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Abstract Isolation mechanisms between mosquito species of the Anopheles gambiae complex, which includes major malaria vectors, remain poorly understood. In some cases, pre-zygotic barriers have been shown to limit gene flow between species of the complex, leading to a low level of hybridisation in nature. Post-zygotic mechanisms manifest with F1 hybrid males fully sterile and F1 hybrid females with reduced fertility. Genetic approaches combined with DNA sequencing techniques have highlighted the involvement of genomic regions in hybrid incompatibility with a predominant role of the X chromosome. In addition, differences in the phenotype of F1 hybrid males have been identified depending on the directionality of the parental cross used to generate them. All these studies have focused on the interaction of nuclear DNA elements in hybrid individuals. Given the role that mitochondrial DNA plays in genetic incompatibilities within other organisms and its unique inheritance pattern, commonly maternal, we conducted a genetic study that relied on the introgression of mitochondrial DNA between Anopheles gambiae and Anopheles arabiensis. The findings indicate that the mitochondrial switch does not appear to restore the fertility of F1 hybrid males, suggesting that mitochondrial DNA may not play a role in hybrid incompatibilities in these Anopheles species.

Abstract The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with

The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with < 92% mortality in all tests, but varied between collections, with mosquitoes from Kimpese being the most resistant. Whole genome sequencing of 165 An. gambiae revealed evidence for genetic differentiation between Kimpese and Kapolowe/Mikalayi, but not between the latter two sample sites despite separation of approximately 800 km. Surprisingly, there was evidence of population structure at a small spatial scale between collection subsites in Kimpese, despite separation of just tens of kilometres. Intra-population (H12) and inter-population (FST) genome scans identified multiple peaks corresponding to genes associated with insecticide resistance such as the voltage gated sodium channel (Vgsc) target site on chromosome 2L, a Cyp6 cytochrome P450 cluster on chromosome arm 2R, and the Cyp9k1 P450 gene on chromosome X. In addition, in the Kimpese subsites, the P450 redox partner gene Cpr showed evidence for contemporary selection (H12) and population differentiation (FST) meriting further exploration as a potential resistance associated marker. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA has been a popular marker in phylogeography, phylogeny, and molecular ecology, but its complex evolution is increasingly recognized. Here, we investigated mitochondrial DNA variation in Anopheles gambiae and Anopheles coluzzii , in relation to other species in the Anopheles gambiae complex, by assembling the mitogenomes of 1,219 mosquitoes across Africa. The mitochondrial DNA phylogeny of the Anopheles gambiae complex was consistent with previously reported highly reticulated evolutionary history, revealing important discordances with the species tree. The three most widespread species (An. gambiae, An. coluzzii , and Anopheles arabiensis), known for extensive historical introgression, could not be discriminated based on mitogenomes. Furthermore, a monophyletic clustering of the three saltwater-tolerant species (Anopheles merus , Anopheles melas , and Anopheles bwambae) in the Anopheles gambiae complex also suggested that introgression and possibly selection shaped mitochondrial DNA evolution. Mitochondrial DNA variation in An. gambiae and An. coluzzii across Africa revealed significant partitioning among populations and species. A peculiar mitochondrial DNA lineage found predominantly in An. coluzzii and in the hybrid taxon of the African " far-west " exhibited divergence comparable to the interspecies divergence in the Anopheles gambiae complex, with a geographic distribution matching closely An. coluzzii 's geographic range. This phylogeographic relict of the An. coluzzii and An. gambiae split was associated with population and species structure, but not with the rare Wolbachia occurrence. The lineage was significantly associated with single nucleotide polymorphisms in the nuclear genome, particularly in genes associated with pathogen and insecticide resistance. These findings underline potential mitonuclear coevolution history and the role played by mitochondria in shaping metabolic responses to pathogens and insecticides in Anopheles. [ABSTRACT FROM AUTHOR]

Abstract Background Chromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological, morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles gambiae. The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R, is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping the 2Ru inversion in individual specimens of both mosquito species. Methods We designed tetra-primer amplification refractory mutation system (ARMS)-PCR assays based on five tag single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation. The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-throughput approach that employs targeted sequencing of multiplexed PCR amplicons. Results A successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis, produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting 2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed. Conclusions The tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already available for the other common polymorphic inversions, 2La, 2Rb and 2Rc, this assay will allow investigations of the adaptive value of the complex set of inversion systems observed in the two major malaria vectors in the Afrotropical region. Graphical Abstract

The molecular characterization of Anopheles gambiae complex mosquitoes in rice agroecosystems in Anambra State was studied from January to December 2020. The study was carried out in rice farms randomly selected from four local government areas in Anambra State. Mosquito larvae were collected with a WHO (350ml) standard dipper at the sampling point of each of the selected rice farms twice every month. The larval samples were taken to the insectary of the National Arbovirus and Vectors Research Centre (NAVRC) to be reared into adults. The molecular characterization of sibling species of Anopheles gambiae complex was accomplished with Polymerase Chain Reaction (PCR). A total of 289 An. gambiae complex mosquitoes were subjected to PCR and identified. The species were identified as An. gambiae s.s., 144 (49.8%) and An. coluzzii. 145 (50.1%). These observations indicated that malaria transmission in the Anambra state is mainly carried out by the Anopheles sibling species. This enhances, sustains, and extends the malaria disease transmission in this geographical area. [ABSTRACT FROM AUTHOR]

Background Chromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological, morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles gambiae. The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R, is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping the 2Ru inversion in individual specimens of both mosquito species. Methods We designed tetra-primer amplifcation refractory mutation system (ARMS)-PCR assays based on five tag single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation. The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-through-put approach that employs targeted sequencing of multiplexed PCR amplicons. Results A successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis, produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting 2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed. Conclusions The tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already available for the other common polymorphic inversions, 2La, 2Rb and 2Rc, this assay will allow investigations of the adaptive value of the complex set of inversion systems observed in the two major malaria vectors in the Afrotropical region. [ABSTRACT FROM AUTHOR]

Abstract Background Vector control is considered to be the most successful component of malaria prevention programs and a major contributor to the reduction of malaria incidence over the last two decades. However, the success of this strategy is threatened by the development of resistance to insecticides and behavioural adaptations of vectors. The aim of this study was to monitor malaria transmission and the distribution of insecticide resistance genes in Anopheles populations from three rural areas of the Moyen Ogooué Province of Gabon. Methods Anopheles spp. were collected using human landing catches in Bindo, Nombakélé and Zilé, three villages located in the surroundings of Lambaréné, during both the rainy and dry seasons. Mosquitoes were identified morphologically, and DNA was extracted from heads and thoraces. Members of the Anopheles gambiae complex were identified by molecular methods using the PCR SINE200 protocol and by sequencing of the internal transcribed spacer 2 region. Taqman assays were used to determine Plasmodium infection and the presence of resistance alleles. Results Anopheles gambiae sensu lato (97.7%), An. moucheti (1.7%) and An. coustani (0.6%) were the three groups of species collected. Anopheles gambiae sensu stricto (98.5%) and An. coluzzii (1.5%) were the only species of the An. gambiae complex present in the collection. Of the 1235 Anopheles collected, 1193 were collected during the rainy season; these exhibited an exophagic behaviour, and consistently more mosquitoes were collected outdoor than indoor in the three study areas. Of the 1166 Anopheles screened, 26 (2.2%) were infected with Plasmodium species, specifically Plasmodium falciparum (66.7%), P. malariae (15.4%), P. ovale curtisi (11.5%) and P. ovale wallikeri (3.8%). Malaria transmission intensity was high in Zilé, with an average annual entomological inoculation rate (aEIR) of 243 infective bites per year, while aEIRs in Bindo and Nombakélé were 80.2 and 17 infective bites per year, respectively. Both the L1014F and L1014S mutations were present at frequencies > 95% but no Ace1G119S mutation was found. Conclusion Our results demonstrate that malaria transmission intensity is heterogeneous in these three rural areas of Moyen Ogooué Province, with areas of high transmission, such as Zilé. The exophagic behaviour of the mosquitoes as well as the high frequency of resistance mutations are serious challenges that need to be addressed by the deployment of control measures adapted to the local setting. Graphical Abstract

BackgroundChemical-based malaria vector control interventions are threatened by the development of insecticide resistance and changes in the behavior of the vectors, and thus require the development of alternative control methods. Bacterial-based larvicides have the potential to target both insecticide resistant and outdoor-biting mosquitoes and are safe to use in the environment. However, the currently available microbial larvicide formulations have a short duration of activity requiring frequent re-applications which increase the cost of control interventions. This study was designed to evaluate the efficacy and duration of activity of two long-lasting formulations of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) (LL3 and FourStar®) under field conditions in western Kenya highlands.MethodsThree sites were selected for this study in the highlands of western Kenya. In each site, one hundred anopheline larval habitats were selected and assigned to one of three arms: (i) LL3; (ii) FourStar®; and (iii) untreated control larval habitats. Four types of larval habitats were surveyed: abandoned gold mines, drainage canals, fish ponds and non-fish ponds. The habitats were sampled for mosquito larvae by using a standard dipping technique and collected larvae were recorded according to the larval stages of the different Anopheles species. The larvicides were applied at manufacturers' recommended dosage of 1 briquette per 100 square feet. Both treatment and control habitats were sampled for mosquito larvae immediately before treatment (day 0), and then at 24 hours, 3 days and weekly post-treatment for 5 months.ResultsOverall larval density in treatment habitats was significantly reduced after application of the two microbial larvicides as compared to the control habitats. Post-intervention reduction in anopheline larval density by LL3 was 65, 71 and 84% for 1 day, 2 weeks and 4 weeks, respectively. FourStar® reduced anopheline larval density by 60, 66 and 80% for 1 day, 2 weeks and 4 weeks, respectively. Comparisons between the treatments reveal that LL3 and FourStar® were similar in efficacy. A higher reduction in Anopheles larval density was observed in the abandoned goldmines, while drainage canals had the lowest reduction.ConclusionsBoth LL3 and FourStar® long-lasting microbial larvicides were effective in reducing immature stages of An. gambiae complex and An. funestus group species, with significant reductions lasting for three months post-application.

Lymphatic filariasis (LF) elimination activities started in Mali in 2005 in the most endemic areas and reached countrywide coverage in 2009. In 2004, the district of Bamako was endemic for LF with a prevalence of 1.5%. The current study was designed to determine LF endemicity level in the urban area of Bamako after three rounds of ivermectin and albendazole mass drug administration (MDA). A cross-sectional study was conducted in 2011 in Bamako city, consisting of human prevalence and entomological surveys. Volunteers aged 14 years and above were invited to participate and tested for evidence of Wuchereria bancrofti using night time blood thick smear microfilarial count and blood spots for LF antibodies using the SD BIOLINE Oncho/LF IgG4 Biplex rapid test (Ov16/Wb123). Mosquitoes were collected using CDC light and gravid traps and tested using molecular methods. Poolscreen software v2.0 was used to estimate vector transmission potential. Of the 899 volunteers, one (0.11%) was found to be positive for LF using the Oncho/LF IgG4 Biplex rapid test, and none was found to have Wuchereria bancrofti microfilariae. No mosquitoes were found infected among 6174 Culex spp. (85.2%), 16 Anopheles gambiae s.l. (An. gambiae s.l.) (0.2%), 26 Aedes spp. (0.4%), 858 Ceratopogonidae (11.8%) and 170 other insects not identified (2.3%) tested. Our data indicate that there was no active LF transmission in the low prevalence urban district of Bamako after three MDA rounds. These data helped the National LF programme move forward towards the elimination goal. [ABSTRACT FROM AUTHOR]

Background: Vector control is considered to be the most successful component of malaria prevention programs and a major contributor to the reduction of malaria incidence over the last two decades. However, the success of this strategy is threatened by the development of resistance to insecticides and behavioural adaptations of vectors. The aim of this study was to monitor malaria transmission and the distribution of insecticide resistance genes in Anopheles populations from three rural areas of the Moyen Ogooué Province of Gabon. Methods: Anopheles spp. were collected using human landing catches in Bindo, Nombakélé and Zilé, three villages located in the surroundings of Lambaréné, during both the rainy and dry seasons. Mosquitoes were identified morphologically, and DNA was extracted from heads and thoraces. Members of the Anopheles gambiae complex were identified by molecular methods using the PCR SINE200 protocol and by sequencing of the internal transcribed spacer 2 region. Taqman assays were used to determine Plasmodium infection and the presence of resistance alleles. Results: Anopheles gambiae sensu lato (97.7%), An. moucheti (1.7%) and An. coustani (0.6%) were the three groups of species collected. Anopheles gambiae sensu stricto (98.5%) and An. coluzzii (1.5%) were the only species of the An. gambiae complex present in the collection. Of the 1235 Anopheles collected, 1193 were collected during the rainy season; these exhibited an exophagic behaviour, and consistently more mosquitoes were collected outdoor than indoor in the three study areas. Of the 1166 Anopheles screened, 26 (2.2%) were infected with Plasmodium species, specifically Plasmodium falciparum (66.7%), P. malariae (15.4%), P. ovale curtisi (11.5%) and P. ovale wallikeri (3.8%). Malaria transmission intensity was high in Zilé, with an average annual entomological inoculation rate (aEIR) of 243 infective bites per year, while aEIRs in Bindo and Nombakélé were 80.2 and 17 infective bites per year, respectively. Both the L1014F and L1014S mutations were present at frequencies > 95% but no Ace1G119S mutation was found. Conclusion: Our results demonstrate that malaria transmission intensity is heterogeneous in these three rural areas of Moyen Ogooué Province, with areas of high transmission, such as Zilé. The exophagic behaviour of the mosquitoes as well as the high frequency of resistance mutations are serious challenges that need to be addressed by the deployment of control measures adapted to the local setting. [ABSTRACT FROM AUTHOR]

Abstract Background Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity. Methods Using a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries. Results Of four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate. Conclusions We have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control.

Bacterial larvicides Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) have been used extensively for mosquito control. However, their efficacy varies greatly mainly due to factors related to target mosquitoes, larval habitat conditions, and inherent larvicide properties. We evaluated the efficacy of Bti (Bactivec®) and Bs (Griselesf®) for control of Anopheles gambiae complex, Culex quinquefasciatus and Aedes aegypti larvae under laboratory and semi-field conditions in northeastern Tanzania. Laboratory bioassays were conducted with five to six different concentrations of Bti and Bs, replicated four times and the experiment repeated on three different days. Larvae mortality was recorded at 24 or 48 h after the application of larvicide and subjected to Probit analysis. Laboratory bioassays were followed by semi-field trials to establish initial and residual activity of Bti and Bs. Semi-field trials were conducted in artificial larval habitats in the open sunlit ground and in “mosquito spheres”. These artificial larval habitats were colonized with mosquito larvae, treated with Bti and Bs, and the impact of treatments on mosquito larvae was monitored daily. Lethal concentration values that caused 50% and 95% mortalities of test larvae (LC50 and LC95) showed that An. gambiae complex and Cx. quinquefasciatus tested were highly susceptible to Bti and Bs under laboratory conditions. Likewise, larvae of Ae. aegypti were highly susceptible to Bti, with LC95 value as low as 0.052 mg/l. However, Ae. aegypti larvae were not susceptible to Bs under practical doses of laboratory settings. In semi-field trials, all treatment dosages for Bti provided 91.0–100% larval mortality within 24 h whereas Bs resulted in 96.8–100% larval mortality within the same time-frame. Bs had a more prolonged residual activity, with pupal reductions range of 55.7–100% for 9 days at all application rates while the corresponding pupal reduction with Bti was 15.4–100% for 5 days. Due to the low residual activity of Bti and Bs tested, weekly application at a maximum label rate would be appropriate to reduce mosquito larvae in natural larval habitats. Based on laboratory findings, Bs product tested would not be recommended for use in the control of Ae. aegypti.

Abstract Background Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species’ spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. Methods We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. Results For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. Conclusions The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.

Haldane’s rule of speciation states that sterility or inviability affects the heterogametic sex of inter-species hybrids. Darwin’s corollary to Haldane’s rule implies that there are asymmetric phenotypes in inter-species hybrids from reciprocal crosses. Studying the phenotypes of F1 hybrids among closely related species of malaria mosquitoes can assist researchers in identifying the genetic factors and molecular mechanisms of speciation. To characterize phenotypes of sterile hybrid males in the Anopheles gambiae complex, we performed crosses between laboratory strains of An. merus and either An. gambiae or An. coluzzii. The reproductive tracts had normal external morphology in hybrid males from crosses between female An. merus and male An. gambiae or An. coluzzii. Despite being sterile, these males could copulate with females for a normal period of time and could transfer a mating plug to induce female oviposition and monogamy. In contrast, the entire reproductive tracts in hybrid males from crosses between female An. gambiae or An. coluzzii and male An. merus were severely underdeveloped. These males had atrophic testes and reduced somatic organs of the reproductive system including male accessary glands and ejaculatory duct. In addition, hybrid males with underdeveloped reproductive tracts displayed a shorter copulation time with females and failed to induce female oviposition and monogamy due to their inability to form and transfer a plug to females during mating. The asymmetry of the phenotypes associated with hybrid male sterility suggests that different genetic factors and molecular mechanisms are responsible for reproductive isolation in reciprocal crosses among species of the An. gambiae complex.

Background The SEMOS gold mine in Sadiola, southwestern Mali, has been implementing a malaria vector control programme for 15 y using indoor residual house spraying and sporadic larval control. Periodic screening of the vector populations have been carried out over the years to provide information to the control programme, mainly on vector species present and their insecticide resistance status. The data from five entomological surveys, carried out in 2006, 2011, 2014, 2016 and 2018, are presented. Methods Adult mosquitoes were collected resting on walls inside houses and on verandas. Insecticide susceptibility assays were carried out and mosquitoes subsequently identified by species using molecular assays. Results The major malaria vector mosquitoes, Anopheles gambiae and Anopheles arabiensis were abundant at each sampling period with Anopheles coluzzii and Anopheles funestus being rare or absent. Anopheles rivulorum was identified in 2006 and Anopheles leesoni in 2016. The presence of Anopheles rivulorum -like, identified for the first time in 2018, was not screened for in previous surveys. Insecticide susceptibility bioassays showed resistance in both A. gambiae and A. arabiensis to pyrethroids, carbamates and dichlorodiphenyltrichloroethane over the 12 y. Conclusions This is the first record of A. rivulorum -like west of Côte d'Ivoire. Resistance levels to the three classes of insecticides were variable but appeared to decrease after pyrethroids were discontinued for house spraying. [ABSTRACT FROM AUTHOR]

Background: Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity. Methods: Using a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries. Results: Of four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate. Conclusions: We have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control. [ABSTRACT FROM AUTHOR]

The Anopheles gambiae complex is comprised of eight morphologically indistinguishable species and has emerged as a model system for the study of speciation genetics due to the rapid radiation of its member species over the past two million years. Male hybrids between most An. gambiae complex species pairs are sterile, and some genotype combinations in hybrid males cause inviability. We investigated the genetic basis of hybrid male inviability and sterility between An. coluzzii and An. quadriannulatus by measuring segregation distortion and performing a QTL analysis of sterility in a backcross population. Hybrid males were inviable if they inherited the An. coluzzii X chromosome and were homozygous at one or more loci in 18.9 Mb region of chromosome 3. The An. coluzzii X chromosome has a disproportionately large effect on hybrid sterility when introgressed into an An. quadriannulatus genetic background. Additionally, an epistatic interaction between the An. coluzzii X and a 1.12 Mb, pericentric region of the An. quadriannulatus 3L chromosome arm has a statistically significant contribution to the hybrid sterility phenotype. This same epistatic interaction occurs when the An. coluzzii X is introgressed into the genetic background of An. arabiensis, the sister species of An. quadriannulatus, suggesting that this may represent one of the first Dobzhansky–Muller incompatibilities to evolve early in the radiation of the Anopheles gambiae species complex. We describe the additive effects of each sterility QTL, epistatic interactions between them, and genes within QTL with protein functions related to mating behavior, reproduction, spermatogenesis, and microtubule morphogenesis, whose divergence may contribute to post-zygotic reproductive isolation between An. coluzzii and An. quadriannulatus.

Abstract Background Members of the Anopheles gambiae (s.l.) complex are one of the major vectors of malaria in Africa. LLINs and IRS are the most effective tools used in vector control of malaria. However, their effectiveness may be hampered by the development and spread of insecticide resistance in the target vectors species. The objective of this study was to assess the susceptibility of Anopheles gambiae (s.l.) mosquitoes from South-West Cameroon to deltamethrin, permethrin and to malathion, four years after the mass deployment of LLINs. Methods Anopheles larvae were collected from Limbe, Tiko and Buea, three cities of the Fako division and reared until adult emergence. Adult mosquitoes from field larvae were identified as belonging to the Anopheles gambiae (s.l.) complex using standard identification keys. Susceptibility of mosquito samples to deltamethrin, permethrin and malathion was assessed using WHO susceptibility tests protocol for adult mosquitoes. Molecular identification of tested samples was performed using the PCR SINE200 protocol and by PCR-RFLP. The kdr alleles were genotyped using the hot ligation oligonucleotide assay (HOLA). Results Two species of the An. gambiae (s.l.) complex, An. coluzzii and An. gambiae (s.s.) were identified in all three study locations with high proportions of An. coluzzii in Limbe (84.06%) and Tiko (92.2%), while in Buea, An. coluzzii (55.6%) and An. gambiae (s.s.) (44.4%) occurred almost in the same proportions. Tested samples were found resistant to pyrethroids (deltamethrin and permethrin) in all locations (< 90% mortality), with > 3-fold increase of KDT50 values compared with the Kisumu susceptible reference strain of An. gambiae (s.s.). However, the mosquito populations from Limbe and Buea were fully susceptible to malathion. The L1014F kdr was found in both An. coluzzii and An. gambiae (s.s.) with the highest frequencies found in An. gambiae (s.l.) populations from Tiko (94%) and Buea (90%) compared with the Limbe population (66%) (P = 0.00063, df = 2). No kdr L1014S was observed in analyzed samples. Conclusions These findings reemphasize the ongoing development of An. gambiae (s.l.) resistance to pyrethroids used in impregnating LLINs and suggest the use of malathion as an alternative insecticide for IRS in complementarity with LLINs.

Abstract Background Chemical-based malaria vector control interventions are threatened by the development of insecticide resistance and changes in the behavior of the vectors, and thus require the development of alternative control methods. Bacterial-based larvicides have the potential to target both insecticide resistant and outdoor-biting mosquitoes and are safe to use in the environment. However, the currently available microbial larvicide formulations have a short duration of activity requiring frequent re-applications which increase the cost of control interventions. This study was designed to evaluate the efficacy and duration of activity of two long-lasting formulations of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) (LL3 and FourStar®) under field conditions in western Kenya highlands. Methods Three sites were selected for this study in the highlands of western Kenya. In each site, one hundred anopheline larval habitats were selected and assigned to one of three arms: (i) LL3; (ii) FourStar®; and (iii) untreated control larval habitats. Four types of larval habitats were surveyed: abandoned gold mines, drainage canals, fish ponds and non-fish ponds. The habitats were sampled for mosquito larvae by using a standard dipping technique and collected larvae were recorded according to the larval stages of the different Anopheles species. The larvicides were applied at manufacturers’ recommended dosage of 1 briquette per 100 square feet. Both treatment and control habitats were sampled for mosquito larvae immediately before treatment (day 0), and then at 24 hours, 3 days and weekly post-treatment for 5 months. Results Overall larval density in treatment habitats was significantly reduced after application of the two microbial larvicides as compared to the control habitats. Post-intervention reduction in anopheline larval density by LL3 was 65, 71 and 84% for 1 day, 2 weeks and 4 weeks, respectively. FourStar® reduced anopheline larval density by 60, 66 and 80% for 1 day, 2 weeks and 4 weeks, respectively. Comparisons between the treatments reveal that LL3 and FourStar® were similar in efficacy. A higher reduction in Anopheles larval density was observed in the abandoned goldmines, while drainage canals had the lowest reduction. Conclusions Both LL3 and FourStar® long-lasting microbial larvicides were effective in reducing immature stages of An. gambiae complex and An. funestus group species, with significant reductions lasting for three months post-application.

The Anopheles gambiae complex is comprised of eight morphologically indistinguishable species and has emerged as a model system for the study of speciation genetics due to the rapid radiation of its member species over the past two million years. Male hybrids between most An. gambiae complex species pairs are sterile, and some genotype combinations in hybrid males cause inviability. We investigated the genetic basis of hybrid male inviability and sterility between An. coluzzii and An. quadriannulatus by measuring segregation distortion and performing a QTL analysis of sterility in a backcross population. Hybrid males were inviable if they inherited the An. coluzzii X chromosome and were homozygous at one or more loci in 18.9 Mb region of chromosome 3. The An. coluzzii X chromosome has a disproportionately large effect on hybrid sterility when introgressed into an An. quadriannulatus genetic background. Additionally, an epistatic interaction between the An. coluzzii X and a 1.12 Mb, pericentric region of the An. quadriannulatus 3L chromosome arm has a statistically significant contribution to the hybrid sterility phenotype. This same epistatic interaction occurs when the An. coluzzii X is introgressed into the genetic background of An. arabiensis , the sister species of An. quadriannulatus , suggesting that this may represent one of the first Dobzhansky–Muller incompatibilities to evolve early in the radiation of the Anopheles gambiae species complex. We describe the additive effects of each sterility QTL, epistatic interactions between them, and genes within QTL with protein functions related to mating behavior, reproduction, spermatogenesis, and microtubule morphogenesis, whose divergence may contribute to post-zygotic reproductive isolation between An. coluzzii and An. quadriannulatus. [ABSTRACT FROM AUTHOR]

Abstract Background A neglected aspect of alien invasive plant species is their influence on mosquito vector ecology and malaria transmission. Invasive plants that are highly attractive to Anopheles mosquitoes provide them with sugar that is critical to their survival. The effect on Anopheles mosquito populations was examined through a habitat manipulation experiment that removed the flowering branches of highly attractive Prosopis juliflora from selected villages in Mali, West Africa. Methods Nine villages in the Bandiagara district of Mali were selected, six with flowering Prosopis juliflora, and three without. CDC-UV light traps were used to monitor their Anopheles spp. vector populations, and recorded their species composition, population size, age structure, and sugar feeding status. After 8 days, all of the flowering branches were removed from three villages and trap catches were analysed again. Results Villages where flowering branches of the invasive shrub Prosopis juliflora were removed experienced a threefold drop in the older more dangerous Anopheles females. Population density dropped by 69.4% and the species composition shifted from being a mix of three species of the Anopheles gambiae complex to one dominated by Anopheles coluzzii. The proportion of sugar fed females dropped from 73 to 15% and males from 77 to 10%. Conclusions This study demonstrates how an invasive plant shrub promotes the malaria parasite transmission capacity of African malaria vector mosquitoes. Proper management of invasive plants could potentially reduce mosquito populations and malaria transmission.

Mosquitoes-borne diseases are major public health issues particularly in Africa. Vector control interventions and human-made environmental/climatic changes significantly affect the distribution and abundance of vector species. We carried out an entomological survey targeting host-seeking mosquitos in two different ecological contexts—coastal and inland—in Senegal, by CDC-light and BG-sentinel traps. Results show high predominance of Culex quinquefasciatus (90%) and of Anopheles arabiensis within malaria vectors (46%), with mean numbers of females/trap/nights =8 and Cx. quinquefasciatus and of CDC traps for An. gambiae s.l. and highlight that, despite both traps target the host-seeking fraction of the population, they provide different patterns of species abundance, temporal dynamics and host-seeking activity, leading to possible misinterpretation of the species bionomics. This draws attention to the need of taking into account trapping performance, in order to provide realistic quantification of the number of mosquitoes per units of space and time, the crucial parameter for evaluating vector–human contact, and estimating risk of pathogen transmission.

Background: Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species' spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. Methods: We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. Results: For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. Conclusions: The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology. [ABSTRACT FROM AUTHOR]

The increase in molecular tools for the genetic engineering of insect pests and disease vectors, such as Anopheles mosquitoes that transmit malaria, has led to an unprecedented investigation of the genomic landscape of these organisms. The understanding of genome variability in wild mosquito populations is of primary importance for vector control strategies. This is particularly the case for gene drive systems, which look to introduce genetic traits into a population by targeting specific genomic regions. Gene drive targets with functional or structural constraints are highly desirable as they are less likely to tolerate mutations that prevent targeting by the gene drive and consequent failure of the technology. In this study we describe a bioinformatic pipeline that allows the analysis of whole genome data for the identification of highly conserved regions that can point at potential functional or structural constraints. The analysis was conducted across the genomes of 22 insect species separated by more than hundred million years of evolution and includes the observed genomic variation within field caught samples of Anopheles gambiae and Anopheles coluzzii, the two most dominant malaria vectors. This study offers insight into the level of conservation at a genome-wide scale as well as at per base-pair resolution. The results of this analysis are gathered in a data storage system that allows for flexible extraction and bioinformatic manipulation. Furthermore, it represents a valuable resource that could provide insight into population structure and dynamics of the species in the complex and benefit the development and implementation of genetic strategies to tackle malaria.

Background: A neglected aspect of alien invasive plant species is their influence on mosquito vector ecology and malaria transmission. Invasive plants that are highly attractive to Anopheles mosquitoes provide them with sugar that is critical to their survival. The effect on Anopheles mosquito populations was examined through a habitat manipulation experiment that removed the flowering branches of highly attractive Prosopis juliflora from selected villages in Mali, West Africa. Methods: Nine villages in the Bandiagara district of Mali were selected, six with flowering Prosopis juliflora, and three without. CDC-UV light traps were used to monitor their Anopheles spp. vector populations, and recorded their species composition, population size, age structure, and sugar feeding status. After 8 days, all of the flowering branches were removed from three villages and trap catches were analysed again. Results: Villages where flowering branches of the invasive shrub Prosopis juliflora were removed experienced a threefold drop in the older more dangerous Anopheles females. Population density dropped by 69.4% and the species composition shifted from being a mix of three species of the Anopheles gambiae complex to one dominated by Anopheles coluzzii. The proportion of sugar fed females dropped from 73 to 15% and males from 77 to 10%. Conclusions: This study demonstrates how an invasive plant shrub promotes the malaria parasite transmission capacity of African malaria vector mosquitoes. Proper management of invasive plants could potentially reduce mosquito populations and malaria transmission. [ABSTRACT FROM AUTHOR]

Background: After seven annual rounds of mass drug administration (MDA) in six Malian villages highly endemic for Wuchereria bancrofti (overall prevalence rate of 42.7%), treatment was discontinued in 2008. Surveillance was performed over the ensuing 5 years to detect recrudescence. Methods: Circulating filarial antigen (CFA) was measured using immunochromatographic card tests (ICT) and Og4C3 ELISA in 6-7 year-olds. Antibody to the W. bancrofti infective larval stage (L3) antigen, Wb123, was tested in the same population in 2012. Microfilaraemia was assessed in ICT-positive subjects. Anopheles gambiae complex specimens were collected monthly using human landing catch (HLC) and pyrethrum spray catch (PSC). Anopheles gambiae complex infection with W. bancrofti was determined by dissection and reverse transcriptase polymerase chain reaction (RT-PCR) of mosquito pools. Results: Annual CFA prevalence rates using ICT in children increased over time from 0% (0/289) in 2009 to 2.7% (8/301) in 2011, 3.9% (11/285) in 2012 and 4.5% (14/309) in 2013 (trend X2 = 11.85, df =3, P = 0.0006). Wb123 antibody positivity rates in 2013 were similar to the CFA prevalence by ELISA (5/285). Although two W. bancrofti-infected Anopheles were observed by dissection among 12,951 mosquitoes collected by HLC, none had L3 larvae when tested by L3-specific RT-PCR. No positive pools were detected among the mosquitoes collected by pyrethrum spray catch. Whereas ICT in 6-7 year-olds was the major surveillance tool, ICT positivity was also assessed in older children and adults (8-65 years old). CFA prevalence decreased in this group from 4.9% (39/800) to 3.5% (28/795) and 2.8% (50/1,812) in 2009, 2011 and 2012, respectively (trend X2 = 7.361, df =2, P = 0.0067). Some ICT-positive individuals were microfilaraemic in 2009 [2.6% (1/39)] and 2011 [8.3% (3/36)], but none were positive in 2012 or 2013. Conclusion: Although ICT rates in children increased over the 5-year surveillance period, the decrease in ICT prevalence in the older group suggests a reduction in transmission intensity. This was consistent with the failure to detect infective mosquitoes or microfilaraemia. The threshold of ICT positivity in children may need to be re-assessed and other adjunct surveillance tools considered. [ABSTRACT FROM AUTHOR]

Background: Mosquito habitat-association studies are an important basis for disease control programmes and/or vector distribution models. However, studies do not explicitly account for incomplete detection during larval presence and abundance surveys, with potential for significant biases because of environmental influences on larval behaviour and sampling efficiency. Methods: Data were used from a dip-sampling study for Anopheles larvae in Ethiopia to evaluate the effect of six factors previously associated with larval sampling (riparian vegetation, direct sunshine, algae, water depth, pH and temperature) on larval presence and detectability. Comparisons were made between: (i) a presence-absence logistic regression where samples were pooled at the site level and detectability ignored, (ii) a success versus trials binomial model, and (iii) a presence-detection mixture model that separately estimated presence and detection, and fitted different explanatory variables to these estimations. Results: Riparian vegetation was consistently highlighted as important, strongly suggesting it explains larval presence (-). However, depending on how larval detectability was estimated, the other factors showed large variations in their statistical importance. The presence-detection mixture model provided strong evidence that larval detectability was influenced by sunshine and water temperature (+), with weaker evidence for algae (+) and water depth (-). For larval presence, there was also some evidence that water depth (-) and pH (+) influenced site occupation. The number of dip-samples needed to determine if larvae were likely present at a site was condition dependent: with sunshine and warm water requiring only two dips, while cooler water and cloud cover required 11. Conclusions: Environmental factors influence true larval presence and larval detectability differentially when sampling in field conditions. Researchers need to be more aware of the limitations and possible biases in different analytical approaches used to associate larval presence or abundance with local environmental conditions. These effects can be disentangled using data that are routinely collected (i.e., multiple dip samples at each site) by employing a modelling approach that separates presence from detectability. [ABSTRACT FROM AUTHOR]

Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high-throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole-genome amplification ( WGA) by primer-extension preamplification. We used the Agena Biosciences MassARRAY® platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high-throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species. [ABSTRACT FROM AUTHOR]

Background: The SEMOS gold mine's malaria vector control programme forms part of the company's community responsibilities with the programme being managed by the mine's health department since 2005. Methods: Data from approximately 10 years of malaria vector control for the Sadiola District are given: namely malaria vector control methods used by the control programme, positive malaria case data and entomological surveys from 2006, 2011 and 2014. Results: Distribution of pyrethroid-treated bed nets and indoor residual spraying (IRS) with deltamethrin were implemented by the programme from 2005-2011. No IRS was done in 2012. Spraying with the organophosphate, pirimiphos-methyl resumed in 2013 and 2014 and was followed by a 70% drop in malaria cases in 2014. Anopheles arabiensis was the major vector present in 2006 and was susceptible to deltamethrin. In 2011 and 2014, An. gambiae s.s. was the most abundant vector with deltamethrin 24 h mortality of 68% and 19%, respectively. Conclusions: Resistance to the pyrethroid deltamethrin has increased in An. gambiae s.s. since 2011, possibly due to the scale-up in distribution of long-lasting insecticide-treated bed nets. Resistance management strategies are recommended using different classes of insecticides for IRS, and including the distribution of new-generation bed nets. [ABSTRACT FROM AUTHOR]

A strategy to decrease the vector competence of Anopheles gambiae sensu stricto (Diptera: Culicidae), the most efficient malaria vector in Africa, may consist of exploiting the genes involved in zoophily. Crossing and backcrossing experiments were performed between An. gambiae s.s. and the zoophilic sibling species Anopheles quadriannulatus. Mosquito strains were tested in a dual-choice olfactometer to investigate their responses to cow odour. Totals of 12% of An. gambiae s.s. and 59% of An. quadriannulatus selected the port with the cow odour. Crosses and backcrosses did not show a significant preference for the cow-baited port. The results indicated that anthropophilic behaviour in An. gambiae s.s. is a dominant or partially dominant trait, which, in conjunction with the unstable zoophilic behaviour observed in An. quadriannulatus, poses a serious obstacle to plans to decrease vector competence by modifying the anthropophilic trait. [ABSTRACT FROM AUTHOR]

Background: The first part of this study aimed to develop a model for Anopheles gambiae s.l. with separate parametrization schemes for Anopheles gambiae s.s. and Anopheles arabiensis. The characterizations were constructed based on literature from the past decades. This part of the study is focusing on the model's ability to separate the mean state of the two species of the An. gambiae complex in Africa. The model is also evaluated with respect to capturing the temporal variability of An. arabiensis in Ethiopia. Before conclusions and guidance based on models can be made, models need to be validated. Methods: The model used in this paper is described in part one (Malaria Journal 2013, 12:28). For the validation of the model, a data base of 5,935 points on the presence of An. gambiae s.s. and An. arabiensis was constructed. An additional 992 points were collected on the presence An. gambiae s.l.. These data were used to assess if the model could recreate the spatial distribution of the two species. The dataset is made available in the public domain. This is followed by a case study from Madagascar where the model's ability to recreate the relative fraction of each species is investigated. In the last section the model's ability to reproduce the temporal variability of An. arabiensis in Ethiopia is tested. The model was compared with data from four papers, and one field survey covering two years. Results: Overall, the model has a realistic representation of seasonal and year to year variability in mosquito densities in Ethiopia. The model is also able to describe the distribution of An. gambiae s.s. and An. arabiensis in sub-Saharan Africa. This implies this model can be used for seasonal and long term predictions of changes in the burden of malaria. Before models can be used to improving human health, or guide which interventions are to be applied where, there is a need to understand the system of interest. Validation is an important part of this process. It is also found that one of the main mechanisms separating An. gambiae s.s. and An. arabiensis is the availability of hosts; humans and cattle. Climate play a secondary, but still important, role. [ABSTRACT FROM AUTHOR]

Background: Most of the current biophysical models designed to address the large-scale distribution of malaria assume that transmission of the disease is independent of the vector involved. Another common assumption in these type of model is that the mortality rate of mosquitoes is constant over their life span and that their dispersion is negligible. Mosquito models are important in the prediction of malaria and hence there is a need for a realistic representation of the vectors involved. Results: We construct a biophysical model including two competing species, Anopheles gambiae s.s. and Anopheles arabiensis. Sensitivity analysis highlight the importance of relative humidity and mosquito size, the initial conditions and dispersion, and a rarely used parameter, the probability of finding blood. We also show that the assumption of exponential mortality of adult mosquitoes does not match the observed data, and suggest that an age dimension can overcome this problem. Conclusions: This study highlights some of the assumptions commonly used when constructing mosquito-malaria models and presents a realistic model of An. gambiae s.s. and An. arabiensis and their interaction. This new mosquito model, OMaWa, can improve our understanding of the dynamics of these vectors, which in turn can be used to understand the dynamics of malaria. [ABSTRACT FROM AUTHOR]

Anopheles gambiae and An. coluzzii are very closely related and recently differentiated species representing the main malaria vectors in the Afrotropical region and responsible of up to >3 infective bites/person/night in Côte D'Ivoire, where prevention and control has stagnated in recent years. The aim of the present study was to genetically and ecologically characterize An. gambiae and An. coluzzii populations from two villages of Côte D'Ivoire, lying in the coastal forest belt and 250 km inland in the Guinean savannah mosaic belt, respectively. Results reveal high frequencies of both species in both study sites and high frequencies of hybrids (4–33%) along the whole year of sampling. Consistently with observations for the well-known high hybridization zone at the far-west of the species range, hybrid frequencies were higher in the coastal village and highest when the two species occurred at more balanced frequencies, supporting the "frequency-dependent hybridization" ecological speciation theory. Pilot genotyping revealed signatures of genomic admixture in both chromosome-X and −3. Coupled with previous reports of hybrids in the region, the results point to the coastal region of Côte D'Ivoire as a possible regions of high hybridization. Preliminary characterization of parameters relevant for malaria transmission and control (e.g. possibly higher sporozoite rates and indoor biting preferences in hybrids than in the parental species) highlight the possible relevance of the breakdown of reproductive barriers between An. gambiae and An. coluzzii not only in the field of ecological evolution, but also in malaria epidemiology and control. • Anopheles gambiae and An. coluzzii are main malaria vectors in Côte D'Ivoire. • The two species are reproductively isolated in most of their range in Africa. • Coastal and inland populations of Côte D'Ivoire show high levels of hybridization. • Results confirm the "frequency-dependent hybridization" ecological speciation theory. • Preliminary data of genomic and phenotypic characterization of hybrids are provided. [ABSTRACT FROM AUTHOR]

Malaria vector control targeting the larval stages of mosquitoes was applied successfully against many species of Anopheles (Diptera: Culicidae) in malarious countries until the mid-20th Century. Since the introduction of DDT in the 1940s and the associated development of indoor residual spraying (IRS), which usually has a more powerful impact than larval control on vectorial capacity, the focus of malaria prevention programmes has shifted to the control of adult vectors. In the Afrotropical Region, where malaria is transmitted mainly by Anopheles funestus Giles and members of the Anopheles gambiae Giles complex, gaps in information on larval ecology and the ability of An. gambiae sensu lato to exploit a wide variety of larval habitats have discouraged efforts to develop and implement larval control strategies. Opportunities to complement adulticiding with other components of integrated vector management, along with concerns about insecticide resistance, environmental impacts, rising costs of IRS and logistical constraints, have stimulated renewed interest in larval control of malaria vectors. Techniques include environmental management, involving the temporary or permanent removal of anopheline larval habitats, as well as larviciding with chemical or biological agents. This present review covers large-scale trials of anopheline larval control methods, focusing on field studies in Africa conducted within the past 15 years. Although such studies are limited in number and scope, their results suggest that targeting larvae, particularly in human-made habitats, can significantly reduce malaria transmission in appropriate settings. These approaches are especially suitable for urban areas, where larval habitats are limited, particularly when applied in conjunction with IRS and other adulticidal measures, such as the use of insecticide treated bednets. [ABSTRACT FROM AUTHOR]

Objectives: To determine the hourly density of vector mosquitoes in coastal Nigeria, compare seasonal human-biting and sporozoite rates in the vector density, locate breeding sites of mosquitoes, and determine larval population at breeding sites. Materials and Methods: Indoor and outdoor mosquitoes of a coastal Nigerian community were caught during early and late wet seasons and in the harmattan period, a time of dusty wind from the Sahara on the western coast of Africa. Larvae were collected from various locations during the study period. The mosquitoes were physically characterized and their salivary glands dissected for sporozoite rate. Larvae density was calculated. Human-biting rate was calculated for Anopheles gambiae complex. Results: Of the 4,317 female A. gambiae complex collected during the night bait catches, 3,543 (82.1%) were from outdoors and 774 (17.9%) from indoors during the three seasons. The maximum human-biting rate approached 25/h and the sporozoite rate was almost 3.0%. These vector mosquitoes were mainly outdoor biting and midnight feeding. Of the 1,269 Anopheles mosquitoes collected with pyrethrum spray catches, 1,245 (98.1%) were A. gambiae complex. There was no significant difference in the entomological inoculation or sporozoite rates during the three seasons of study. There was a preponderance of A. gambiae complex larvae from larval collection. Conclusion: Findings from this study should be useful in the implementation of Integrated Vector Management for the control of malaria in coastal and noncoastal areas of Nigeria. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We conducted laboratory tests to assess the sensitivity to the insect repellent 1-piperidinecarboxylic acid, 2-(2-hydroxyethyl)-, 1-methylpropylester (known as KBR 3023 or Picaridin, trade name Bayrepel) of West African strains of the yellow fever mosquito Aedes aegypti and of malaria vectors of the Anopheles gambiae complex, in comparison with the standard repellent N,N-diethyl-3-methyl-benzamide (DEET). Test mosquitoes were exposed according to a 'separate arms' protocol to logarithmic dose increments applied on one arm of human subjects to evaluate the relative potency, and the median effective dosages (ED50 and ED90). According to a logistic regression model fitted to the experimental data, the dose-response relationship for the two repellents was the same within each species, thus pooled ED values were assessed for each mosquito separately. The median ED of KBR 3023 and DEET was estimated at 0.78 (95% confidence limits (CI): 0.57-1.04) and at 0.018 microg/cm2 (0.004-0.052) for mosquitoes of the An. gambiae complex and Ae. aegypti, respectively. ED90 values were 125.6 (81.4-201.3) and 24.0 microg/cm2 (5.7-208.5) for An. gambiae s.l. and Ae. aegypti, respectively. The relative potency of KBR 3023 was not significantly different from that of DEET for An. gambiae s.l. (95% confidence limits 0.7-1.0), whereas in the case of Ae. aegypti it was with 95% probability 1.1-2.0 times more potent than DEET. On the basis of available evidence, KBR 3023 represents a promising alternative to DEET for personal protection against bites of these important vectors of disease in the Afrotropical region. [ABSTRACT FROM AUTHOR]

Abstract For differential identification of sibling species in the Anopheles gambiae Giles complex (Diptera: Culicidae), including simultaneous separation of M and S molecular forms within An. gambiae Giles sensu stricto , we describe a PCR-RFLP method. This procedure is more efficient, faster and cheaper than those used before, so is recommended for large-scale processing of field-collected larval and adult specimens to be identified in malaria vector studies. [ABSTRACT FROM AUTHOR]