidase I (COI) sequences were obtained from Iranian specimens of An. hyrcanus, An. pseudopictus, Cx. theileri and Oc. caspiuss.l. Culex theileri and An. maculipennis were found naturally infected with third-stage (infective) larvae of Dirofilaria immitis (Leidy) and Setaria labiatopapillosa (Alessandrini) (Spirurida: Onchocercidae), respectively, for the first time in Iran.

Bionomics, taxonomy, and distribution of the major malaria vector taxa of Anopheles subgenus Cellia in Southeast Asia: an updated review.

There is high diversity of Anopheles mosquitoes in Southeast Asia and the main vectors of malaria belong to complexes or groups of species that are difficult or impossible to distinguish due to overlapping morphological characteristics. Recent advances in molecular systematics have provided simple and reliable methods for unambiguous species identification. This review summarizes the latest information on the seven taxonomic groups that include principal malaria vectors in Southeast Asia, i.e. the Minimus, Fluviatilis, Culicifacies, Dirus, Leucosphyrus, and Sundaicus Complexes, and the Maculatus Group. Main issues still to be resolved are highlighted. The growing knowledge on malaria vectors in Southeast Asia has implications for vector control programs, the success of which is highly dependant on precise information about the biology and behavior of the vector species. Acquisition of this information, and consequently the application of appropriate, sustainable control measures, depends on our ability to accurately identify the specific vectors.

Presence of Aedes albopictus in Lebanon and Syria.

The occurrence of Aedes albopictus in Lebanon and Syria is reported for the first time. Larvae were found in 4 localities in Lebanon, and 1 female was captured inside a house located in a coastal locality in Syria. The potential of the species to vector arboviral disease in the region is noted.

Molecular identification of mosquito species in the Anopheles annularis group in southern Asia.

The Anopheles annularis group of subgenus Cellia Theobald (Diptera: Culicidae) includes five currently recognized species in southern Asia: An. annularis Van der Wulp, Anopheles nivipes (Theobald) and Anopheles philippinensis Ludlow, which are widespread in the region, Anopheles pallidus Theobald, which is known in Sri Lanka, India and Myanmar, and Anopheles schueffneri Stanton, which occurs in Java and Sumatra. Identification of the four mainland species based on morphology is problematic. In view of the fact that the three widespread species are variously involved in malaria transmission in different parts of the region, we developed a species-specific polymerase chain reaction assay based on rDNA internal transcribed spacer 2 (ITS2) sequences to facilitate entomological and epidemiological studies of the four species. The method proved to be reliable when tested over a wide geographical area.

Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA.

The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are either connected by gene flow and/or share a recent common history.

Molecular variation, systematics and distribution of the Anopheles fluviatilis complex in southern Asia.

Species of the Anopheles fluviatilis James and Anopheles minimus complexes (Diptera: Culicidae) are difficult to distinguish morphologically. Members of the two complexes have been confused and, consequently, their distributions and roles in malaria transmission are uncertain. We identified numerous mosquitoes from China, Thailand, Vietnam, India, Nepal, Pakistan and Iran by single-strand conformation polymorphism (SSCP) and/or sequencing, and analysed the variation in the 28S D3 region of ribosomal DNA for members of the Minimus Subgroup and also the internal transcribed spacer region 2 (ITS2) for members of the An. fluviatilis complex. The D3 region is highly conserved between taxa and therefore could serve as a standard for molecular diagnosis of the subgroup members. D3 sequence, bionomics and malaria transmission data provide further evidence that An. fluviatilis S in India is conspecific with An. minimus C in South-east Asia. An. fluviatilis T has three ITS2 haplotypes (designated T1, T2 and Y) and its distribution in India, Nepal, Pakistan and Iran is confirmed. An. fluviatilis U is well defined on cytogenetic grounds in Uttar Pradesh, India, but is very close to An. fluviatilis T and the two species may hybridize in some regions. Variant ITS2 sequences suggest the possible existence of two additional taxa within the An. fluviatilis complex, one in Iran and another in India, provisionally designated An. fluviatilis forms V and X, respectively. The distributions of members of the An. fluviatilis and An. minimus complexes in south-central Asia are summarized.

Anopheles (Cellia) epiroticus (Diptera: Culicidae), a new malaria vector species in the Southeast Asian Sundaicus Complex.

Anopheles sundaicus species A of the Southeast Asian A. sundaicus complex is formally named Anopheles epiroticus Linton & Harbach based on DNA sequence differentiation of the whole nuclear ITS2 region and a portion of both the cytochrome b and cytochrome c oxidase I mitochondrial genes. Detailed comparative morphological studies of the adult, larval and pupal stages did not reveal any differential or diagnostic differences that reliably distinguish A. epiroticus from A. sundaicus s.s. Information is provided on the bionomics and systematics of the new species.

Mosquitoes of the Anopheles maculipennis group (Diptera: Culicidae) in Romania, with the discovery and formal recognition of a new species based on molecular and morphological evidence.

Mosquitoes of the Anopheles maculipennis group were collected in five districts of Romania (Constant,a, Giurgiu, Ilfov, Mehedint,i and Suceava) between March 2000 and June 2003. Two hundred and ninety-seven specimens were identified by molecular methods. Nuclear rDNA ITS2 sequences of 178 specimens were compared with GenBank sequences for nine known Palaearctic species of the group, and 119 specimens were identified using an ITS2 PCR-RFLP assay developed during the study. Five genetically distinct species of the group were identified: A. atroparvus van Thiel, A. maculipennis Meigen, A. melanoon Hackett and A. messeae Falleroni and a previously unrecognized species. The new species, herein formally described and named A. daciae sp. n., was collected in the Black Sea coastal region and plains adjacent to the Danube River in southern Romania. Anopheles daciae is most similar to and sympatric with A. messeae. It is contrasted with A. messeae and characterized on the basis of unique nuclear ITS2 and mitochondrial COI DNA sequences and morphological characters of the eggs. The larval, pupal and adults stages of the two species were also compared, but no reliable characters were found to distinguish them. It seems likely that A. daciae is more widespread in eastern Europe and the Balkan States, and could be responsible for malaria transmission in these regions that is currently attributed to A. messeae. Anopheles melanoon is reported from Romania for the first time.

The classification of genus Anopheles (Diptera: Culicidae): a working hypothesis of phylogenetic relationships.

The internal classification of genus Anopheles is updated to reflect taxonomic actions published since the classification was last reviewed in 1994. Both formal and informal taxa are included. The classification is intended to aid researchers and students who are interested in analysing species relationships, making group comparisons and testing phylogenetic hypotheses. The genus includes 444 formally named and 40 provisionally designated extant species divided between six subgenera: Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia. Subgenera Anopheles, Cellia and Nyssorhynchus are subdivided hierarchically into nested informal groups of morphologically similar species that are believed to represent monophyletic lineages based on morphological similarity. Changes to the classification include additional species, eliminated species and changes to the hierarchical organization and composition of supraspecific groups, some as a result of molecular studies.

Genetic variation and population structure of the mosquito Anopheles jeyporiensis in southern China.

Genetic differentiation among populations of Anopheles jeyporiensis was examined using 76 mtDNA COII sequences from 16 sites throughout southern China and northern Vietnam. The COII sequences are AT-rich (74.58%) and reveal high levels of diversity with 39 of 685 sites polymorphic and 50 different haplotypes present. Genetic variation is high within populations and significant geographical structure was detected at both population and regional levels. In the larger samples, the distributions of haplotypes suggest recent population expansion.

The Anopheles maculipennis complex (Diptera: Culicidae) in Iran: molecular characterization and recognition of a new species.

Mosquitoes of the Anopheles maculipennis complex were collected in nine provinces of Iran (Esfahan, Fars, Gilan, Golestan, Kohkiluyeh va Boyerahmad, Mazandaran, Tehran, Azarbaijan-e Gharbi and Zanjan) between June 1983 and September 2002. The nuclear rDNA ITS2 sequences of 86 specimens were compared with those of seven species of the complex available in GenBank. Three genetically distinct species of the complex were distinguished: A. maculipennis Meigen, A. sacharovi Favre and a previously unrecognized species. The last species is most similar to, but clearly distinct from, A. martinius Shingarev and A. sacharovi. The taxonomy of A. martinius and A. sacharovi is critically reviewed, and justification is provided for formally recognizing the third species as Anopheles persiensis sp.n. The new species is the first culicid to be characterized and named principally on the basis of DNA evidence. Anopheles persiensis was collected only in the northern Caspian Sea littoral provinces of Gilan and Mazandaran, and it seems likely that this species could be responsible for malaria transmission in this region that was previously attributed to A. maculipennis. A species-specific RFLP-PCR assay based on ITS2 sequences was developed to facilitate further studies of the three species in Iran.

Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review, distribution and malaria vector status.

Mosquitoes of the Anopheles minimus group (Diptera: Culicidae) from nine Provinces of southern China were identified morphologically and by molecular characterization, using single-strand conformation polymorphisms (SSCPs) and sequence data for the D3 region of the 28S ribosomal DNA and the mitochondrial COII locus. Species A and C (sensu Green et al., 1990) of the An. minimus complex were found to be sympatric in Yunnan Province. Species A occurs eastward from Yunnan through southern Guangxi, Hainan, Guangdong and Taiwan Provinces, whereas species C occurs northward to northern Guangxi, Guizhou and Sichuan Provinces. Morphological and molecular evidence (based on specimens from the field and four isofemale lines) shows that An. minimus forms A and B (sensu Yu & Li, 1984) are morphological variants of species A, which is accepted as An. minimus Theobald sensu stricto (type-locality: Pokfulam, Hong Kong). The so-called subspecies x of An. minimus (sensu Baba, 1950) is reinterpreted as An. aconitus Dönitz. The distribution and vector status of members of the An. minimus group are discussed in relation to the historical and current transmission of malaria and filariasis in China. Both An. minimus A and C have been implicated as widespread vectors of malaria, whereas only species A has been found in Hainan, where An. minimus s.l. was a vector of Bancroftian filariasis. The presence of An. aconitus in Hainan and Yunnan Provinces is confirmed, but the occurrence of An. varuna Iyengar and An. fluviatilis James, which were previously recorded in China, could not be verified.

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north-eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR-RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR-RFLP assay can detect species not included in the initial set-up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non-vector anophelines in South-east Asia.