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

Correlations Between Household Occupancy and Malaria Vector Biting Risk in Rural Tanzanian Villages: Implications for High-resolution Spatial Targeting of Control Interventions.

Fine-scale targeting of interventions is increasingly important where epidemiological disease profiles depict high geographical stratifications. This study verified correlations between household biomass and mosquito house-entry using experimental hut studies, and then demonstrated how geographical foci of mosquito biting risk can be readily identified based on spatial distributions of household occupancies in villages. A controlled 4 × 4 Latin square experiment was conducted in rural Tanzania, in which no, one, three or six adult male volunteers slept under intact bed nets, in experimental huts. Mosquitoes entering the huts were caught using exit interception traps on eaves and windows. Separately, monthly mosquito collections were conducted in 96 randomly selected households in three villages using CDC light traps between March-2012 and November-2013. The number of people sleeping in the houses and other household and environmental characteristics were recorded. ArcGIS 10 (ESRI-USA) spatial analyst tool, Gi* Ord Statistic was used to analyse clustering of vector densities and household occupancy. The densities of all mosquito genera increased in huts with one, three or six volunteers, relative to huts with no volunteers, and direct linear correlations within tested ranges (P < 0.001). Significant geographical clustering of indoor densities of malaria vectors, Anopheles arabiensis and Anopheles funestus, but not Culex or Mansonia species occurred in locations where households with highest occupancy were also most clustered (Gi* P ≤ 0.05, and Gi* Z-score ≥1.96). This study demonstrates strong correlations between household occupancy and malaria vector densities in households, but also spatial correlations of these variables within and between villages in rural southeastern Tanzania. Fine-scale clustering of indoor densities of vectors within and between villages occurs in locations where houses with highest occupancy are also clustered. The study indicates potential for using household census data to preliminarily identify households with greatest Anopheles mosquito biting risk

Outdoor Mosquito Control Using Odour-Baited Devices: Development and Evaluation of a Potential New Strategy to Complement Indoor Malaria Prevention Methods

A considerable effort is currently underway to develop a malaria vaccine based on live Plasmodium falciparum sporozoites. The first requisite of a sporozoite vaccine is the guarantee of parasite arrest prior to the onset of the pathogenic blood stage. Immunisation with genetically attenuated parasites (GAP) that arrest in the liver forms a promising approach. Work in this thesis describes the development and characterisation of a P. berghei Δb9Δslarp GAP that fully arrests in the liver. Immunisation of multiple mouse strains with low numbers of Δb9Δslarp GAP resulted in sterile protection. The Δb9Δslarp GAP is there- fore the leading GAP vaccine candidate. Work in this the- sis further describes the effect of varying the parameters of sporozoite inoculation on parasite liver load. These findings provide a rationale for the design of clinical trials aimed at the administration of live attenuated P. falciparum sporozoites

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=&#60;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=&#60;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

Effects of a new outdoor mosquito control device, the mosquito landing box, on densities and survival of the malaria vector, Anopheles arabiensis, inside controlled semi-field settings

Background The significance of malaria transmission occurring outdoors has risen even in areas where indoor interventions such as long-lasting insecticidal nets and indoor residual spraying are common. The actual contamination rates and effectiveness of recently developed outdoor mosquito control device, the mosquito landing box (MLB), on densities and daily survival of host-seeking laboratory Anopheles arabiensis, which readily bites humans outdoors was demonstrated. Methods Experiments were conducted in large semi-field systems (SFS) with human volunteers inside, to mimic natural ecosystems, and using MLBs baited with natural or synthetic human odours and carbon dioxide. The MLBs were dusted with 10 % pyriproxyfen (PPF) or entomopathogenic fungi (Metarhizium anisopliae) spores to mark mosquitoes physically contacting the devices. Each night, 400 laboratory-reared An. arabiensis females were released in one SFS chamber with two MLBs, and another chamber without MLBs (control). Mosquitoes were individually recaptured while attempting to bite volunteers inside SFS or by aspiration from SFS walls. Mosquitoes from chambers with PPF-treated MLBs and respective controls were individually dipped in water-filled cups containing ten conspecific third-instar larvae, whose subsequent development was monitored. Mosquitoes recaptured from chambers with fungi-treated MLBs were observed for fungal hyphal growth on their cadavers. Separately, effects on daily survival were determined by exposing An. arabiensis in chambers having MLBs treated with 5 % pirimiphos methyl compared to chambers without MLBs (control), after which the mosquitoes were recaptured and monitored individually until they died. Results Up to 63 % (152/240) and 43 % (92/210) of mosquitoes recaptured inside treatment chambers were contaminated with pyriproxyfen and M. anisopliae, respectively, compared to 8 % (19/240) and 0 % (0/164) in controls. The mean number of larvae emerging from cups in which adults from chambers with PPF-treated MLBs were dipped was significantly lower [0.75 (0.50–1.01)], than in controls [28.79 (28.32–29.26)], P < 0.001). Daily survival of mosquitoes exposed to 5 % pirimiphos methyl was nearly two-fold lower than controls [hazard ratio (HR) = 1.748 (1.551–1.920), P < 0.001]. Conclusion High contamination rates in exposed mosquitoes even in presence of humans, demonstrates potential of MLBs for controlling outdoor-biting malaria vectors, either by reducing their survival or directly killing host-seeking mosquitoes. The MLBs also have potential for dispensing filial infanticides, such as PPF, which mosquitoes can transmit to their aquatic habitats for mosquito population control. Keywords: Mosquito landing box; Malaria; Elimination; Anopheles arabiensis ; Pirimiphos methyl; Outdoor biting; Pyriproxyfen; Metarhizium anisopliae ; Semi-field syste

Evaluation of an ultraviolet LED trap for catching Anopheles and Culex mosquitoes in south-eastern Tanzania

Background: Improved surveillance techniques are required to accelerate efforts against major arthropod-borne diseases such as malaria, dengue, filariasis, Zika and yellow-fever. Light-emitting diodes (LEDs) are increasingly used in mosquito traps because they improve energy efficiency and battery longevity relative to incandescent bulbs. This study evaluated the efficacy of a new ultraviolet LED trap (Mosclean) against standard mosquito collection methods. Methods: The study was conducted in controlled semi-field settings and in field conditions in rural south-eastern Tanzania. The Mosclean trap was compared to commonly used techniques, namely CDC-light traps, human landing catches (HLCs), BG-Sentinel traps and Suna traps. Results: When simultaneously placed inside the same semi-field chamber, the Mosclean trap caught twice as many Anopheles arabiensis as the CDC-light trap, and equal numbers to HLCs. Similar results were obtained when traps were tested individually in the chambers. Under field settings, Mosclean traps caught equal numbers of An. arabiensis and twice as many Culex mosquitoes as CDC-light traps. It was also better at trapping malaria vectors compared to both Suna and BG-Sentinel traps, and was more efficient in collecting mosquitoes indoors than outdoors. The majority of An. arabiensis females caught by Mosclean traps were parous (63.6%) and inseminated (89.8%). In comparison, the females caught by CDC-light traps were 43.9% parous and 92.8% inseminated. Conclusions: The UV LED trap (Mosclean trap) was efficacious for sampling Anopheles and Culex mosquitoes. Its efficacy was comparable to and in some instances better than traps commonly used for vector surveillance. The Mosclean trap was more productive in sampling mosquitoes indoors compared to outdoors. The trap can be used indoors near human-occupied nets, or outdoors, in which case additional CO2 improves catches. We conclude that this trap may have potential for mosquito surveillance. However, we recommend additional field tests to validate these findings in multiple settings and to assess the potential of LEDs to attract non-target organisms, especially outdoors

Persistently high proportions of plasmodium-infected Anopheles funestus mosquitoes in two villages in the Kilombero valley, South-Eastern Tanzania

Background: In south-eastern Tanzania where insecticide-treated nets have been widely used for &gt;20 years, malaria transmission has greatly reduced but remains highly heterogenous over small distances. This study investigated the seasonal prevalence of Plasmodium sporozoite infections in the two main malaria vector species, Anopheles funestus and Anopheles arabiensis for 34 months, starting January 2018 to November 2020. Methods: Adult mosquitoes were collected using CDC-light traps and Prokopack aspirators inside local houses in Igumbiro and Sululu villages, where earlier surveys had found very high densities of An. funestus. Collected females were sorted by taxa, and the samples examined using ELISA assays for detecting Plasmodium circumsporozoite protein in their salivary glands. Results: Of 7859 An. funestus tested, 4.6% (n = 365) were positive for Pf sporozoites in the salivary glands. On the contrary, only 0.4% (n = 9) of the 2382 An. arabiensis tested were positive. The sporozoite prevalence did not vary significantly between the villages or seasons. Similarly, the proportions of parous females of either species were not significantly different between the two villages (p &gt; 0.05) but was slightly higher in An. funestus (0.50) than in An. arabiensis (0.42). Analysis of the 2020 data determined that An. funestus contributed 97.7% of all malaria transmitted in households in these two villages. Conclusions: In contexts where individual vector species mediate most of the pathogen transmission, it may be most appropriate to pursue a species-focused approach to better understand the ecology of the dominant vectors and target them with effective interventions to suppress transmission. Despite the ongoing efforts on tackling malaria in the two study villages, there is still persistently high Plasmodium infection prevalence in local populations of An. funestus, which now carry ~97% of all malaria infections and mediates intense year-round transmission. Further reduction in malaria burden in these or other similar settings requires effective targeting of An. funestus