Using Bayesian state-space models to understand the population dynamics of the dominant malaria vector, Anopheles funestus in rural Tanzania

Background: It is often assumed that the population dynamics of the malaria vector Anopheles funestus, its role in malaria transmission and the way it responds to interventions are similar to the more elaborately characterized An. gambiae. However, An. funestus has several unique ecological features that could generate distinct transmission dynamics and responsiveness to interventions. The objectives of this work were to develop a model which will; 1) reconstruct the population dynamics, survival, and fecundity of wild An. funestus populations in southern Tanzania, 2) quantify impacts of density dependence on the dynamics, and 3) assess seasonal fluctuations in An. funestus demography. Through quantifying the population dynamics of An. funestus, this model will enable analysis of how their stability and response to interventions may different from that of An. gambiae s.l.Methods: A Bayesian State Space Model (SSM) based on mosquito life history was fit to time series data on the abundance of female An. funestus s.s. collected over 2 years in southern Tanzania. Prior values of fitness and demography were incorporated from empirical data on larval development, adult survival and fecundity from laboratory-reared first generation progeny of wild caught An. funestus. The model was structured to allow larval and adult fitness traits to vary seasonally in response to environmental covariates (i.e. temperature and rainfall), and for density dependency in larvae. We measured the effects of density dependence and seasonality through counterfactual examination of model fit with or without these covariates.Results: The model accurately reconstructed the seasonal population dynamics of An. funestus and generated biologically-plausible values of their survival larval, development and fecundity in the wild. This model suggests that An-funestus survival and fecundity annual pattern was highly variable across the year, but did not show consistent seasonal trends either rainfall or temperature. While the model fit was somewhat improved by inclusion of density dependence, this was a relatively minor effect and suggests that this process is not as important for An. funestus as it is for An. gambiae populations.Conclusion: The model's ability to accurately reconstruct the dynamics and demography of An. funestus could potentially be useful in simulating the response of these populations to vector control techniques deployed separately or in combination. The observed and simulated dynamics also suggests that An. funestus could be playing a role in year-round malaria transmission, with any apparent seasonality attributed to other vector species.

Entomological Indicators of Malaria Transmission Prior to a Cluster Randomised Controlled Trial of a “Lethal House Lure” Intervention in Central Côte D’Ivoire

Background: A study was conducted prior to implementing a cluster randomised controlled trial (CRT) of a lethal house lure strategy in central Côte d’Ivoire and aimed to provide baseline information on malaria vectors in 40 village clusters. Methods: Human landing catches (HLC) was performed between November-December 2016, capturing mosquitoes indoor and outdoor between 18.00-08.00. Mosquitoes were processed for entomological indicators of malaria transmission (human biting rates, parity rates, sporozoite infection rates and the entomological inoculation rates (EIR)). Species composition and allelic frequencies of Kdr-w and Ace-1R mutations were also investigated within the Anopheles gambiae complex. Results: Overall, 15,632 mosquitoes were captured. Anopheles gambiae s.l. and Anopheles funestus were the two malaria vectors found during the survey period, with predominance for Anopheles gambiae s.l. (66.2%) compared to Anopheles funestus (10.3%). The mean biting rate for An. gambiae s.l. was almost 5 times higher than that for An. funestus s.l.(19.8 bites per person per night for An. gambiae s. l. vs 4.3 bites per person per night for An. funestus s. l.) and this was evident indoor and outdoor. An. funestus was more competent to transmit malaria parasites in the study area, despite relatively lower number tested for sporozoite index (1.6% (1,373) for An. gambiae vs 4.7 % (722) for An. funestus s.l.). There was no significant difference between the proportion infected outdoor and indoor for An. gambiae s.l. (1.6% vs 1.5%; OR=1.11[0.65-1.9]; P=0.676), but for An. funestus, more mosquitoes were infected outdoor (6.4%) than indoor (3.5%) (OR=1.86 [1.07-3.23]; P=0.0249). The majority of both infected vectors with malaria parasites harboured P. falciparum (90.6% for An. gambiae s. l. and 97, 8% for An. funestus s. l.). The EIR for both vectors (0.43 infected bites per night) were similar and there were no significant differences for transmission occurring outdoor and indoor for both species. Of the An. gambiae s.l. analysed, only An. gambiae (14.1%) and An. coluzzii (85.9%) were found. The allelic frequencies of Kdr and Ace-1R were higher in An. gambiae (0.97 for Kdr and 0.19 for Ace-1R) than in An. coluzzii (0.86 for Kdr and 0.10 for Ace-1R) (P<0.001).Conclusion: Despite universal coverage of long-lasting insecticidal nets (LLINs) in the area, there was an abundance of malaria vectors in the study in area in central Côte d’Ivoire, specifically highly resistant An. gambiae s.l. as well as An. funestus s.l.. The malaria sporozoite rate was higher in An. funestus s.l than An. gambiae s.l.. but EIR rates in these two species were similarly high, both indoor and outdoor. Novel tools or strategies are urgently needed to further reduce malaria transmission in this area.

Gene conversion explains elevated diversity in the immunity modulating APL1 gene of the malaria vector Anopheles funestus

The leucine rich repeat gene APL1 is a key component of immunity to Plasmodium and other microbial pathogens in Anopheles mosquitoes. In the malaria vector Anopheles funestus the APL1 gene has four paralogues which occur along the same chromosome arm. We show that APL1 has exceptional levels of non-synonymous polymorphism across the range of An. funestus with an average πn of 0.027 versus a genome-wide average of 0.002, and πn (and πs) is consistently high in populations across Africa. The pattern of APL1 diversity was consistent between independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide-resistance was observed with the phenotyped target-enrichment dataset. Two further innate immunity genes of the gambicin anti-microbial peptide family had πn/πs ratios greater than one, possibly driven by either positive or balancing selection. Cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, an observation discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogs, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. Gene conversion at APL1 versus alternative explanations is concordant with similarly elevated diversity in APL1 and TEP1 loci in An. gambiae. In contrast, the more closely related An. stephensi which also encodes a single-copy of APL1 does not show this elevated diversity. We hypothesise that a more open chromatin formation at the APL1 locus due to higher gene expression than its paralogues enhances gene conversion, and therefore increased polymorphism, at APL1.

Technical Workflow Development for Integrating Drone Surveys and Entomological Sampling to Characterise Aquatic Larval Habitats of Anopheles funestus in Agricultural Landscapes in Côte d’Ivoire

Land-use practices such as agriculture can impact mosquito vector breeding ecology, resulting in changes in disease transmission. The typical breeding habitats of Africa’s second most important malaria vector Anopheles funestus are large, semipermanent water bodies, which make them potential candidates for targeted larval source management. This is a technical workflow for the integration of drone surveys and mosquito larval sampling, designed for a case study aiming to characterise An. funestus breeding sites near two villages in an agricultural setting in Côte d’Ivoire. Using satellite remote sensing data, we developed an environmentally and spatially representative sampling frame and conducted paired mosquito larvae and drone mapping surveys from June to August 2021. To categorise the drone imagery, we also developed a land cover classification scheme with classes relative to An. funestus breeding ecology. We sampled 189 potential breeding habitats, of which 119 (63%) were positive for the Anopheles genus and nine (4.8%) were positive for An. funestus. We mapped 30.42 km2 of the region of interest including all water bodies which were sampled for larvae. These data can be used to inform targeted vector control efforts, although its generalisability over a large region is limited by the fine-scale nature of this study area. This paper develops protocols for integrating drone surveys and statistically rigorous entomological sampling, which can be adjusted to collect data on vector breeding habitats in other ecological contexts. Further research using data collected in this study can enable the development of deep-learning algorithms for identifying An. funestus breeding habitats across rural agricultural landscapes in Côte d’Ivoire and the analysis of risk factors for these sites.

Larval ecology and bionomics of Anopheles funestus in highland and lowland sites in western Kenya

Background An. funestus is a major Afrotropical vector of human malaria. This study sought to investigate the larval ecology, sporozoite infection rates and blood meal sources of An. funestus in western Kenya. Methods Larval surveys were carried out in Bungoma (Highland) and Kombewa (lowland) of western Kenya. Aquatic habitats were identified, characterized, georeferenced and carefully examined for mosquito larvae and predators. Indoor resting mosquitoes were sampled using pyrethrum spray catches. Adults and larvae were morphologically and molecularly identified to species. Sporozoite infections and blood meal sources were detected using real-time PCR and ELISA respectively. Results Of the 151 aquatic habitats assessed, 62/80 (78%) in Bungoma and 58/71(82%) in Kombewa were positive for mosquito larvae. Of the 3,193 larvae sampled, An. funestus larvae constitute 38% (1224/3193). Bungoma recorded a higher number of An. funestus larvae (85%, 95%, CI, 8.722–17.15) than Kombewa (15%, 95%, CI, 1.33–3.91). Molecular identification of larvae showed that 89% (n = 80) were An. funestus. Approximately 59%, 35% and 5% of An. funestus larvae co-existed with An. gambiae s.l, Culex spp and An. coustani in the same habitats respectively. Of 1,221 An. funestus s.l adults sampled, molecular identifications revealed that An. funestus constituted 87% (n = 201) and 88% (n = 179) in Bungoma and Kombewa, respectively. The Plasmodium falciparum sporozoite rate of An. funestus in Bungoma and Kombewa was 2% (3/174) and 1% (2/157), respectively, and the human blood index of An. funestus was 84% (48/57) and 89% (39/44) and for Bungoma and Kombewa, respectively. Conclusion Man-made ponds had the highest abundance of An. funestus larvae. Multiple regression and principal component analyses identified the distance to the nearest house as the key environmental factor associated with the abundance of An. funestus larvae in aquatic habitats. This study serves as a guide for the control of An. funestus and other mosquito species to complement existing vector control strategies.

RNAseq-based gene expression profiling of the Anopheles funestus pyrethroid resistant strain FUMOZ highlights the predominant role of the duplicated CYP6P9a/b cytochrome P450s

Insecticide-based interventions, notably long-lasting insecticidal nets (LLINs), against mosquito vectors of malaria are currently threatened by pyrethroid resistance. Here, we contrasted RNAseq-based gene expression profiling of laboratory resistant (FUMOZ) and susceptible (FANG) strains of the major malaria vector Anopheles funestus. Cytochrome P450 genes were the predominant over-expressed detoxification genes in FUMOZ, with high expression of the duplicated CYP6P9a (fold-change of 82.23 versus FANG) and CYP6P9b (FC 11.15). Other over-expressed P450s belonged to the same cluster of P450s corresponding to the resistance to pyrethroid 1 (rp1) QTL on chromosome 2R. Several Epsilon class glutathione s-transferases were also over-expressed in FUMOZ, as was the ATP-binding cassette transporter AFUN019220 (ABCA) which also exhibited between-strain alternative splicing events at exon 7. Significant differences in SNP frequencies between strains occurred in resistance QTLs rp1 (CYP6P9a/b, CYP6AA1), rp2 on chromosome 2 L (CYP6Z1, CYP6M7, CYP6Z3) and rp3 on chromosome 3R (CYP9J5, CYP9J4 and CYP9J3). Differences were also detected in CYP4G17 and CYP4G16 genes on the X chromosome, both of which are associated with cuticular resistance in An. gambiae. A close analysis of nonsynonymous diversity at the CYP6P9a/b loci revealed a drastic loss of diversity in FUMOZ with only a single polymorphism and 2 haplotypes vs 18 substitutions and 8 haplotypes in FANG. By contrast, a lowly expressed cytochrome P450 (CYP4C36) did not exhibit diversity differences between strains. We also detected the known pyrethroid resistance conferring amino acid change N384S in CYP6P9b. This study further elucidates the molecular bases of resistance in An. funestus, informing strategies to better manage widespread resistance across Africa.

Wild populations of malaria vectors can mate both inside and outside human dwellings

Background Wild populations of Anopheles mosquitoes are generally thought to mate outdoors in swarms, although once colonized, they also mate readily inside laboratory cages. This study investigated whether the malaria vectors Anopheles funestus and Anopheles arabiensis can also naturally mate inside human dwellings. Method Mosquitoes were sampled from three volunteer-occupied experimental huts in a rural Tanzanian village at 6:00 p.m. each evening, after which the huts were completely sealed and sampling was repeated at 11:00 p.m and 6 a.m. the next morning to compare the proportions of inseminated females. Similarly timed collections were done inside local unsealed village houses. Lastly, wild-caught larvae and pupae were introduced inside or outside experimental huts constructed inside two semi-field screened chambers. The huts were then sealed and fitted with exit traps, allowing mosquito egress but not entry. Mating was assessed in subsequent days by sampling and dissecting emergent adults caught indoors, outdoors and in exit traps. Results Proportions of inseminated females inside the experimental huts in the village increased from approximately 60% at 6 p.m. to approximately 90% the following morning despite no new mosquitoes entering the huts after 6 p.m. Insemination in the local homes increased from approximately 78% to approximately 93% over the same time points. In the semi-field observations of wild-caught captive mosquitoes, the proportions of inseminated An. funestus were 20.9% (95% confidence interval [CI]: ± 2.8) outdoors, 25.2% (95% CI: ± 3.4) indoors and 16.8% (± 8.3) in exit traps, while the proportions of inseminated An. arabiensis were 42.3% (95% CI: ± 5.5) outdoors, 47.4% (95% CI: ± 4.7) indoors and 37.1% (CI: ± 6.8) in exit traps. Conclusion Wild populations of An. funestus and An. arabiensis in these study villages can mate both inside and outside human dwellings. Most of the mating clearly happens before the mosquitoes enter houses, but additional mating happens indoors. The ecological significance of such indoor mating remains to be determined. The observed insemination inside the experimental huts fitted with exit traps and in the unsealed village houses suggests that the indoor mating happens voluntarily even under unrestricted egress. These findings may inspire improved vector control, such as by targeting males indoors, and potentially inform alternative methods for colonizing strongly eurygamic Anopheles species (e.g. An. funestus) inside laboratories or semi-field chambers. Graphical Abstract