Yvonne-Marie Linton

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.

Morphological and molecular identity of Anopheles (Cellia) sundaicus (Diptera: Culicidae), the nominotypical member of a malaria vector species complex in Southeast Asia

A neotype for Anopheles (Cellia) sundaicus (Rodenwaldt), the nominotypical member of a malaria vector species complex, is selected from the progeny of a female collected at Pandan Beach near Lundu, Sarawak, Malaysia. Siblings of the neotype and other topotypic specimens are used to characterize and fix the morphological and molecular identity of the species as a foundation for systematic studies of the species complex. The species is described and illustrated in the adult, pupal and larval stages, and DNA sequence data are included for the mitochondrial cytochrome c oxidase I (COI) gene and the nuclear ITS‐2 region.

Making Mosquito Taxonomy Useful: A Stable Classification of Tribe Aedini that Balances Utility with Current Knowledge of Evolutionary Relationships.

The tribe Aedini (Family Culicidae) contains approximately one-quarter of the known species of mosquitoes, including vectors of deadly or debilitating disease agents. This tribe contains the genus Aedes, which is one of the three most familiar genera of mosquitoes. During the past decade, Aedini has been the focus of a series of extensive morphology-based phylogenetic studies published by Reinert, Harbach, and Kitching (RH&K). Those authors created 74 new, elevated or resurrected genera from what had been the single genus Aedes, almost tripling the number of genera in the entire family Culicidae. The proposed classification is based on subjective assessments of the “number and nature of the characters that support the branches” subtending particular monophyletic groups in the results of cladistic analyses of a large set of morphological characters of representative species. To gauge the stability of RH&K’s generic groupings we reanalyzed their data with unweighted parsimony jackknife and maximum-parsimony analyses, with and without ordering 14 of the characters as in RH&K. We found that their phylogeny was largely weakly supported and their taxonomic rankings failed priority and other useful taxon-naming criteria. Consequently, we propose simplified aedine generic designations that 1) restore a classification system that is useful for the operational community; 2) enhance the ability of taxonomists to accurately place new species into genera; 3) maintain the progress toward a natural classification based on monophyletic groups of species; and 4) correct the current classification system that is subject to instability as new species are described and existing species more thoroughly defined. We do not challenge the phylogenetic hypotheses generated by the above-mentioned series of morphological studies. However, we reduce the ranks of the genera and subgenera of RH&K to subgenera or informal species groups, respectively, to preserve stability as new data become available.

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.

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.

Incrimination of Anopheles (Nyssorhynchus) rangeli and An. (Nys.) oswaldoi as natural vectors of Plasmodium vivax in Southern Colombia

Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1% of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1%) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION ANOPHELES (ANOPHELES) SACHAROVI FAVRE, A PRIMARY VECTOR OF MALARIA IN THE MIDDLE EAST

Abstract. Combined morphological and molecular studies were conducted on Anopheles (Anopheles) sacharovi Favre, an important malaria vector of the Palaearctic Maculipennis Complex. Specimens collected in Greece and Iran were identified on the basis of morphology and confirmed by correlation of their nuclear internal transcribed spacer 2 (ITS2) rDNA sequences with those available in GenBank. The progeny of females collected in Greece were used for detailed morphological study. The morphology of the adults, pupa, fourth‐instar larva and egg are described and distinguished from those of An. maculipennis, the nominotypical member of the Maculipennis Complex. In addition to sequence data for the nuclear ITS2 region, partial sequence data are provided for the mitochondrial cytochrome oxidase I gene. The taxonomy, systematics, bionomics and distribution of the species are reviewed. This work provides the first fully integrated assessment of An. sacharovi as a basis for comparative studies of the Maculipennis Complex.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Ribosomal ITS2 sequence data for Anopheles maculipennis and An. messeae in northern Greece, with a critical assessment of previously published sequences

DNA sequences were generated for eight specimens of the Anopheles maculipennis complex from Florina in NW Greece, and identified to species on the basis of comparison with ITS2 sequences for members of the complex already in GenBank. The sequences revealed the presence of An. maculipennis and An. messeae in Florina. Problems with sequence reliability and accessibility of sequences generated in earlier studies of Palaearctic members of the complex are discussed.

Molecular comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon.

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.