Occurrence and host preferences of Anopheles maculipennis group mosquitoes in England and Wales

Mosquitoes of the Anopheles maculipennis Meigen (Diptera: Culicidae) group are of public health concern: five of the 11 morphologically indistinct species have been historically considered as vectors of malaria in Europe. Three members of the An. maculipennis group have been reported in the U.K.: Anopheles atroparvus van Thiel; Anopheles messeae Falleroni, and Anopheles daciae Linton, Nicolescu & Harbach. To study the distribution of the three U.K. species, particularly that of An. daciae, we developed a polymerase chain reaction–Restriction fragment length polymorphism (PCR‐RFLP) assay using the nuclear ribosomal internal transcribed spacer 2 (ITS‐2) gene. Anopheles daciae was found to be widespread, occurring in four of the five counties surveyed in southern England and on the Welsh island of Anglesey, often in sympatry with the closely related species An. messeae. The host preferences of 237 blood‐fed females were determined using either direct sequencing or PCR‐based fragment analysis of the mitochondrial cytochrome oxidase b gene with DNA from females' abdomens. All three species were found to be opportunistic, having fed on at least three different hosts. Seventeen individuals contained multiple bloodmeals, including two An. daciae that had fed on humans and birds. Our results show that An. daciae is widespread in England and Wales, occurs in sympatry with other members of the An. maculipennis group, and feeds on humans, which suggests it is a potential vector of disease in the U.K.     

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.     

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.     

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.     

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.     

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).     

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.     

Larval salinity tolerance of two members of the Anopheles funestus group

The Anopheles funestus group (Diptera: Culicidae) is one of the main species groups involved in malaria transmission in the Afrotropical regions. Basic research into this group has been limited because its members are eurygamic (they tend not to mate in confined spaces), which makes laboratory colonization difficult. Currently, only a few An. funestus Giles colonies are available and no colonies of other members of the group have been established. As information on the larval biology of members of the An. funestus group is limited, the present study aims to determine the effects of different salt concentrations on survival rates of the aquatic stages of two members of the An. funestus group, Anopheles funestus and Anopheles rivulorum Leeson. There were statistically significant negative trends in hatch rate and larval survival rate in An. funestus with increasing salt concentrations, with no larvae surviving to pupae at concentrations that included > 15% seawater. Anopheles rivulorum, by contrast, showed no significant trends in hatch rate or larval survival with increasing salt concentrations. This is the first report on salinity tolerance in An. rivulorum. A basic understanding of these variations in salinity tolerance provides vital information on the biology, ecology and colony rearing of members of the An. funestus group.     

A five-year monitoring study of the chlorinated hydrocarbons in the fish of a Finnish lake ecosystem

The present study, concerning the chlorinated hydrocarbon contents, PCB- and DDT-related compounds in the Finnish inland water fish, was the biggest ever carried out in Finland. During five years 1723 specimens and twelve species were analysed, the material being obtained from Lake Päijänne, the second largest lake in Finland. The lake consists of five limnologically different areas. The results of the study were statistically tested by t-test, one way analysis of variance and regression analysis. The results showed that the average PCB concentration mg/kg on a wet weight basis was 0·056 and ΣDDT (DDT + metabolites) 0·010, respectively. The concentrations of lindane, aldrin and dieldrin were under 0·0005 mg/kg. The differences between the species were greater than those between the areas. The regression analysis showed that in the commonest fish, pike, perch and bream, mostly no statistically significant differences were observed in the concentrations between different areas and research years.     

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.     

Studies on the resting behaviour and host choice of Anopheles gambiae and An. arabiensis from Muleba,Tanzania

The relative efficacy of a mechanical (Prokopack) collection method vs. manual aspiration in the collection of resting mosquitoes was evaluated in northern Tanzania before and after an intervention using indoor residual spraying and longlasting insecticide‐treated nets. In smoke‐free houses mosquitoes were collected from the roof and walls, but in smoky houses mosquitoes were found predominantly on the walls. Anopheles gambiae (Diptera: Culicidae) constituted 97.7% of the 312 An. gambiae complex specimens identified before but only 19.3% of the 183 identified after the intervention. A single sampling with the Prokopack collected a third of the available insects. Anopheles gambiae completed its gonotrophic development indoors, whereas Anopheles arabiensis did so outdoors. In both species gonotrophic development took 2 days. Most unfed resting An. arabiensis collected outdoors were virgins, whereas the majority of engorged insects were parous (with well‐contracted sacs). Daily survival was estimated to be 80.0%. Only 9.4% of the engorged An. arabiensis collected outdoors and 47.1% of those collected indoors had fed on humans. Using the Prokopack sampler is more efficient than manual methods for the collection of resting mosquitoes. Malaria transmission may have been affected by a change in vector composition resulting from a change in feeding, rather than reduced survival. Monitoring the proportions of members of the An. gambiae complex may provide signals of an impending breakdown in control.     

Transcriptomic differences between euryhaline and stenohaline malaria vector sibling species in response to salinity stress

Evolution of osmoregulatory systems is a key factor in the transition of species between fresh‐ and saltwater habitats. Anopheles coluzzii and Anopheles merus are stenohaline and euryhaline malaria vector mosquitoes belonging to a larger group of sibling species, the Anopheles gambiae complex, which radiated in Africa within the last 2 million years. Comparative ecological genomics of these vector species can provide insight into the mechanisms that permitted the rapid radiation of this species complex into habitats of contrasting salinity. Here, we use RNA‐Seq to investigate gene expression differences between An. coluzzii and An. merus after briefly exposing both young and old larval instars of each species to either saltwater (SW) or freshwater (FW). Our study aims to identify candidate genes and pathways responsible for the greater SW tolerance of An. merus. Our results are congruent with the ability of gene induction to mediate salinity tolerance, with both species showing increasing amounts of differential gene expression between SW and FW as salt concentrations increase. Besides ion transporters such as AgAE2 that may serve as effectors for osmoregulation, we also find mitogen‐activated protein kinases that may serve in a phosphorylation signalling pathway responding to salinity, and report potential cross‐talk between the mosquito immune response and osmoregulation. This study provides a key step towards applying the growing molecular knowledge of these malaria vectors to improve understanding of their ecological tolerances and habitat occupancy.     

Bionomics of Anopheles spp. (Diptera: Culicidae) in a malaria endemic region of Sukabumi,West Java,Indonesia

A 15‐month bionomic study of Anopheles species was conducted in two ecologically distinct villages (coastal and upland) of Sukabumi District, West Java, Indonesia from June 2006 to September 2007. Mosquitoes were captured using human‐landing collections at both sites. During the study, a total of 17,100 Anopheles mosquitoes comprising 13 Anopheles species were caught: 9,151 at the coastal site and 7,949 at the upland site. Anopheles barbirostris, Anopheles maculatus, and Anopheles vagus were the predominant species caught at the coastal site, and Anopheles aconitus, Anopheles barbirostris, and An. maculatus predominated in the upland site. Overall, species were exophagic at both sites, but there was variation between species. Anopheles aconitus was endophagic at the coastal site, exophagic at the upland site, collected most often in April 2007 and had a peak landing time between 22:00 and 23:00. Anopheles sundaicus was only collected at the coastal site, exophagic, collected most often in October 2006, and had a peak landing time between 19:00 and 20:00. Potential malaria vector species such An. aconitus, An. maculatus, and An. sundaicus were present throughout the year. None of the 7,770 Anopheles tested using CSP‐ELISA were positive for malaria, although the risk for malaria outbreaks in Sukabumi district remains high.     

Distribution and larval habitats of Anopheles species in northern Gyeonggi Province,Republic of Korea

A total of 180 larval collection sites (e.g., rice paddies, marshes, ground pools, ponds, stream margins, and irrigation and drainage ditches) was surveyed within a 2 km radius from Warrior Base training area, 5 km south of Panmunjeom (Joint Security Area, demilitarized zone), Gyeonggi Province, Republic of Korea (ROK), from May through October, 2007 to characterize larval habitat distributions of members of the Anopheles Hyrcanus Group (An. sinensis, An. lesteri, An. pullus, An. belenrae, An. kleini, and An. sineroides). A total of 5,859 anopheline larvae was collected from 84.4% of the sites surveyed, of which 4,071 were identified to species by polymerase chain reaction (PCR) using the ribosomal DNA internal transcribed spacer 2 (rDNA ITS2). Anopheles sinensis (52.6%) was the most frequently collected, followed by An. kleini (29.4%), An. sineroides (9.8%), An. pullus (6.7%), An. belenrae (1.1%), and An. lesteri (0.5%). Anopheles pullus and An. kleini were collected in greater proportions in May and from May – July, respectively. Few An. sinensis were collected from May – June, but it was the predominant species collected by August, and accounted for >80% of all larvae from September – October. Anopheles kleini was found in all habitats sampled; however, it was collected most frequently in young growth rice paddies, while An. sinensis was collected more frequently in mature and post‐harvest paddies. Anopheles pullus was associated with pre‐cultivated rice paddies, including water‐filled tire ruts left from the previous fall's harvest.     

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.     

Molecular cytogenetic analysis of DNA from pericentric heterochromatin of chromosome 2L of malaria mosquito <Emphasis Type="Italic">Anopheles beklemishevi</Emphasis> (culicidae,Diptera)

Using the method of microdissection of polytene chromosomes, followed by in situ hybridization, chromosomal localization of region-specific DNA probe from pericentic heterochromatin of chromosome 2L of Anopheles beklemishevi Stegnii et Kabanova was examined on polytene chromosomes of Anopheles atroparvus van Thiel, An. messeae Fall, and An. beklemishevi. DNA sequences homologous to the probe used were found in all species examined on chromosomes 2 and 3 in pericentric regions and in attachment regions. The exclusion were the attachment regions of chromosome XL in An. beklemishevi and An. messeae, and pericentric region of arm 2R in An. messeae. Pericentric α -heterochromatin of arm 2L in An. messeae and arm 3R in An. atroparvus also contained no sequences homologous to the DNA probe. The data obtained were compared with the earlier obtained data on localization of species-specific probe from the segment of chromosome 2R of An. atroparvus on chromosomes of An. artoparvus, An. messeae, and An. beklemishevi. The differences between the species in the sites of probes localization and fluorescence intensity revealed pointed to the existence of individual sequence associations in the regions of chromosomes attachment.     

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.     

Characterization of larval habitats of Anopheles albimanus, Anopheles pseudopunctipennis, Anopheles punctimacula, and Anopheles oswaldoi s.l. populations in lowland and highland Ecuador

Recent collection data indicate that at least four potential malaria vectors occupy more widespread distributions within the Andean highlands than in the past. Since habitat elimination is an important aspect of malaria control, it is vital to characterize larval habitats for Anopheles species within both lowland and highland sites. To that end, 276 sites within Ecuador were surveyed between 2008 and 2010. Characteristics of Anopheles‐present sites for four species were compared to Anopheles‐absent sites within the same geographical range and also to Anopheles‐absent sites within a highland range representing potential future habitats. Thermochron iButtons^© were used to describe the daily temperature variation within a subset of potential habitats. Anopheles albimanus (W.) was positively associated with permanent habitats, sand substrates, floating algae (cyanobacterial mats), and warmer temperatures in both comparisons. Anopheles pseudopunctipennis (T.) was associated with floating algae (cyanobacterial mats), warmer temperatures, and higher water clarity in both comparisons. Anopheles punctimacula (D.&K.) was negatively associated with floating algae and positively associated with dissolved oxygen in both comparisons. Anopheles oswaldoi s.l. (P.) was not significantly associated with any parameters more often than expected given larval‐absent sites. The results indicate that minimum water temperatures might limit the upper altitudinal distribution of An. albimanus (18.7° C) and An. pseudopunctipennis (16.0° C).     

Interactions and competition processes among tree species in young experimental mixed forests,assessed with chlorophyll fluorescence and leaf morphology

Chlorophyll a fluorescence (ChlF) and leaf morphology were assessed in two sites in Europe (Kaltenborn, Germany, and Satakunta, Finland) within a forest diversity experiment. Trees at Satakunta, planted in 1999, form a stratified canopy, while in Kaltenborn the trees are 7 years old, with no apparent canopy connection among broadleaf species. The following ChlF parameters from measured OJIP transient curves were examined: F_V/F_M (a proxy for maximum quantum yield); Ψ_Eo (a proxy for efficiency in transferring an electron from reduced QA to the electron transport chain); I‐P phase (a proxy for efficiency of reducing final acceptors beyond PSI); and PI_tot (total performance index for potential energy conservation from photons absorbed by PSII to reduction of PSI end acceptors). At Satakunta F_V/F_M and Ψ_Eo in Betula pendula were higher in monocultures and lower in mixed plots, perhaps due to increasing light availability in mixed plots, which can induce photoinhibition. The opposite trend was observed in Picea abies, which was shaded in mixed plots. At Kaltenborn F_V/F_M decreased in Fagus sylvatica and P. abies in mixed plots due to competition both above‐ and belowground. At Satakunta LMA increased in B. pendula leaves with increasing species richness. Leaf area of ten leaves was reduced in F. sylvatica in mixed plots at Kaltenborn. By up‐scaling the overall fluorescence response to plot level (PI_{tot_plot}), a significant positive correlation with tree diversity was found at Kaltenborn, but not at Satakunta. This could suggest that competition/facilitation processes in mixed stands play a significant role in the early stages of forest establishment, but then tend to be compensated in more mature stands.