Single nucleotide polymorphism analysis of the ITS2 region of two sympatric malaria mosquito species in Sweden: Anopheles daciae and Anopheles messeae

Four species of the Anopheles maculipennis complex have previously been recorded in Sweden. A recent addition to the complex is Anopheles daciae, which is considered to be closely related to, but distinct from Anopheles messeae. The original designation of An. daciae was based on five genetic differences (161, 165, 167, 362 and 382) in the second internal transcribed spacer (ITS) 2 of the ribosomal RNA. Further studies have shown that only two nucleotide differences (362 and 382) robustly separate the species. Thirty-three An. maculipennis complex mosquitoes were collected in the province of Uppland, Sweden. All were An. daciae but showed double peaks for three variable positions (161, 165 and 167). When cloned, the intra-individual nucleotide variation was almost exclusively fixed with either TTC or AAT, originally diagnostic for An. messae and An. daciae, respectively. To further investigate the intra-individual variation, nine An. daciae and 11 An. messeae were collected in southern Sweden and their ITS2 fragments were amplified and sequenced using Illumina MiSeq sequencing (Illumina, Inc., San Diego, CA, USA). For the diagnostic nucleotide 382 no intra-individual variation could be detected. However, although each An. daciae specimen carried several ITS2 sequence variants for the four other nucleotides, there was no intra-individual variation in the An. messeae specimens.     

Estimated resistance of the malaria mosquito Anopheles messeae s.l. to the insecticide malathion

Resistance to agricultural pesticides is an important and insufficiently studied concern for pest and disease vector research. We determined the malathion resistance of species in the Anopheles maculipennis mosquito group in a habitat near Novosibirsk, Russia. Most of the 851 individuals we measured were members of the Anopheles messeae s.l. complex (An. messeae and An. daciae species). The LC₅₀ value for malathion was 0.052 mg/L for the mixed specimens, and we failed to find any differences between species. The LC₅₀ value was within the range of values for malathion resistance of Anopheles stephensi and Culex quinquefasciatus. As the main resistance mechanism to organophosphate and carbamate insecticides is a single mononucleotide substitution in the ace‐1 gene, we searched for this mutation in An. messeae s.l. and An. beklemishevi by restriction analysis. This mutation was not found in 347 of the specimens. We sequenced the ace‐1 gene fragment for 24 specimens from four species of the Anopheles maculipennis group, including An. messeae, An. daciae, An. atroparvus, and An. beklemishevi. These specimens harbored a nucleotide substitution in the triplet where a mutation can lead to insecticide resistance, but this substitution would make it difficult for the resistance to develop. Since the studied specimens belong to branches of the Palearctic portion of the Anopheles maculipennis group, we suspect that all other Palearctic species of this group would have difficulties harboring the ace‐1 mutation that would lead to organophosphate and carbamate resistance.     

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.     

Dynamics of Malaria Mosquito Species Composition in Siberian Populations Detected by Restriction Analysis

The peculiarities of the dynamics of malaria mosquito species proportions were studied in natural populations. Twenty-one collections from five larval habitats of the Anopheles maculipennis complex malaria mosquitoes were taken in the vicinity of Novosibirsk (Russia). It was detected that the Anopheles messeae and An. daciae are dominant species in the collected samples. Three An. beklemishevi individuals were also detected. The dynamics of the species proportions within the reproduction seasons for a number of years and also the differences between localities in the species composition were studied. It was revealed that the An. daciae proportion is maximal in July and falls in August. The species proportions can change sharply from year to year. For example, the An. messeae species prevailed in localities on the left bank of Novosibirsk Reservoir in 2013 and 2014 with the frequency of 54–68%, while An. daciae began to prevail on this territory in 2016 with the frequency of 73–85%. Our data on the proportions of species in different reservoirs were compared with data of other authors for the collections of malaria mosquitoes of the Anopheles maculipennis complex in Russia (Tomsk oblast) and in Germany. A high correspondence of models provided by these authors for the territory that we studied was demonstrated. Thus, the ecological peculiarities of the An. messeae and An. daciae species are highly stable even in geographically and climatically distant localities. An increase in the portion of relatively anthropophilic An. daciae in the middle of summer can be a risk factor relative to malaria transmission in this period of time.     

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real‐time PCR assays

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species‐specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles maculipennis

The Anopheles maculipennis complex includes the most important malaria vectors of the western Palearctic. Anopheles maculipennis s.s., one member of this complex, is a reported vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the An. maculipennis s.s. genome, displaying a high among individual diversity (0.37–0.77) in a sample from France. Three loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining 12 loci are good candidates for further genetic studies in this species.     

Malaria and the Anopheles mosquitoes of Tajikistan

Surveys of Anopheles mosquitoes were conducted in urban, rural, and natural areas of Tajikistan to obtain updated information on their distributions, especially in southern districts of the country where malaria is a prevalent disease. Nine species of Anopheles are found in Tajikistan. Anopheles superpictus, An. claviger, An. hyrcanus, and An. pulcherrimus are the most widespread and abundant species. Investigations in northern Tajikistan confirmed the presence of An. artemievi and the absence of An. martinius, both members of the An. maculipennis complex of malaria vectors. Anopheles barianensis, An. lindesayi, and An. marteri sogdianus, species previously recorded in the country, were not encountered during our surveys. The history of Anopheles and malaria research in Tajikistan is reviewed and bionomical and distributional information is provided for each of the nine species.     

Chromosomal polymorphism in the populations of malaria mosquito <Emphasis Type="Italic">Anopheles messeae</Emphasis> (Diptera,Culicidae) in the Volga region

We studied the species composition and chromosomal variability of malaria mosquitoes in the Volga Basin (Upper, Middle, and Lower Volga regions). We investigated larvae karyotypes of sibling species of the Anopheles maculipennis group. We calculated the frequencies of chromosomal inversions in the local populations of the dominant species An. messeae. We discovered that karyotypic structure of An. messeae populations depends on landscape-climatic zones. Populations of the Upper, Middle and Lower Volga differ in frequency of chromosome inversions XL, 2R, 3R, and 3L.     

Application of a reverse dot blot DNA–DNA hydridization method to quantify host‐feeding tendencies of two sibling species in the Anopheles gambiae complex

A DNA–DNA hybridization method, reverse dot blot analysis (RDBA), was used to identify Anopheles gambiae s.s. and Anopheles arabiensis (Diptera: Culicidae) hosts. Of 299 blood‐fed and semi‐gravid An. gambiae s.l. collected from Kisian, Kenya, 244 individuals were identifiable to species; of these, 69.5% were An. arabiensis and 29.5% were An. gambiae s.s. Host identifications with RDBA were comparable with those of conventional polymerase chain reaction (PCR) followed by direct sequencing of amplicons of the vertebrate mitochondrial cytochrome b gene. Of the 174 amplicon‐producing samples used to compare these two methods, 147 were identifiable by direct sequencing and 139 of these were identifiable by RDBA. Anopheles arabiensis bloodmeals were mostly (94.6%) bovine in origin, whereas An. gambiae s.s. fed upon humans more than 91.8% of the time. Tests by RDBA detected that two of 112 An. arabiensis contained blood from more than one host species, whereas PCR and direct sequencing did not. Recent use of insecticide‐treated bednets in Kisian is likely to have caused the shift in the dominant vector species from An. gambiae s.s. to An. arabiensis. Reverse dot blot analysis provides an opportunity to study changes in host‐feeding by members of the An. gambiae complex in response to the broadening distribution of vector control measures targeting host‐selection behaviours.     

On the ecology and range of Anopheles beklemishevi (Diptera: Culicidae) with reference to the taxonomy of An. lewisi

The ecological features and geographic distribution of Anopheles beklemishevi have not been studied extensively. These studies are important in connection with the validity of the ‘Anopheles lewisi’ taxon. The materials were collected in Russia and Kazakhstan from 1973 to 2012, and species identity was defined by cytogenetic analysis of polytene chromosomes of larvae and adult females. A total of 7,896 specimens from 34 geographic locations was included in the analysis. It was established that An. beklemishevi is distributed from the east coast of the Baltic Sea to the basin of the Lena River, and from the forest‐tundra zone to the Altai and Sayan Mountain systems. This species is exophilic and is confined to high and/or swampy terrains found in the zone of conifer and mixed forests. The frequency of An. beklemishevi in the southwestern area, where it is sympatric with An. messeae s.l., has significantly decreased over the past decades. The results of the study indirectly suggest that An. beklemishevi does not play a significant role as a vector of malaria. It is highly improbable that An. beklemishevi and An. lewisi are the same species. Changes in the proportions of the species of the Maculipennis complex, as well as a shift of their ranges, will significantly impact the epidemiology of malaria over large areas of northern Eurasia.     

Characterization and Comparative Analysis of DNA from the Pericentric Heterochromatin of Chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The library containing DNA sequences from the diffuse pericentric heterochromatin from the right arm ofAnopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the library fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the A. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of A. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific library. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

Biting activity and host preference of the malaria vectors Anopheles maculatus and Anopheles sawadwongporni (Diptera: Culicidae) in Thailand

Adult mosquitoes in the Anopheles maculatus group were surveyed from different regions of Thailand and five different species were morphologically identified, including Anopheles maculatus, Anopheles sawadwongporni, Anopheles notanandai, Anopheles dravidicus, and Anopheles willmori. Blood‐feeding activity and host preference of two species, Anopheles maculatus and Anopheles sawadwongporni, were observed during a one‐year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, west‐central Thailand. Both species were more prevalent during the wetter period of the year and each had a greater predilection to feed on cattle than humans. Primary feeding activity occurred between 20:00‐23:00 and a smaller peak at 01:00‐03:00. Findings are discussed relative to the importance of these two vectors for malaria transmission in Pu Teuy.     

Inversion polymorphism and ecological specialization of malaria mosquitoes (Diptera, Culicidae) in Karelia

The species composition and inversion polymorphism were studied in malaria mosquito larva hemipopulations of Karelia. Three malaria mosquito species??Anopheles messeae, A. beklemishevi, and A. maculipennis??were found in the region. The northern boundary of their range is at 65°N. The greatest species diversity was observed in biotopes of the central region. Within-species chromosome polymorphism was observed in larva populations of all three species. For A. messeae, maximum karyotype diversity indices were established for the southern and northern regions of Karelia.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles sacharovi

The mosquito Anopheles sacharovi, a member of the A. maculipennis complex, is an important malaria vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the A. sacharovi genome, displaying a high among‐individual diversity (0.30–0.92) in a sample from Turkey. Seven loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining eight loci are good candidates for further genetic studies in this species.     

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex

Abstract. Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described. Genomic libraries were prepared for four members of the An.gambiae complex. These were screened using radiolabeled DNA from different species of An. gambiae sensu lato and a number of clones selected on the basis of their species specificity. These clones could be divided into two groups, each containing homologous sequences. Sequences homologous to group 1 inserts are highly reiterated in the genomes of Anopheles arabiensis Patton and Anopheles merus Dönitz, present in low copy number in Anopheles melas Theobald, but were not detected in Anopheles gambiae sensu stricto. Studies on the organization of this sequence in the genome of An.arabiensis show that homologous sequences are male specific and interspersed within the chromatin. Sequences homologous to group 2 inserts are highly repeated in the genomes of An.merus and An.melas, but present in low copy number in An.gambiae s.s. and An.arabiensis. Group 2 homologous sequences are not sex-specific in the species tested and appear to be tandemly repeated. When used as hybridization probes, these sequences provide a sensitive means for the identification of species within the Anopheles gambiae complex.     

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.     

Identification of bloodmeals in Anopheles quadrimaculatus and Anopheles punctipennis from eastern equine encephalitis virus foci in northeastern U.S.A.

The host‐feeding patterns of Anopheles quadrimaculatus Say and Anopheles punctipennis (Say) were examined in order to evaluate their potential contributions to the transmission of eastern equine encephalitis virus (EEEv) and other arboviruses in the northeastern U.S.A. Engorged mosquitoes of the two species were collected from EEEv foci in central New York (NY) and throughout New Jersey (NJ), and their bloodmeals were identified using a polymerase chain reaction (PCR)‐based assay and sequencing portions of the mitochondrial cytochrome b gene. Analysis of 131 An. quadrimaculatus and 107 An. punctipennis from NY revealed that 97.7% and 97.2%, respectively, had acquired blood solely from mammalian hosts. Similarly, examination of 288 An. quadrimaculatus and 127 An. punctipennis from NJ showed 100% and 96.0%, respectively, contained mammalian‐derived bloodmeals. Mosquitoes containing mixed bloodmeals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus from NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White‐tailed deer (Odocoileus virginianus) constituted the most common vertebrate host for these anopheline mosquitoes, accounting for 85.8–97.7% of all bloodmeals identified. The predominance of white‐tailed deer as a source of bloodmeals supports enzootic amplification of deer‐associated arboviruses in this region, including Jamestown Canyon, Cache Valley and Potosi viruses. One horse‐ and two human‐derived bloodmeals were also detected in An. quadrimaculatus collected in NJ. Limited avian‐derived bloodmeals were detected from mourning dove (Zenaida macroura), sharp‐shinned hawk (Accipiter striatus) and house finch (Carpodacus mexicanus), mostly in mixed bloodmeals. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epizootic–epidemic bridge vectors of EEEv from viraemic birds to mammalian hosts of concern, including horses and humans. An isolate of EEEv was recovered from the head and thorax of an An. punctipennis mosquito collected in NY.     

Differential ecology of Anopheles punctulatus and three members of the Anopheles farauti complex of mosquitoes on Guadalcanal, Solomon Islands, identified by PCR-RFLP analysis

From a series of larval collections made across northern Guadalcanal during the dry season, October–November 1997, four members of the Anopheles punctulatus group of mosquitoes (Diptera: Culicidae) were identified using PCR‐RFLP analysis. Anopheline larvae were found in 54/57 (95%) of the sites sampled, comprising An. farauti Laveran sensu stricto (32 sites), An. farauti species no. 2 (39 sites), An. farauti no. 7 (36 sites) and An. punctulatus Dönitz (10 sites). Anopheles punctulatus occurred only on the coastal plain, where it was associated with the more transient sites. Anopheles farauti sensu lato was more widespread throughout the survey region, with similar proportions of all three sibling species in both transient and permanent sites. Two members of the An. farauti complex, An. farauti s.s. and species no. 2, were found in brackish water. All breeding sites of An. punctulatus were cohabited by An. farauti s.l., sometimes by all three sibling species. Anopheles farauti s.s. was the only species collected on human bait, with a much higher biting rate early in the evening (57 bites/human/hour at 18.30–20.00 hours) than later (0.8 bites/human/hour at 21.00–24.00 hours).     

Spatio‐temporal variations of Anopheles coluzzii and An. gambiae and their Plasmodium infectivity rates in Lobito,Angola

From 2003 to 2007, entomological surveys were conducted in Lobito town (Benguela Province, Angola) to determine which Anopheles species were present and to identify the vectors responsible for malaria transmission in areas where workers of the Sonamet Company live. Two types of surveys were conducted: (1) time and space surveys in the low and upper parts of Lobito during the rainy and dry periods; (2) a two‐year longitudinal study in Sonamet workers' houses provided with long‐lasting insecticide‐treated nets (LLIN), “PermaNet,” along with the neighboring community. Both species, An. coluzzii (M molecular form) and An. gambiae (S molecular form), were collected. Anopheles coluzzii was predominant during the dry season in the low part of Lobito where larvae develop in natural ponds and temporary pools. However, during the rainy season, An. gambiae was found in higher proportions in the upper part of the town where larvae were collected in domestic water tanks built near houses. Anopheles melas and An. listeri were captured in higher numbers during the dry season and in the low part of Lobito where larvae develop in stagnant brackish water pools. The infectivity rates of An. gambiae s.l. varied from 0.90% to 3.41%.