Anopheles gambiae on remote islands in the Indian Ocean: origins and prospects for malaria elimination by genetic modification of extant populations.

The mosquito Anopheles gambiae s.s. is a primary malaria vector throughout sub-Saharan Africa including the islands of the Comoros archipelago (Anjouan, Grande Comore, Mayotte and Mohéli). These islands are located at the northern end of the Mozambique Channel in eastern Africa. Previous studies have shown a relatively high degree of genetic isolation between the Comoros islands and mainland populations of A. gambiae, but the origin of the island populations remains unclear. Here, we analyzed phylogenetic relationships among island and mainland populations using complete mitochondrial genome sequences of individual A. gambiae specimens. This work augments earlier studies based on analysis of the nuclear genome. We investigated the source population of A. gambiae for each island, estimated the number of introductions, when they occurred and explored evidence for contemporary gene flow between island and mainland populations. These studies are relevant to understanding historical patterns in the dispersal of this important malaria vector and provide information critical to assessing their potential for the exploration of genetic-based vector control methods to eliminate this disease. Phylogenetic analysis and haplotype networks were constructed from mitogenome sequences of 258 A. gambiae from the four islands. In addition, 112 individuals from seven countries across sub-Saharan Africa and Madagascar were included to identify potential source populations. Our results suggest that introduction events of A. gambiae into the Comoros archipelago were rare and recent events and support earlier claims that gene flow between the mainland and these islands is limited. This study is concordant with earlier work suggesting the suitability of these oceanic islands as appropriate sites for conducting field trial releases of genetically engineered mosquitoes (GEMs).

Genomic signatures of local adaptation in recent invasive Aedes aegypti populations in California.

BACKGROUND: Rapid adaptation to new environments can facilitate species invasions and range expansions. Understanding the mechanisms of adaptation used by invasive disease vectors in new regions has key implications for mitigating the prevalence and spread of vector-borne disease, although they remain relatively unexplored. RESULTS: Here, we integrate whole-genome sequencing data from 96 Aedes aegypti mosquitoes collected from various sites in southern and central California with 25 annual topo-climate variables to investigate genome-wide signals of local adaptation among populations. Patterns of population structure, as inferred using principal components and admixture analysis, were consistent with three genetic clusters. Using various landscape genomics approaches, which all remove the confounding effects of shared ancestry on correlations between genetic and environmental variation, we identified 112 genes showing strong signals of local environmental adaptation associated with one or more topo-climate factors. Some of them have known effects in climate adaptation, such as heat-shock proteins, which shows selective sweep and recent positive selection acting on these genomic regions. CONCLUSIONS: Our results provide a genome wide perspective on the distribution of adaptive loci and lay the foundation for future work to understand how environmental adaptation in Ae. aegypti impacts the arboviral disease landscape and how such adaptation could help or hinder efforts at population control.

Field Evaluation of In2Care Mosquito Traps to Control Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Hawai'i Island.

Aedes aegypti Linnaeus and Aedes albopictus Skuse are vectors of dengue virus and responsible for multiple autochthonous dengue outbreaks in Big Island, Hawai'i. Control of Ae. aegypti and Ae. albopictus has been achieved in In2Care trap trials, which motivated us to investigate this potential control approach in the Big Island. Our In2Care trial was performed in the coastal settlement of Miloli'i in the southwest of Big Island where both Ae. aegypti and Ae. albopictus are found. This trial starting in the second week of July and ending in the last week of October 2019 fell within the traditional wet season in Miloli'i. No significant reduction in egg or adult counts in our treatment areas following 12 wk of two In2Care trap placements per participating household were observed. In fact, an increase in numbers of adults during the trial reached levels that required the local mosquito abatement program to stop the In2Care trap trial and institute a thorough source reduction and treatment campaign. The source reduction campaign revealed a large variety and quantity of water sources competed with the oviposition cups we had placed, which likely lowered the chances of our oviposition cups being visited by pyriproxyfen-contaminated Aedes adults exiting the In2Care traps.

A highly expressed odorant receptor from the yellow fever mosquito, AaegOR11, responds to (+)- and (-)-fenchone and a phenolic repellent.

The cornerstone of the reverse chemical ecology approach is the identification of odorant receptors (OR) sensitive to compounds in a large panel of odorants. In this approach, we de-orphanize ORs and, subsequently, measure behaviors elicited by these semiochemicals. After that, we evaluate behaviorally active compounds for applications in insect vector management. Intriguingly, multiple ORs encoded by genes highly expressed in mosquito antennae do not respond to any test odorant. One such case is CquiOR125 from the southern house mosquito, Culex quinquefasciatus Say. To better understand CquiOR125's role in Culex mosquito olfaction, we have cloned a CquiOR125 orthologue in the genome of the yellow fever mosquito, Aedes aegypti (L.), AaegOR11. Unlike the unresponsive nature of the orthologue in Cx. quinquefasciatus, oocytes co-expressing AaegOR11 and AaegOrco elicited robust responses when challenged with fenchone, 2,3-dimethylphenol, 3,4-dimethylphenol, 4-methycyclohexanol, and acetophenone. Interestingly, AaegOR11 responded strongly and equally to (+)- and (-)-fenchone, with no chiral discrimination. Contrary to reports in the literature, fenchone did not show any repellency activity against Ae. aegypti or Cx. quinquefasciatus. Laboratory and field tests did not show significant increases in egg captures in cups filled with fenchone solutions compared to control cups. The second most potent ligand, 2,3-dimethylphenol, showed repellency activity stronger than that elicited by DEET at the same dose. We, therefore, concluded that AaegOR11 is a mosquito repellent sensor. It is feasible that CquiOR125 responds to repellents that remain elusive.

Population Genetics of Anopheles pretoriensis in Grande Comore Island.

Anopheles pretoriensis is widely distributed across Africa, including on oceanic islands such as Grande Comore in the Comoros. This species is known to be mostly zoophylic and therefore considered to have low impact on the transmission of human malaria. However, A. pretoriensis has been found infected with Plasmodium, suggesting that it may be epidemiologically important. In the present study, we sequenced and assembled the complete mitogenome of A. pretoriensis and inferred its phylogenetic relationship among other species in the subgenus Cellia. We also investigated the genetic structure of A. pretoriensis populations on Grande Comore Island, and between this island population and sites in continental Africa, using partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Seven haplotypes were found on the island, one of which was ubiquitous. There was no clear divergence between island haplotypes and those found on the continent. The present work contributes knowledge on this understudied, yet abundant, Anopheles species.

The origin of island populations of the African malaria mosquito, Anopheles coluzzii.

Anopheles coluzzii is a major malaria vector throughout its distribution in west-central Africa. Here we present a whole-genome study of 142 specimens from nine countries in continental Africa and three islands in the Gulf of Guinea. This sample set covers a large part of this species' geographic range. Our population genomic analyses included a description of the structure of mainland populations, island populations, and connectivity between them. Three genetic clusters are identified among mainland populations and genetic distances (FST) fits an isolation-by-distance model. Genomic analyses are applied to estimate the demographic history and ancestry for each island. Taken together with the unique biogeography and history of human occupation for each island, they present a coherent explanation underlying levels of genetic isolation between mainland and island populations. We discuss the relationship of our findings to the suitability of São Tomé and Príncipe islands as candidate sites for potential field trials of genetic-based malaria control strategies.

Frequency of sodium channel genotypes and association with pyrethrum knockdown time in populations of Californian Aedes aegypti.

BACKGROUND: Since their detection in 2013, Aedes aegypti has become a widespread urban pest in California. The availability of cryptic larval breeding sites in residential areas and resistance to insecticides pose significant challenges to control efforts. Resistance to pyrethroids is largely attributed to mutations in the voltage gated sodium channels (VGSC), the pyrethroid site of action. However, past studies have indicated that VGSC mutations may not be entirely predictive of the observed resistance phenotype. METHODS: To investigate the frequencies of VGSC mutations and the relationship with pyrethroid insecticide resistance in California, we sampled Ae. aegypti from four locations in the Central Valley, and the Greater Los Angeles area. Mosquitoes from each location were subjected to an individual pyrethrum bottle bioassay to determine knockdown times. A subset of assayed mosquitoes from each location was then analyzed to determine the composition of 5 single nucleotide polymorphism (SNP) loci within the VGSC gene. RESULTS: The distribution of knockdown times for each of the five Californian populations sampled was non-parametric with potentially bimodal distributions. One group succumbs to insecticidal effects around 35-45 min and the second group lasts up to and beyond the termination of the assay (120+ min). We detected 5 polymorphic VGSC SNPs within the sampled California populations. One is potentially new and alternatively spliced (I915K), and four are documented and associated with resistance: F1534C, V1016I, V410L and S723T. The Central Valley populations (Clovis, Dinuba, Sanger and Kingsburg) are fairly homogenous with only 5% of the mosquitoes showing heterozygosity at any given position. In the Greater LA mosquitoes, 55% had at least one susceptible allele at any of the five SNP loci. The known resistance allele F1534C was detected in almost all sampled mosquitoes (99.4%). We also observe significant heterogeneity in the knockdown phenotypes of individuals with the identical VGSC haplotypes suggesting the presence of additional undefined resistance mechanisms. CONCLUSIONS: Resistance associated VGSC SNPs are prevalent, particularly in the Central Valley. Interestingly, among mosquitoes carrying all 4 resistance associated SNPs, we observe significant heterogeneity in bottle bioassay profiles suggesting that other mechanisms are important to the individual resistance of Ae. aegypti in California.

Evidence for Divergent Selection on Immune Genes between the African Malaria Vectors, Anopheles coluzzii and A. gambiae.

During their life cycles, microbes infecting mosquitoes encounter components of the mosquito anti-microbial innate immune defenses. Many of these immune responses also mediate susceptibility to malaria parasite infection. In West Africa, the primary malaria vectors are Anopheles coluzzii and A. gambiae sensu stricto, which is subdivided into the Bamako and Savanna sub-taxa. Here, we performed whole genome comparisons of the three taxa as well as genotyping of 333 putatively functional SNPs located in 58 immune signaling genes. Genome data support significantly higher differentiation in immune genes compared with a randomly selected set of non-immune genes among the three taxa (permutation test p < 0.001). Among the 58 genes studied, the majority had one or more segregating mutations (72.9%) that were significantly diverged among the three taxa. Genes detected to be under selection include MAP2K4 and Raf. Despite the genome-wide distribution of immune genes, a high level of linkage disequilibrium (r2 > 0.8) was detected in over 27% of SNP pairs. We discuss the potential role of immune gene divergence as adaptations to the different larval habitats associated with A. gambiae taxa and as a potential force driving ecological speciation in this group of mosquitoes.

Malaria Vectors and Vector Surveillance in Limpopo Province (South Africa): 1927 to 2018.

Despite the annual implementation of a robust and extensive indoor residual spraying programme against malaria vectors in Limpopo Province (South Africa), significant transmission continues and is a serious impediment to South Africa's malaria elimination objectives. In order to gain a better understanding regarding possible causes of this residual malaria, we conducted a literature review of the historical species composition and abundance of malaria vector mosquitoes in the Limpopo River Valley region of the Vhembe District, northern Limpopo Province, the region with the highest remaining annual malaria cases in South Africa. In addition, mosquito surveys were carried out in the same region between October 2017 and October 2018. A total of 2225 adult mosquitoes were collected using CO2-baited tent and light traps, human landing catches and cow-baited traps. Of the 1443 Anopheles collected, 516 were members of the An. gambiae complex and 511 An. funestus group. In the malaria endemic rural areas outside the Kruger National Park, one specimen each of An. gambiae s.s. and An. funestus and only three of An. arabiensis were collected. The latter species was abundant at a remote hot spring in the neighboring Kruger National Park. Eighteen other species of Anopheles were collected. Our survey results support the historical findings that An. arabiensis, the species widely held to be the prime malaria vector in South Africa, is a rare species in the malaria endemic Limpopo River Valley. The implications of the mosquito surveys for malaria transmission, elimination and vector control in northern Limpopo Province and neighboring regions are discussed.

Complete mitogenome sequence of Anopheles coustani from São Tomé island.

We report the first complete mitogenome (Mt) sequence of Anopheles coustani, an understudied malaria vector in Africa. The sequence was extracted from one individual mosquito from São Tomé island. The length of the A. coustani Mt genome was 15,408 bp with 79.3% AT content. Phylogenetic analysis revealed that A. coustani is most closely related to A. sinensis (93.5% of identity); and 90.1% identical to A. gambiae complex members.

Transcontinental dispersal of Anopheles gambiae occurred from West African origin via serial founder events.

The mosquito Anopheles gambiae s.s. is distributed across most of sub-Saharan Africa and is of major scientific and public health interest for being an African malaria vector. Here we present population genomic analyses of 111 specimens sampled from west to east Africa, including the first whole genome sequences from oceanic islands, the Comoros. Genetic distances between populations of A. gambiae are discordant with geographic distances but are consistent with a stepwise migration scenario in which the species increases its range from west to east Africa through consecutive founder events over the last ~200,000 years. Geological barriers like the Congo River basin and the East African rift seem to play an important role in shaping this process. Moreover, we find a high degree of genetic isolation of populations on the Comoros, confirming the potential of these islands as candidate sites for potential field trials of genetically engineered mosquitoes for malaria control.

Odorant Inhibition in Mosquito Olfaction.

How chemical signals are integrated at the peripheral sensory system of insects is still an enigma. Here we show that when coexpressed with Orco in Xenopus oocytes, an odorant receptor from the southern house mosquito, CquiOR32, generated inward (regular) currents when challenged with cyclohexanone and methyl salicylate, whereas eucalyptol and fenchone elicited inhibitory (upward) currents. Responses of CquiOR32-CquiOrco-expressing oocytes to odorants were reduced in a dose-dependent fashion by coapplication of inhibitors. This intrareceptor inhibition was also manifested in vivo in fruit flies expressing the mosquito receptor CquiOR32, as well in neurons on the antennae of the southern house mosquito. Likewise, an orthologue from the yellow fever mosquito, AaegOR71, showed intrareceptor inhibition in the Xenopus oocyte recording system and corresponding inhibition in antennal neurons. Inhibition was also manifested in mosquito behavior. Blood-seeking females were repelled by methyl salicylate, but repellence was significantly reduced when methyl salicylate was coapplied with eucalyptol.

Genome-wide divergence among invasive populations of Aedes aegypti in California.

BACKGROUND: In the summer of 2013, Aedes aegypti Linnaeus was first detected in three cities in central California (Clovis, Madera and Menlo Park). It has now been detected in multiple locations in central and southern CA as far south as San Diego and Imperial Counties. A number of published reports suggest that CA populations have been established from multiple independent introductions. RESULTS: Here we report the first population genomics analyses of Ae. aegypti based on individual, field collected whole genome sequences. We analyzed 46 Ae. aegypti genomes to establish genetic relationships among populations from sites in California, Florida and South Africa. Based on 4.65 million high quality biallelic SNPs, we identified 3 major genetic clusters within California; one that includes all sample sites in the southern part of the state (South of Tehachapi mountain range) plus the town of Exeter in central California and two additional clusters in central California. CONCLUSIONS: A lack of concordance between mitochondrial and nuclear genealogies suggests that the three founding populations were polymorphic for two main mitochondrial haplotypes prior to being introduced to California. One of these has been lost in the Clovis populations, possibly by a founder effect. Genome-wide comparisons indicate extensive differentiation between genetic clusters. Our observations support recent introductions of Ae. aegypti into California from multiple, genetically diverged source populations. Our data reveal signs of hybridization among diverged populations within CA. Genetic markers identified in this study will be of great value in pursuing classical population genetic studies which require larger sample sizes.

The fate of genes that cross species boundaries after a major hybridization event in a natural mosquito population.

Animal species are able to acquire new genetic material via hybridization and subsequent introgression. However, little is known about how foreign genomic material is incorporated into a population over time and what genes are susceptible to introgression. Here, we follow the closely related mosquito sister species Anopheles coluzzii and Anopheles gambiae in a sympatric natural population in Mali at multiple time points spanning a period of 25 years. During this period, we observed the temporary breakdown of mating barriers, which allowed us to explore the fate of alleles that crossed the species boundary in a natural population. Whole genome sequencing of 74 individuals revealed introgression within only 34 genes (0.26% of total genes) from A. gambiae to A. coluzzii, the majority contained within a 4 Mb region on the 2L chromosome which includes the insecticide resistance gene (AGAP004707). We designed a genotyping assay to follow 25 of the 34 introgressed alleles over time and found that all A. gambiae alleles, except four, reached a frequency of 50% in the A. coluzzii population within 4 years (~50 generations) and increased to ~80% within 6 years (~75 generations). However, the frequency of all introgressed alleles, except three, decreased to ~60% in 2016. This suggests an ongoing process of purifying selection in the population against DNA of foreign ancestry, except for alleles that are under positive selection, resulting in a complex genomic landscape. This study shows that stable introgression is limited to only specific genes even within closely related species.

Complete Anopheles funestus mitogenomes reveal an ancient history of mitochondrial lineages and their distribution in southern and central Africa.

Anopheles funestus s.s. is a primary vector of malaria in sub-Saharan Africa. Despite its important role in human Plasmodium transmission, evolutionary history, genetic diversity, and population structure of An. funestus in southern and central Africa remains understudied. We deep sequenced, assembled, and annotated the complete mitochondrial genome of An. funestus s.s. for the first time, providing a foundation for further genetic research of this important malaria vector species. We further analyzed the complete mitochondrial genomes of 43 An. funestus s.s. from three sites in Zambia, Democratic Republic of the Congo, and Tanzania. From these 43 mitogenomes we identified 41 unique haplotypes that comprised 567 polymorphic sites. Bayesian phylogenetic reconstruction confirmed the co-existence of two highly divergent An. funestus maternal lineages, herein defined as lineages I and II, in Zambia and Tanzania. The estimated coalescence time of these two mitochondrial lineages is ~500,000 years ago (95% HPD 426,000-594,000 years ago) with subsequent independent diversification. Haplotype network and phylogenetic analysis revealed two major clusters within lineage I, and genetic relatedness of samples with deep branching in lineage II. At this time, data suggest that the lineages are partially sympatric. This study illustrates that accurate retrieval of full mitogenomes of Anopheles vectors enables fine-resolution studies of intraspecies genetic relationships, population differentiation, and demographic history. Further investigations on whether An. funestus mitochondrial lineages represent biologically meaningful populations and their potential implications for malaria vector control are warranted.

Mosquito community composition in South Africa and some neighboring countries.

BACKGROUND: A century of studies have described particular aspects of relatively few mosquito species in southern Africa, mostly those species involved with disease transmission, specifically malaria and arboviruses. Patterns of community composition such as mosquito abundance and species diversity are often useful measures for medical entomologists to guide broader insights and projections regarding disease dynamics and potential introduction, spread or maintenance of globally spreading pathogens. However, little research has addressed these indicators in southern Africa. RESULTS: We collected 7882 mosquitoes from net and light traps at 11 localities comprising 66 species in 8 genera. We collected an additional 8 species using supplementary collection techniques such as larval sampling, sweep-netting and indoor pyrethrum knockdown catches. Highest diversity and species richness was found in the Okavango Delta of Botswana and in South Africa's Kruger National Park, while the lowest diversity and abundances were in the extreme southern tip of South Africa and in semi-desert Kalahari close to the South Africa border with Botswana. Species composition was more similar between proximal localities than distant ones (Linear model P-value = 0.005). Multiple arbovirus vector species were detected in all localities we surveyed (proportion of vector mosquito numbers were > 0.5 in all locations except Shingwedzi). Their proportions were highest (> 90%) in Vilankulo and Kogelberg. CONCLUSIONS: Multiple known arbovirus vector species were found in all study sites, whereas anopheline human malaria vector species in only some sites. The combination of net traps and light traps effectively sampled mosquito species attracted to carbon-dioxide or light, accounting for 89% of the 74 species collected. The 11% remaining species were collected using supplementary collection techniques mentioned above. The diversity of species weas highest in savanna type habitats, whereas low diversities were found in the drier Kalahari sands regions and the southern Cape fynbos regions.

Detection of Anopheles rivulorum-like, a member of the Anopheles funestus group, in South Africa.

BACKGROUND: The Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. species complexes contain the most important malaria vectors in Africa. Within the An. funestus group of at least 11 African species, the vector status of all but the nominal species An. funestus appears poorly investigated, although evidence exists that Anopheles rivulorum and Anopheles vaneedeni may play minor roles. A new species, An. rivulorum-like, was described from Burkina Faso in 2000 and subsequently also found in Cameroon and Zambia. This is the first paper reporting the presence of this species in South Africa, thereby significantly extending its known range. METHODS: Mosquitoes were collected using dry-ice baited net traps and CDC light traps in the Kruger National Park, South Africa. Sixty-four An. funestus s.l. among an overall 844 mosquitoes were captured and identified to species level using the polymerase chain reaction assay. All samples were also analysed for the presence of Plasmodium falciparum circumsporozoite protein using the enzyme-linked-immunosorbent assay. RESULTS: Four members of the An. funestus group were identified: An. rivulorum-like (n = 49), An. rivulorum (n = 11), Anopheles parensis (n = 2) and Anopheles leesoni (n = 1). One mosquito could not be identified. No evidence of P. falciparum was detected in any of the specimens. CONCLUSION: This is the first report of An. rivulorum-like south of Zambia, and essentially extends the range of this species from West Africa down to South Africa. Given the continental-scale drive towards malaria elimination and the challenges faced by countries in the elimination phase to understand and resolve residual transmission, efforts should be directed towards determining the largely unknown malaria vector potential of members of the An. funestus group and other potential secondary vectors.

Avian malaria co-infections confound infectivity and vector competence assays of Plasmodium homopolare.

Currently, there are very few studies of avian malaria that investigate relationships among the host-vector-parasite triad concomitantly. In the current study, we experimentally measured the vector competence of several Culex mosquitoes for a newly described avian malaria parasite, Plasmodium homopolare. Song sparrow (Melospiza melodia) blood infected with a low P. homopolare parasitemia was inoculated into a naïve domestic canary (Serinus canaria forma domestica). Within 5 to 10 days post infection (dpi), the canary unexpectedly developed a simultaneous high parasitemic infection of Plasmodium cathemerium (Pcat6) and a low parasitemic infection of P. homopolare, both of which were detected in blood smears. During this infection period, PCR detected Pcat6, but not P. homopolare in the canary. Between 10 and 60 dpi, Pcat6 blood stages were no longer visible and PCR no longer amplified Pcat6 parasite DNA from canary blood. However, P. homopolare blood stages remained visible, albeit still at very low parasitemias, and PCR was able to amplify P. homopolare DNA. This pattern of mixed Pcat6 and P. homopolare infection was repeated in three secondary infected canaries that were injected with blood from the first infected canary. Mosquitoes that blood-fed on the secondary infected canaries developed infections with Pcat6 as well as another P. cathemerium lineage (Pcat8); none developed PCR detectable P. homopolare infections. These observations suggest that the original P. homopolare-infected songbird also had two un-detectable P. cathemerium lineages/strains. The vector and host infectivity trials in this study demonstrated that current molecular assays may significantly underreport the extent of mixed avian malaria infections in vectors and hosts.

Genetic variation associated with increased insecticide resistance in the malaria mosquito, Anopheles coluzzii.

BACKGROUND: Malaria mortality rates in sub-Saharan Africa have declined significantly in recent years as a result of increased insecticide-treated bed net (ITN) usage. A major challenge to further progress is the emergence and spread of insecticide resistance alleles in the Anopheles mosquito vectors, like An. coluzzii. A non-synonymous mutation in the para voltage-gated sodium channel gene reduces pyrethroid-binding affinity, resulting in knockdown resistance (kdr). Metabolic mechanisms of insecticide resistance involving detoxification genes like cytochrome P450 genes, carboxylesterases, and glutathione S-transferases are also important. As some gene activity is tissue-specific and/or environmentally induced, gene regulatory variation may be overlooked when comparing expression from whole mosquito bodies under standard rearing conditions. RESULTS: We detected complex insecticide resistance in a 2014 An. coluzzii colony from southern Mali using bottle bioassays. Additional bioassays involving recombinant genotypes from a cross with a relatively susceptible 1995 An. coluzzii colony from Mali confirmed the importance of kdr and associated increased permethrin resistance to the CYP9K1 locus on the X chromosome. Significant differential expression of CYP9K1 was not observed among these colonies in Malpighian tubules. However, the P450 gene CYP6Z1 was overexpressed in resistant individuals following sublethal permethrin exposure and the carboxylesterase gene COEAE5G was constitutively overexpressed. CONCLUSIONS: The significant P450-related insecticide resistance observed in the 2014 An. coluzzii colony indicates that ITNs treated with the P450 inhibitor piperonyl butoxide (PBO) would be more effective in this region. The known insecticide resistance gene CYP6Z1 was differentially expressed exclusively in the context of sublethal permethrin exposure, highlighting the importance of tissue-specificity and environmental conditions in gene expression studies. The increased activity of the carboxylesterase COEAE5G in the resistant An. coluzzii colony suggests resistance to other insecticides like organophosphates. Additional gene expression studies involving other tissues (e.g. fat body) would provide a more comprehensive view of genes underlying metabolic insecticide resistance in An. coluzzii from Mali. Identifying genetic markers linked to these regulatory alleles is an important next step that would substantially improve insecticide resistance surveillance and population genetic studies in this important vector species.

Mosquito-borne arboviruses of African origin: review of key viruses and vectors.

Key aspects of 36 mosquito-borne arboviruses indigenous to Africa are summarized, including lesser or poorly-known viruses which, like Zika, may have the potential to escape current sylvatic cycling to achieve greater geographical distribution and medical importance. Major vectors are indicated as well as reservoir hosts, where known. A series of current and future risk factors is addressed. It is apparent that Africa has been the source of most of the major mosquito-borne viruses of medical importance that currently constitute serious global public health threats, but that there are several other viruses with potential for international challenge. The conclusion reached is that increased human population growth in decades ahead coupled with increased international travel and trade is likely to sustain and increase the threat of further geographical spread of current and new arboviral disease.