Anopheline Mosquito Species Research Articles

Molecular characterization of anopheline mosquitoes from the goat malaria-endemic areas of Thailand.

Despite the fact that over a 100 anopheline mosquito species have been identified as human malaria vectors, little is known about ungulate malaria vectors. Consequently, we focused on investigating the bionomics and genetic characterizations of anopheline mosquitoes in goat malaria-endemic regions. We also attempted to screen for ungulate malaria potential vectors. A total of 1019 female anopheline mosquitoes were collected from six goat farms in four provinces of Thailand from 2020 to 2021. Mosquitoes were morphologically identified and subsequently confirmed using the mitochondrial DNA barcoding region-cytochrome oxidase c subunit I (MtDNA-COI), mitochondrial DNA-cytochrome c oxidase subunit II (MtDNA-COII), and ribosomal DNA internal transcribed spacer 2 (rDNA-ITS2) sequences. The current study reveals the genetic characteristics and distribution of nine mosquito species within the Anopheles and Cellia subgenera. Four dominant species, including Anopheles peditaeniatus, Anopheles subpictus, Anopheles vagus, and Anopheles aconitus exhibited significant intraspecific gene flow within their corresponding species. Although malaria parasites were not found in 126 mosquito pools, meaning more investigation is necessary, the current study adds to the existing DNA barcoding data collection and improves the current understanding of the genetic structure and distribution of anopheline mosquito species, which could be useful for effective control of mosquito-borne diseases.

The Possible Time and Region of Host Switches of Ancient Malaria Parasites with Reference to the Pliocene–Quaternary Archaeological Sites in Africa

AbstractAbout 96% of all malaria deaths occur in Africa, and the malignant falciparum malaria also originated on the continent. Although falciparum malaria only appeared in the Holocene period, it can be hypothesized that the transfer of malaria parasites from other primates to humans occurred several times in history parallel to human evolution. This study develops the model that examines the possible coexistence of the potential original host apes, human ancestors, and the diverse anopheline mosquito species; and how, where, and when the host switch of these parasites from great apes to humans occurred. Based on the Pliocene-early Pleistocene archaeological sites, it was found that certain early hominin populations could have lived in malaria areas where the anopheline mosquito fauna was moderately diverse. The people of the Lupemban Culture, as well as the Greenlandian and Northgrippian human populations of East and West-Central Africa, lived close to the high diversity of anopheline fauna and the territories of such great apes as Gorilla gorrilla. African mid-Holocene cultures likely came in contact with gorilla populations — the original hosts of Plasmodium falciparum — along the coasts of the Gulf of Guinea and the East African Rift Valley during their migration to southern Africa. The host switch of the ancestor of the falciparum malaria parasite likely occurred in these regions.

Myzorhynchus series of Anopheles mosquitoes as potential vectors of Plasmodium bubalis in Thailand

Ungulate malaria parasites and their vectors are among the least studied when compared to other medically important species. As a result, a thorough understanding of ungulate malaria parasites, hosts, and mosquito vectors has been lacking, necessitating additional research efforts. This study aimed to identify the vector(s) of Plasmodium bubalis. A total of 187 female mosquitoes (133 Anopheles spp., 24 Culex spp., 24 Aedes spp., and 6 Mansonia spp. collected from a buffalo farm in Thailand where concurrently collected water buffalo samples were examined and we found only Anopheles spp. samples were P. bubalis positive. Molecular identification of anopheline mosquito species was conducted by sequencing of the PCR products targeting cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2), and internal transcribed spacer 2 (ITS2) markers. We observed 5 distinct groups of anopheline mosquitoes: Barbirostris, Hyrcanus, Ludlowae, Funestus, and Jamesii groups. The Barbirostris group (Anopheles wejchoochotei or Anopheles campestris) and the Hyrcanus group (Anopheles peditaeniatus) were positive for P. bubalis. Thus, for the first time, our study implicated these anopheline mosquito species as probable vectors of P. bubalis in Thailand.

Discrimination of 15 Amazonian Anopheline Mosquito Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism.

Precise identification of anopheline species is paramount for incrimination of malaria vectors and implementation of a sustainable control program. Anopheline mosquitoes are routinely identified morphologically, a technique that is time-consuming, needs high level of expertise, and prone to misidentifications especially when considering Amazonian species. The aim of this study was therefore to develop a DNA-based identification technique to supplement traditional morphological identification methods for the discrimination of anopheline mosquitoes collected in French Guiana. The internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA) for anopheline species was amplified by polymerase chain reaction (PCR), and digested with AluI/MspI restriction enzymes. PCR-restriction fragments length polymorphism (RFLP) assay was compared to sequencing of the ITS2 region for validation. Fifteen Anopheles species have shown distinct PCR-RFLP profiles. A concordance of 100% was obtained when identification by PCR-RFLP was compared to sequencing of ITS2. A high throughput, fast, and cost-effective PCR-RFLP assay has been developed for unambiguous discrimination of fifteen anopheline mosquito species from French Guiana including primary and suspected secondary malaria vectors.

Attraction of the Mosquitoes Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to a 3-Part Phytochemical Blend in a Mesocosm.

Aedes aegypti (L.) and Aedes albopictus (Skuse) mosquitoes of both sexes were attracted to a 3-part volatile synthetic phytochemical blend but differed according to their component ratios, 7:3:2 or 1:1:1, and their initial concentrations. These arbovirus vectors were presented with the blends as baits in paired baited and blank CFG traps in a large greenhouse mesocosm. Ae. aegypti attraction was highest at a 7:3:2 blend ratio, but at a concentration half that found most effective for an anopheline mosquito species in outdoor screenhouses. Both lower and higher concentrations yielded substantially lower attraction scores for Ae. aegypti. By contrast, the few tests conducted on Ae. albopictus showed that it was not as sensitive to concentration, but again it was more responsive to the 7:3:2 ratio of components than to the 1:1:1 ratio. The two sexes of both species were represented equally in the trap catches, indicating the potential value of this and similar attractive blends for population surveillance and control of Aedes mosquitoes.

Anopheline Diversity in Indonesia: An Evaluation of Animal-Baited Sampling Techniques.

Indonesia has rich Anopheline (Diptera: Culicidae) mosquito species living in various types of ecosystems. The study was conducted to profile and compare Anopheles diversity, equitability, and dominance in various ecosystems using different animal-based sampling techniques. The present study analyzed a subset of data collected from a nation-wide vector and animal reservoirs survey in 2016. Analyses were restricted to three ecosystem types (forest, nonforest, and coastal areas) in Java and Sumatera Islands. A total of 5,477 Anopheles were collected by using animal-baited (n = 1,909) and animal-baited trap nets (n = 1,978), consisting of 23 Anopheline species. Overall, Anopheles vagus was the most abundant species, followed by An. subpictus and An. barbirostris. Among the three ecosystems, the forest had a higher diversity index (H' = 1.98), but each ecosystem has its specific predominant species. Compared with the animal-baited method, the Anopheles abundance collected by animal-baited trap nets was two-fold higher. Ecosystem, elevation, and sampling methods were associated with the abundance of female Anopheles (P-value < 0.001). Our findings revealed that Anopheles were found in a different ecosystem, indicating the potential of malaria transmission. This suggests that improved malaria vector surveillance is essential in all types of ecosystem. Furthermore, the study suggested that animal-baited trap nets could be used as the standard method of outdoor resting sampling in Indonesia in addition to the traditional human landing collection approach.

Malaria vector dynamics and utilization of insecticide-treated nets in low-transmission setting in Southwest Ethiopia: implications for residual transmission

BackgroundUnderstanding the behaviour of local malaria vectors is essential as effectiveness of the commonly used vector-targeted malaria control tools heavily relies on behaviour of the major malaria vectors. This study was conducted to determine species composition, biting behaviour, host preference and infectivity of anopheline mosquitoes, and assess utilization of insecticide-treated nets (ITNs) in a low transmission setting in Southwest Ethiopia.MethodsAdult anopheline mosquitoes were collected using human landing catches (HLCs), Centers for Disease Control and Prevention (CDC) light traps (LTs) and Pyrethrum Spray Catches (PSCs) from June 2016 to May 2018 in Kishe, Jimma Zone, Southwest Ethiopia. The anopheline mosquitoes were morphologically identified. Moreover, sub-sample of An. gambiae s.l. was identified to species using polymerase chain reaction (PCR). Circum-sporozoite proteins (CSPs) and blood meal sources of the anopheline mosquitoes were tested using enzyme-linked immunosorbent assay (ELISA). In addition, a cross-sectional survey was conducted to assess ITN utilization by the inhabitants.ResultsA total of 3659 anopheline mosquitoes comprising An. coustani complex (84.4%), An. gambiae s.l. (11.3%), and An. pharoensis and An. squamosus comprising less than 5% were collected. The anopheline mosquitoes showed marked outdoor (67%) and early evening (63%) biting behaviour. An. coustani complex and An. gambiae s.l. were predominantly zoophilic and anthropophilic, respectively. None of the sampled anopheline were CSP-positive. Most of the households (97.8%) owned at least one ITN, with modest usage by the inhabitants (73.4%). ITN usage was significantly higher among under-five children (AOR = 7.9, 95% CI: 4.41–14.03), household heads and spouses (AOR = 4.8, 95% CI: 3.0–7.59), those with sufficient access to ITNs (AOR = 1.8, 95% CI: 1.39–2.35), and who were not utilizing alternative mosquito repellents (AOR = 2.2, 95% CI: 1.58–2.99).ConclusionThe anopheline mosquito species exhibited predominantly outdoor and early evening biting activity. Household ITN coverage was high with slight gap in usage. Vector control interventions should target outdoor and early biting vectors to further suppress the local mosquito population. Moreover, sensitization of the community on consistent use of ITNs is required.

Living with predators at the larval stage has differential long-lasting effects on adult life history and physiological traits in two anopheline mosquito species

1.Under predation stress, individuals increase their metabolic rate and adopt different resource allocation strategies to favor their direct survival to the detriment of growth, reproduction and self-maintenance. Species and sexes are both faced with different physiological and life history demands and should adopt different strategies which lead to carry-over effects on adult metabolic reserves and on oxidative status.2.Malaria mosquitoes Anopheles coluzzii and An. gambiae are sibling species. In nature, their larvae are exposed to different levels of predation risk and An. coluzzii larvae are more capable of surviving predation threats than An. gambiae, because of a better ability to gauge the threat level and to produce an adapted behavioral response. Impact of this response on adult physiology is however unknown.3.Here, we investigated the carry-over effects of larval predation stress: first, on life history traits such as development length, size and weight at emergence, fecundity and survival; second, on metabolic resources at emergence such as lipids, proteins, carbohydrates and amino acids; and third, on oxidative status with the total anti-oxidant capacity and the oxidation of proteins in the two species and in both sexes.4.We found that on one hand, it took more time for An. coluzzii to develop in presence of a predator and they emerged with a smaller size but a similar weight as control individuals. On the other hand, An. gambiae exposed to predators developed as fast as controls but with both a smaller size and a smaller weight. Fecundity was negatively impacted in both species following exposure to predation threats. Teneral reserves were affected depending on the species and the sex. The total anti-oxidant capacity of individuals exposed to stress was lower than in the controls but a significant level of damages to proteins was detected in An. gambiae males only.5.Our findings suggest that predation stress induces very different physiological responses even in closely related species and between sexes. Allocation of resources in larvae may differ and depend on the species’ aptitude to develop in presence of predators and on the sex, since males and females might adopt different strategies to fulfill their fitness.

Clock genes and environmental cues coordinate Anopheles pheromone synthesis, swarming, and mating.

Anopheles mating is initiated by the swarming of males at dusk followed by females flying into the swarm. Here, we show that mosquito swarming and mating are coordinately guided by clock genes, light, and temperature. Transcriptome analysis shows up-regulation of the clock genes period (per) and timeless (tim) in the head of field-caught swarming Anopheles coluzzii males. Knockdown of per and tim expression affects Anopheles gambiae s.s. and Anopheles stephensi male mating in the laboratory, and it reduces male An. coluzzii swarming and mating under semifield conditions. Light and temperature affect mosquito mating, possibly by modulating per and/or tim expression. Moreover, the desaturase gene desat1 is up-regulated and rhythmically expressed in the heads of swarming males and regulates the production of cuticular hydrocarbons, including heptacosane, which stimulates mating activity.

Cuticular pheromone circadian regulation

Mosquito Biology Several species of anopheline mosquitoes are important malaria vectors in Africa. Male mosquitoes show species-specific swarming behaviors at certain times of the day to attract females for mating. Wang et al. found that transcriptional patterns of metabolic and immune function genes apparently showed a diurnal rhythm that correlated with the physiological demands of mating flight swarming (see the Perspective by Manoukis). By altering temperature and light regimes and by knocking out the master genes period and timeless for circadian clock regulation, the authors disrupted mating flight behavior in a combination of cage experiments and enclosed field conditions. Knocking out the rhythmically expressed desaturase enzyme reduced cuticular hydrocarbon pheromone production and limited mating success. These key interacting components of the diurnal regulation of mosquito mating behavior are potential targets for alternative malaria control strategies. Science , this issue p. [411][1]; see also p. [340][2] [1]: /lookup/doi/10.1126/science.abd4359 [2]: /lookup/doi/10.1126/science.abf7917

Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae)

BackgroundIn 1987, Gillies and Coetzee published a pictorial key for the morphological identification of adult female mosquitoes. Since then, several new species of anopheline mosquitoes have been described.MethodsThe 1987 key to adult female mosquitoes was used as the template for the current key.ResultsNew species described in the literature over the past 32 years have been included. A list of all currently known Afrotropical species is provided. Anopheles stephensi is included for the first time as occurring on the African continent.ConclusionsAn updated key for the morphological identification of Afrotropical anopheline species is presented.

Anopheline Mosquito Species Composition, Kdr Mutation Frequency, and Parasite Infectivity Status in Northern Tanzania.

The scaling-up of malaria control interventions in northern Tanzania has resulted in a decline in malaria prevalence and vector species composition. Despite this achievement, residual malaria transmission remains a concern in the area. The main aim of this study was to investigate malaria vector species composition, parasite infectivity rates, and the presence of insecticide knockdown resistance (kdr) mutations in three sites that have experienced a significant decline in malaria in northern Tanzania. Adult mosquitoes were sampled using light traps in houses and hand-aspirators in cowsheds, whereas the standard dipping method was used for sampling mosquito larvae. Adult mosquitoes identified as Anopheles gambiae s.l. and An. funestus s.l. and larval stages III and IV of An. gambiae s.l. were stored in absolute ethanol for further laboratory molecular identification. The identified species in the An. gambiae complex were An. gambiae s.s., An. merus, An. quadriannulatus, and An. arabiensis, whereas the An. funestus group comprised An. funestus s.s., An. rivulorum, and An. leesoni. For An. gambiae s.s. analyzed from Zeneth, 47.6% were kdr-East homozygous susceptible, 35.7% kdr-East heterozygous resistant, 9.6% kdr-East homozygous resistant, and 7.1% undefined, whereas specimens from Kwakibuyu were 45.5% kdr-East homozygous susceptible, 32.7% kdr-East heterozygous resistant, 16.3% kdr-East homozygous resistant, and 5.5% undefined. There were no kdr-West alleles identified from any specimen. The overall malaria parasite infectivity rate was 0.75%. No infections were found in Moshi. The findings indicate that populations of the major malaria vector mosquitoes are still present in the study area, with An. funestus taking a lead in malaria transmission.

Prevalence of human and non-human primate Plasmodium parasites in anopheline mosquitoes: a cross-sectional epidemiological study in Southern Vietnam

BackgroundHuman malaria is a major threat in rural communities of central Vietnam. Anopheles dirus and Anopheles minimus species are critical malaria vectors in Vietnam, which transmit Plasmodium parasites. However, the entomological aspects of malaria transmission in some of the central provinces of Vietnam remain unexplored. Hence, a cross-sectional entomological survey was carried out to identify the malaria vector species and the transmission of Plasmodium parasites in seven endemic provinces of Vietnam.MethodsMosquitoes were collected from seven provinces, Gia Lai, Khanh Hoa, Phu Yen, Ninh Thuan, Binh Thuan, Dong Nai, and Binh Phuoc. The collection was conducted for four to eight consecutive nights using three established methods, indoor and outdoor human landing catches and light trap method. Nested-PCR analysis was performed to detect the Plasmodium species in the separated thorax and the abdomen of the individual mosquitoes.ResultsA total of 2278 mosquitoes belonging to one of the four species of anopheline mosquitoes, An. dirus, An. maculatus, An. aconitus, and An. minimus were collected. Among the collected mosquitoes, 1398 were analysed using nested-PCR, of which, 40 mosquitoes were positive for Plasmodium parasites. Most of these parasites were detected in the samples from the thorax region, followed by the abdominal portion. The parasites were detected in both the thorax and abdomen of An. dirus. Seven species of Plasmodium parasites were detected during the analysis, of which, Plasmodium inui was the most common species, followed by Plasmodium falciparum, Plasmodium vivax, Plasmodium cynomolgi, Plasmodium coatneyi, Plasmodium knowlesi, and Plasmodium fieldi. Out of the 49 positive samples, 12 showed mixed infections. Co-infection of P. inui with human and other non-human primate Plasmodium species was common.ConclusionsThis study demonstrated the presence of human and non-human primate Plasmodium infection in An. dirus, a predominant malarial vector. Further, we showed that An. maculatus and An. minimus species also take part in malarial transmission. This might potentially lead to an alarming situation conducive for the emergence of novel zoonotic malaria.

Distribution and seasonal abundance of Anopheline mosquitoes and their association with rainfall around irrigation and non-irrigation areas in Nigeria

The abundance of Anopheline mosquito species is the most common entomological measurement to determine the relationship between vectors and malaria incidence. We conducted an entomological survey to determine mosquito species diversity and abundance in relation to rainfall in Omi reservoir irrigation area, Nigeria. We collected adult mosquitoes from 10 randomly selected residential houses using Pyrethrum spray sheet and Human Landing Catch methods. We grouped the samples into irrigated (intervention) and non-irrigated (control) communities. During the 12-month sampling period, we collected a total of 4 285 mosquitoes belonging to 10 species in one family. The three most common species during this study were Anopheles gambiae, Anopheles funestus and Culex quinquefasciatus. Irrigated community has higher numbers of mosquitoes (69,4%) compared to those collected in non-irrigated community (32,0%). Comparing the two collection methods used, Pyrethrum spray sheet has a greater number 2 225(75,4%) of mosquitoes than those with Human Landing Catch method 724(24,6%). During dry season, we collected fewer mosquitoes. The lowest number was collected in February (114) and the highest occurring during the wet season in July (445).

Phylogeny of Anophelinae using mitochondrial protein coding genes.

Malaria is a vector-borne disease that is a great burden on the poorest and most marginalized communities of the tropical and subtropical world. Approximately 41 species of Anopheline mosquitoes can effectively spread species of Plasmodium parasites that cause human malaria. Proposing a natural classification for the subfamily Anophelinae has been a continuous effort, addressed using both morphology and DNA sequence data. The monophyly of the genus Anopheles, and phylogenetic placement of the genus Bironella, subgenera Kerteszia, Lophopodomyia and Stethomyia within the subfamily Anophelinae, remain in question. To understand the classification of Anophelinae, we inferred the phylogeny of all three genera (Anopheles, Bironella, Chagasia) and major subgenera by analysing the amino acid sequences of the 13 protein coding genes of 150 newly sequenced mitochondrial genomes of Anophelinae and 18 newly sequenced Culex species as outgroup taxa, supplemented with 23 mitogenomes from GenBank. Our analyses generally place genus Bironella within the genus Anopheles, which implies that the latter as it is currently defined is not monophyletic. With some inconsistencies, Bironella was placed within the major clade that includes Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia, which were found to be monophyletic groups within Anophelinae. Our findings provided robust evidence for elevating the monophyletic groupings Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia to genus level; genus Anopheles to include subgenera Anopheles, Baimaia, Cellia and Christya; Anopheles parvus to be placed into a new genus; Nyssorhynchus to be elevated to genus level; the genus Nyssorhynchus to include subgenera Myzorhynchella and Nyssorhynchus; Anopheles atacamensis and Anopheles pictipennis to be transferred from subgenus Nyssorhynchus to subgenus Myzorhynchella; and subgenus Nyssorhynchus to encompass the remaining species of Argyritarsis and Albimanus Sections.

Anopheline mosquitoes behaviour and entomological monitoring in southwestern Ethiopia.

Despite a tremendous expansion in the financing and coverage of malaria control programmes, the disease continues to be a global health threat. This study was conducted to assess the entomological parameters of anopheline mosquitoes, viz. species composition, abundance, longevity, behaviour and infectivity rates in Kersa district, Jimma zone, southwestern Ethiopia. Mosquito collection was carried out from each selected household in each of the nine selected study villages of Kersa district, using CDC light-traps and pyrethrum spray catches (PSCs) for seven months (June to December 2014). Mosquito count data were log transformed before analysis and the data were analyzed using SPSS software package version 16.0. Analysis of variance (ANOVA) was employed to compare means and Tukey's post-hoc test was used for mean separation. In total, 1559 adult female anopheline mosquitoes, representing at least three species were collected from the study villages. Of these, 1122 were collected by CDC light-traps and the rest 437 were collected by PSCs. Anopheles gambiae s.1. (71.8%) was the most abundant species, followed by An. coustani s.1. (22%) and An. pharoensis (6.2%). The mean monthly density of anopheline mosquito species was highly significant (p < 0.001). Significantly (p <0.05) higher population of An. gambiae s.1. were trapped indoor than outdoor. However, outdoor mean densities of An. pharoensis and An. coustani s.1. were significantly (p < 0.001) higher than indoor mean densities. The longevity of An. gambiae s.1. was higher in the months of June, July and August (mean 7.32 days) and lower in the months of October, November and December (mean 2.94 days). Two An. gambiae s.1. specimens were found positive for Plasmodium vivax 210 polymorphs and the overall infectivity rate was estimated to be 1.04%. This study could contribute to the understanding of anopheline mosquitoes with respect to their composition, dynamics, distribution and behaviour in Kersa district, for evidence based malaria vector control programmes, mainly in the appropriate timing of the indoor residual spray programme.

A whole genome gene content phylogenetic analysis of anopheline mosquitoes

Construction of stringent gene content matrices was accomplished for 21 Anopheline mosquito species and strains and four outgroups species. The presence absence matrix using e−75 as a cutoff in single linkage clustering had over 17,000 ortholog groups. We used the gene content matrix to generate a phylogenetic hypothesis that is in general agreement with gene sequence based phylogenies. In addition to establishing a congruent gene content phylogeny we examined the consistency of three methods for analyzing presence absence data – unweighted parsimony, dollo parsimonly and maximum likelihood using a BINGAMMA model. An examination of the chromosomal location of the gains and losses in the presence absence matrix revealed a low frequency of gains and losses at centromeres and tips of chromosomes.

Mosquito Vectors and the Globalization of Plasmodium falciparum Malaria.

Plasmodium falciparum malaria remains a devastating public health problem. Recent discoveries have shed light on the origin and evolution of Plasmodium parasites and their interactions with their vertebrate and mosquito hosts. P. falciparum malaria originated in Africa from a single horizontal transfer between an infected gorilla and a human, and became global as the result of human migration. Today, P. falciparum malaria is transmitted worldwide by more than 70 different anopheline mosquito species. Recent studies indicate that the mosquito immune system can be a barrier to malaria transmission and that the P. falciparum Pfs47 gene allows the parasite to evade mosquito immune detection. Here, we review the origin and globalization of P. falciparum and integrate this history with analysis of the biology, evolution, and dispersal of the main mosquito vectors. This new perspective broadens our understanding of P. falciparum population structure and the dispersal of important parasite genetic traits.

Infection of laboratory colonies of Anopheles mosquitoes with Plasmodium vivax from cryopreserved clinical isolates

Plasmodium vivax is the most geographically widespread malaria parasite. Unique features of transmission biology complicate P. vivax control. Interventions targeting transmission are required for malaria eradication. In the absence of an in vitro culture, transmission studies rely on live isolates from non-human primates or endemic regions. Here, we demonstrate P. vivax gametocytes from both India and Brazil are stable during cryopreservation. Importantly, cryopreserved gametocytes from Brazil were capable of infecting three anopheline mosquito species in feedings done in the United States. These findings create new opportunities for transmission studies in diverse locales.

Phylogeny of anopheline (Diptera: Culicidae) species in southern Africa, based on nuclear and mitochondrial genes.

A phylogeny of anthropophilic and zoophilic anopheline mosquito species was constructed, using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes. The ITS2 alignment, typically difficult due to its noncoding nature and large size variations, was aided by using predicted secondary structure, making this phylogenetically useful gene more amenable to investigation. This phylogeny is unique in explicitly including zoophilic, non-vector anopheline species in order to illustrate their relationships to malaria vectors. Two new, cryptic species, Anopheles funestus-like and Anopheles rivulorum-like, were found to be present in Zambia for the first time. Sequences from the D3 region of the 28S rDNA suggest that the Zambian An. funestus-like may be a hybrid or geographical variant of An. funestus-like, previously reported in Malawi. This is the first report of An. rivulorum-like sympatric with An. rivulorum (Leeson), suggesting that these are separate species rather than geographic variants.