Quantifying the ecology of Anopheles funestus and its implications for improved malaria control

The malaria burden is highest in African countries where more than 95% of deaths and cases occur. There was a consistent decline in malaria deaths and cases in Africa between 2000 and 2015 but progress has since stalled. Due to biological changes in vector populations, notably insecticide resistance and behavioral adaptations such as outdoor-biting, the primary vector control measures are no longer as successful as they once were. In recent years, mosquito species considered to be the primary vector of malaria, (e.g. Anopheles gambiae s.s) have declined, and even disappeared from some communities. In settings such as rural south-eastern Tanzania, the residual transmission is now being maintained by An. funestus followed by An. arabiensis. Currently, An. funestus mediates a high proportion of malaria transmission events in east and southern Africa. The resilience of this vector may be linked to its high insecticide resistance; though recent evidence suggests that it may also be capable of shifting its biting behaviors to avoid contact with insecticidal interventions. Yet, our ability to tackle this vector species is impeded by the limited knowledge of its basic ecology and population dynamics, the difficulties in colonizing it under laboratory conditions and the many uncertainties about appropriate surveillance approaches. The overall aim of this PhD project was to quantify the ecology of An. funestus mosquitoes in Tanzania and assess the implications of its key attributes for improved malaria control in settings such as Tanzania where the vector species dominates. The work involved the following steps: 1) quantifying the fitness and behavioral attributes of wild An. funestus and their offspring during repeated colonization attempts under standard laboratory conditions, 2) developing and validating a framework for predicting human biting exposures from different exposure-free sampling methods, 3) developing and testing a population dynamics model to describe the ecology of the wild An. funestus populations, and 4) assessing the generalizability of the population dynamics model and its ability to reconstruct missing data. To achieve the first objective, I attempted to colonize a local population of An. funestus s.s. from southeastern Tanzania and assessed the key barriers which hinder laboratory establishment. Adult females (F0) from three wild An. funestus populations were brought into the laboratory for rearing. Their fecundity, and the development, survival, body size and mating success of their F1 offspring were measured to evaluate their fitness under laboratory conditions. While adult survival was relatively high, the mating success, poor hatching rate and poor larval survival and extended larval development periods were identified as key barriers to establishing a colony in the laboratory. Due to these factors, this colony was not sustained beyond the F1 generation in the laboratory, but the lessons were deployed for a subsequent and more successful colonization effort. To address the second objective, I analyzed data comparing the outdoor catch rates of An. funestus using six exposure-free trapping methods relative to the human landing catches (HLC), the gold standard method for estimating human exposures to mosquito bites. I tested different models for the relationship between HLC and other trapping methods while allowing flexibility for associations to be impacted by interspecific and intraspecific density dependence. This analysis indicated that that the association between catches in alternative traps and the HLC can best be explained by simple linear models; with minimal impact of intra and inter specific density dependence. A shiny app interface was developed to allow expanded use of this statistical calibration framework for future estimations of malaria vector biting risk in communities. For the third objective, I used the demographic parameters generated from the colonization attempt (described above) and published literature, to develop the first population dynamics model of wild An. funestus in Tanzania. I used a Bayesian framework to develop a state-space model and reconstruct the observed population dynamics of this species. I then used this model to assess the strength of evidence for intrinsic (density dependence) and extrinsic (environmental covariates) drivers of An. funestus population dynamics and how they drive seasonality in abundance and demographic variables (development periods and survival). This analysis indicated that density dependence has a minimal contribution on the overall dynamics of An. funestus in these settings. Daily larval and adult survival probabilities were marginally affected by changes in environmental covariates (temperature and rainfall), suggesting there is little seasonality in these fitness parameters. This study also revealed that An. funestus may be essential for sustaining year-round malaria transmission in settings such as rural south-eastern Tanzania. Finally, I interrogated the generalizability and sensitivity of this modelling framework for An. funestus by assessing its ability to predict missing time series data. Here, I refitted the model to a single population and assessed any unexplained features of population dynamics which is causing the density dependence to have small contributions. I also omitted portions of the time series data to assess model prediction capability. For example I first removed 25% and then 50% of the data, then reconstructed the missing sections. The single population model indicated that An. funestus demographic variables were much more sensitive to changes in environmental covariates compared to the preceding hierarchical model; suggesting that clear signals of environmental drivers may be lost by fitting the model to multiple populations that may have distinct drivers. The model was able to reconstruct the observed population trajectory poorly when 50% of the data was removed as compared to when 25% was removed. Overall, the model was only able to predict for the missing data if the training set included some representation of data from both dry and wet seasons. In conclusion, this PhD work contributes to a general understanding of the key barriers to colonization and the population dynamics of An. funestus. While An. funestus was not successfully colonised in this study, the lessons learned by documenting which fitness traits are impeded in the laboratory led to progress in further work at the Ifakara Health Institute, where a stable colony of An. funestus has now been established. Additionally, the model of An. funestus dynamics and demography developed here will underpin further research to evaluate and select optimal vector control packages for crashing these populations in southern Tanzania and other settings where they are the major source of residual malaria transmission

Variations in household microclimate affect outdoor-biting behaviour of malaria vectors

Background: Mosquito behaviours including the degree to which they bite inside houses or outside is a crucial determinant of human exposure to malaria. Whilst seasonality in mosquito vector abundance is well documented, much less is known about the impact of climate on mosquito behaviour. We investigated how variations in household microclimate affect outdoor-biting by malaria vectors, Anopheles arabiensis and Anopheles funestus. Methods: Mosquitoes were sampled indoors and outdoors weekly using human landing catches at eight households in four villages in south-eastern Tanzania, resulting in 616 trap-nights over 12 months. Daily temperature, relative humidity and rainfall were recorded. Generalized additive mixed models (GAMMs) were used to test associations between mosquito abundance and the microclimatic conditions. Generalized linear mixed models (GLMMs) were used to investigate the influence of microclimatic conditions on the tendency of vectors to bite outdoors (proportion of outdoor biting). Results: An. arabiensis abundance peaked during high rainfall months (February-May), whilst An. funestus density remained stable into the dry season (May-August). Across the range of observed household temperatures, a rise of 1ºC marginally increased nightly An. arabiensis abundance (~11%), but more prominently increased An. funestus abundance (~66%). The abundance of An. arabiensis and An. funestus showed strong positive associations with time-lagged rainfall (2-3 and 3-4 weeks before sampling). The degree of outdoor biting in An. arabiensis was significantly associated with the relative temperature difference between indoor and outdoor environments, with exophily increasing as temperature inside houses became relatively warmer. The exophily of An. funestus did not vary with temperature differences. Conclusions: This study demonstrates that malaria vector An. arabiensis shifts the location of its biting from indoors to outdoors in association with relative differences in microclimatic conditions. These environmental impacts could give rise to seasonal variation in mosquito biting behaviour and degree of protection provided by indoor-based vector control strategies

Interventions that effectively target Anopheles funestus mosquitoes could significantly improve control of persistent malaria transmission in south–eastern Tanzania

Malaria is transmitted by many Anopheles species whose proportionate contributions vary across settings. We re-assessed the roles of Anopheles arabiensis and Anopheles funestus, and examined potential benefits of species-specific interventions in an area in south-eastern Tanzania, where malaria transmission persists, four years after mass distribution of long-lasting insecticide-treated nets (LLINs). Monthly mosquito sampling was done in randomly selected households in three villages using CDC light traps and back-pack aspirators, between January-2015 and January-2016, four years after the last mass distribution of LLINs in 2011. Multiplex polymerase chain reaction (PCR) was used to identify members of An. funestus and Anopheles gambiae complexes. Enzyme-linked immunosorbent assay (ELISA) was used to detect Plasmodium sporozoites in mosquito salivary glands, and to identify sources of mosquito blood meals. WHO susceptibility assays were done on wild caught female An. funestus s.l, and physiological ages approximated by examining mosquito ovaries for parity. A total of 20,135 An. arabiensis and 4,759 An. funestus were collected. The An. funestus group consisted of 76.6% An. funestus s.s, 2.9% An. rivulorum, 7.1% An. leesoni, and 13.4% unamplified samples. Of all mosquitoes positive for Plasmodium, 82.6% were An. funestus s.s, 14.0% were An. arabiensis and 3.4% were An. rivulorum. An. funestus and An. arabiensis contributed 86.21% and 13.79% respectively, of annual entomological inoculation rate (EIR). An. arabiensis fed on humans (73.4%), cattle (22.0%), dogs (3.1%) and chicken (1.5%), but An. funestus fed exclusively on humans. The An. funestus populations were 100% susceptible to organophosphates, pirimiphos methyl and malathion, but resistant to permethrin (10.5% mortality), deltamethrin (18.7%), lambda-cyhalothrin (18.7%) and DDT (26.2%), and had reduced susceptibility to bendiocarb (95%) and propoxur (90.1%). Parity rate was higher in An. funestus (65.8%) than An. arabiensis (44.1%). Though An. arabiensis is still the most abundant vector species here, the remaining malaria transmission is predominantly mediated by An. funestus, possibly due to high insecticide resistance and high survival probabilities. Interventions that effectively target An. funestus mosquitoes could therefore significantly improve control of persistent malaria transmission in south–eastern Tanzania

Semi-field assessment of the BG-Malaria trap for monitoring the African malaria vector, Anopheles arabiensis

Odour-baited technologies are increasingly considered for effective monitoring of mosquito populations and for the evaluation of vector control interventions. The BG-Malaria trap (BGM), which is an upside-down variant of the widely used BG-Sentinel trap (BGS), has been demonstrated to be effective to sample the Brazilian malaria vector, Anopheles darlingi. We evaluated the BGM as an improved method for sampling the African malaria vectors, Anopheles arabiensis. Experiments were conducted inside a large semi-field cage to compare trapping efficiencies of BGM and BGS traps, both baited with the synthetic attractant, Ifakara blend, supplemented with CO2. We then compared BGMs baited with either of four synthetic mosquito lures, Ifakara blend, Mbita blend, BG-lure or CO2, and an unbaited BGM. Lastly, we compared BGMs baited with the Ifakara blend dispensed via either nylon strips, BG cartridges (attractant-infused microcapsules encased in cylindrical plastic cartridge) or BG sachets (attractant-infused microcapsules encased in plastic sachets). All tests were conducted between 6P.M. and 7A.M., with 200–600 laboratory-reared An. arabiensis released nightly in the test chamber. The median number of An. arabiensis caught by the BGM per night was 83, IQR:(73.5–97.75), demonstrating clear superiority over BGS (median catch = 32.5 (25.25–37.5)). Compared to unbaited controls, BGMs baited with Mbita blend caught most mosquitoes (45 (29.5–70.25)), followed by BGMs baited with CO2 (42.5 (27.5–64)), Ifakara blend (31 (9.25–41.25)) and BG lure (16 (4–22)). BGM caught 51 (29.5–72.25) mosquitoes/night, when the attractants were dispensed using BG-Cartridges, compared to BG-Sachet (29.5 (24.75–40.5)), and nylon strips (27 (19.25–38.25)), in all cases being significantly superior to unbaited controls (p < 000.1). The findings demonstrate potential of the BGM as a sampling tool for African malaria vectors over the standard BGS trap. Its efficacy can be optimized by selecting appropriate odour baits and odour-dispensing systems

Fitness characteristics of the malaria vector Anopheles funestus during an attempted laboratory colonization

Background: The malaria vector Anopheles funestus is increasingly recognized as a dominant vector of residual transmission in many African settings. Efforts to better understand its biology and control are significantly impeded by the difficulties of colonizing it under laboratory conditions. To identify key bottlenecks in colonization, this study compared the development and fitness characteristics of wild An. funestus from Tanzania (FUTAZ) and their F1 offspring during colonization attempts. The demography and reproductive success of wild FUTAZ offspring were compared to that of individuals from one of the only An. funestus strains that has been successfully colonized (FUMOZ, from Mozambique) under similar laboratory conditions. Methods: Wild An. funestus (FUTAZ) were collected from three Tanzanian villages and maintained inside an insectary at 70–85% RH, 25–27 °C and 12 h:12 h photoperiod. Eggs from these females were used to establish three replicate F1 laboratory generations. Larval development, survival, fecundity, mating success, percentage pupation and wing length were measured in the F1 -FUTAZ offspring and compared with wild FUTAZ and FUMOZ mosquitoes. Results: Wild FUTAZ laid fewer eggs (64.1; 95% CI [63.2, 65.0]) than FUMOZ females (76.1; 95% CI [73.3, 79.1]). Survival of F1-FUTAZ larvae under laboratory conditions was low, with an egg-to-pupae conversion rate of only 5.9% compared to 27.4% in FUMOZ. The median lifespan of F1-FUTAZ females (32 days) and males (33 days) was lower than FUMOZ (52 and 49 for females and males respectively). The proportion of female F1-FUTAZ inseminated under laboratory conditions (9%) was considerably lower than either FUMOZ (72%) or wild-caught FUTAZ females (92%). This resulted in nearly zero viable F2-FUTAZ eggs produced. Wild FUTAZ wings appear to be larger compared to the lab reared F1-FUTAZ and FUMOZ. Conclusions: This study indicates that poor larval survival, mating success, low fecundity and shorter survival under laboratory conditions all contribute to difficulties in colonizing of An. funestus. Future studies should focus on enhancing these aspects of An. funestus fitness in the laboratory, with the biggest barrier likely to be poor mating

A statistical calibration tool for methods used to sample outdoor-biting mosquitoes

Background: Improved methods for sampling outdoor-biting mosquitoes are urgently needed to improve surveillance of vector-borne diseases. Such tools could potentially replace the human landing catch (HLC), which, despite being the most direct option for measuring human exposures, raises significant ethical and logistical concerns. Several alternatives are under development, but detailed evaluation still requires common frameworks for calibration relative to HLC. The aim of this study was to develop and validate a statistical framework for predicting human-biting rates from different exposure-free alternatives. Methods: We obtained mosquito abundance data (Anopheles arabiensis, Anopheles funestus and Culex spp.) from a year-long Tanzanian study comparing six outdoor traps [Suna Trap (SUN), BG Sentinel (BGS), M-Trap (MTR), M-Trap + CDC (MTRC), Ifakara Tent Trap-C (ITT-C) and Mosquito Magnet-X Trap (MMX)] and HLC. Generalised linear models were developed within a Bayesian framework to investigate associations between the traps and HLC, taking intra- and inter-specific density dependence into account. The best model was used to create a calibration tool for predicting HLC-equivalents. Results: For An. arabiensis, SUN catches had the strongest correlation with HLC (R2 = 19.4), followed by BGS (R2 = 17.2) and MTRC (R2 = 13.1) catches. The least correlated catch was MMX (R2 = 2.5). For An. funestus, BGS had the strongest correlation with the HLC (R2 = 53.4), followed by MTRC (R2 = 37.4) and MTR (R2 = 37.4). For Culex mosquitoes, the traps most highly correlated with the HLC were MTR (R2 = 45.4) and MTRC (R2 = 44.2). Density dependence, both between and within species, influenced the performance of only BGS traps. An interactive Shiny App calibration tool was developed for this and similar applications. Conclusion: We successfully developed a calibration tool to assess the performance of different traps for assessing outdoor-biting risk, and established a valuable framework for estimating human exposures based on the trap catches. The performance of candidate traps varied between mosquito taxa; thus, there was no single optimum. Although all the traps tested underestimated the HLC-derived exposures, it was possible to mathematically define their representativeness of the true biting risk, with or without density dependence. The results of this study emphasise the need to aim for a consistent and representative sampling approach, as opposed to simply seeking traps that catch the most mosquitoes

New evidence of mating swarms of the malaria vector, Anopheles arabiensis in Tanzania

Background: Malaria mosquitoes form mating swarms around sunset, often at the same locations for months or years. Unfortunately, studies of Anopheles swarms are rare in East Africa, the last recorded field observations in Tanzania having been in 1983. Methods: Mosquito swarms were surveyed by trained volunteers between August-2016 and June-2017 in Ulanga district, Tanzania. Identified Anopheles swarms were sampled using sweep nets, and collected mosquitoes killed by refrigeration then identified by sex and taxa. Sub-samples were further identified by PCR, and spermatheca of females examined for mating status. Mosquito ages were estimated by observing female ovarian tracheoles and rotation of male genitalia. GPS locations, types of swarm markers, start/end times of swarming, heights above ground, mosquito counts/swarm, and copulation events were recorded. Results: A total of 216 Anopheles swarms were identified, characterized and mapped, from which 7,142 Anopheles gambiae s.l and 13 Anopheles funestus were sampled. The An. gambiae s.l were 99.6% males and 0.4% females, while the An. funestus were all males. Of all An. gambiae s.l analyzed by PCR, 86.7% were An. arabiensis, while 13.3% returned non-amplified DNA. Mean height (±SD) of swarms was 2.74±0.64m, and median duration was 20 (IQR; 15-25) minutes. Confirmed swarm markers included rice fields (25.5%), burned grounds (17.2%), banana trees (13%), brick piles (8.8%), garbage heaps (7.9%) and ant-hills (7.4%). Visual estimates of swarm sizes by the volunteers was strongly correlated to actual sizes by sweep nets (R=0.94; P=<0.001). All females examined were nulliparous and 95.6% [N=6787] of males had rotated genitalia, indicating sexual maturity. Conclusions: This is the first report of Anopheles swarms in Tanzania in more than three decades. The study demonstrates that the swarms can be identified and characterized by trained community-based volunteers, and highlights potential new interventions, for example targeted aerosol spraying of the swarms to improve malaria control

New evidence of mating swarms of the malaria vector, Anopheles arabiensis in Tanzania

Background: Malaria mosquitoes form mating swarms around sunset, often at the same locations for months or years. Unfortunately, studies of Anopheles swarms are rare in East Africa, the last recorded field observations in Tanzania having been in 1983. Methods: Mosquito swarms were surveyed by trained volunteers between August-2016 and June-2017 in Ulanga district, Tanzania. Identified Anopheles swarms were sampled using sweep nets, and collected mosquitoes killed by refrigeration then identified by sex and taxa. Sub-samples were further identified by PCR, and spermatheca of females examined for mating status. Mosquito ages were estimated by observing female ovarian tracheoles and rotation of male genitalia. GPS locations, types of swarm markers, start/end times of swarming, heights above ground, mosquito counts/swarm, and copulation events were recorded. Results: A total of 216 Anopheles swarms were identified, characterized and mapped, from which 7,142 Anopheles gambiae s.l and 13 Anopheles funestus were sampled. The An. gambiae s.l were 99.6% males and 0.4% females, while the An. funestus were all males. Of all An. gambiae s.l analyzed by PCR, 86.7% were An. arabiensis, while 13.3% returned non-amplified DNA. Mean height (±SD) of swarms was 2.74±0.64m, and median duration was 20 (IQR; 15-25) minutes. Confirmed swarm markers included rice fields (25.5%), burned grounds (17.2%), banana trees (13%), brick piles (8.8%), garbage heaps (7.9%) and ant-hills (7.4%). Visual estimates of swarm sizes by the volunteers was strongly correlated to actual sizes by sweep nets (R=0.94; P=<0.001). All females examined were nulliparous and 95.6% [N=6787] of males had rotated genitalia, indicating sexual maturity. Conclusions: This is the first report of Anopheles swarms in Tanzania in more than three decades. The study demonstrates that the swarms can be identified and characterized by trained community-based volunteers, and highlights potential new interventions, for example targeted aerosol spraying of the swarms to improve malaria control

Creating mosquito-free outdoor spaces using transfuthrin-treated chairs and ribbons

This research article published by Springer Nature, 2020Background: Residents of malaria-endemic communities spend several hours outdoors performing diferent activities, e.g. cooking, story-telling or eating, thereby exposing themselves to potentially-infectious mosquitoes. This compromises efectiveness of indoor interventions, notably long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). This study characterized common peri-domestic spaces in rural south-eastern Tanzania, and assessed protective efcacy against mosquitoes of hessian fabric mats and ribbons treated with the spatial repellent, transfuthrin, and ftted to chairs and outdoor kitchens, respectively. Methods: Two hundred households were surveyed, and their most-used peri-domestic spaces physically characterized. Protective efcacies of locally-made transfuthrin-emanating chairs and hessian ribbons were tested in outdoor environments of 28 households in dry and wet seasons, using volunteer-occupied exposure-free double net traps. CDC light traps were used to estimate host-seeking mosquito densities within open-structure outdoor kitchens. Fieldcollected Anopheles arabiensis and Anopheles funestus mosquitoes were exposed underneath the chairs to estimate 24 h-mortality. Finally, The World Health Organization insecticide susceptibility tests were conducted on wild-caught Anopheles from the villages. Results: Approximately half (52%) of houses had verandas. Aside from these verandas, most houses also had peridomestic spaces where residents stayed most times (67% of houses with verandas and 94% of non-veranda houses). Two-thirds of these spaces were sited under trees, and only one third (34.4%) were built-up. The outdoor structures were usually makeshift kitchens having roofs and partial walls. Transfuthrin-treated chairs reduced outdoor-biting An. arabiensis densities by 70–85%, while transfuthrin-treated hessian ribbons ftted to the outdoor kitchens caused 77–81% reduction in the general peri-domestic area. Almost all the feld-collected An. arabiensis (99.4%) and An. funestus (100%) exposed under transfuthrin-treated chairs died. The An. arabiensis were susceptible to non-pyrethroids (pirimiphos methyl and bendiocarb), but resistant to pyrethroids commonly used on LLINs (deltamethrin and permethrin). Conclusion: Most houses had actively-used peri-domestic outdoor spaces where exposure to mosquitoes occurred. The transfuthrin-treated chairs and ribbons reduced outdoor-biting malaria vectors in these peri-domestic spaces, and also elicited signifcant mortality among pyrethroid-resistant feld-caught malaria vectors. These two new prototype formats for transfuthrin emanators, if developed further, may constitute new options for complementing LLINs and IRS with outdoor protection against malaria and other mosquito-borne pathogens in areas where peri-domestic human activities are common