Background Agricultural pesticides may exert strong selection pressures on malaria vectors during the aquatic life stages and may contribute to resistance in adult mosquitoes. This could reduce the performance of key vector control interventions such as indoor-residual spraying and insecticide-treated nets. The aim of this study was to investigate effects of agrochemicals on susceptibility and fitness of the malaria vectors across farming areas in Tanzania. Methods An exploratory mixed-methods study was conducted to assess pesticide use in four villages (V1–V4) in south-eastern Tanzania. Anopheles gambiae (s.l.) larvae were collected from agricultural fields in the same villages and their emergent adults examined for insecticide susceptibility, egg-laying and wing lengths (as proxy for body size). These tests were repeated using two groups of laboratory-reared An. arabiensis, one of which was pre-exposed for 48 h to sub-lethal aquatic doses of agricultural pesticides found in the villages. Results Farmers lacked awareness about the linkages between the public health and agriculture sectors but were interested in being more informed. Agrochemical usage was reported as extensive in V1, V2 and V3 but minimal in V4. Similarly, mosquitoes from V1 to V3 but not V4 were resistant to pyrethroids and either pirimiphos-methyl or bendiocarb, or both. Adding the synergist piperonyl butoxide restored potency of the pyrethroids. Pre-exposure of laboratory-reared mosquitoes to pesticides during aquatic stages did not affect insecticide susceptibility in emergent adults of the same filial generation. There was also no effect on fecundity, except after pre-exposure to organophosphates, which were associated with fewer eggs and smaller mosquitoes. Wild mosquitoes were smaller than laboratory-reared ones, but fecundity was similar. Conclusions Safeguarding the potential of insecticide-based interventions requires improved understanding of how agricultural pesticides influence important life cycle processes and transmission potential of mosquito vectors. In this study, susceptibility of mosquitoes to public health insecticides was lower in villages reporting frequent use of pesticides compared to villages with little or no pesticide use. Variations in the fitness parameters, fecundity and wing length marginally reflected the differences in exposure to agrochemicals and should be investigated further. Pesticide use may exert additional life cycle constraints on mosquito vectors, but this likely occurs after multi-generational exposures. Graphical Abstract

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

Background: The role that larval predators play in regulating the population of malaria vectors remains relatively unknown. This study aimed to investigate the common predators that co-exist with Anopheles funestus group larvae and evaluate factors that influence their abundance in rural south-eastern Tanzania. Methods: Mosquito larvae and predators were sampled concurrently using standard dipper (350 ml) or 10 L bucket in nine villages in southern Tanzania. Predators were identified using standard identification keys. All positive habitats were geo-located and their physical features characterized. Water physicochemical parameters such as dissolved oxygen (DO), pH, electrical conductivity (EC), total dissolved solids (TDS) and temperature were also recorded. Results: A total of 85 previously identified An. funestus aquatic habitats were sampled for larvae and potential predators. A total of 8,295 predators were sampled. Of these Coenagrionidae 57.7% (n=4785), Corixidae 12.8% (n=1,060), Notonectidae 9.9% (n=822), Aeshnidae 4.9% (n=405), Amphibian 4.5% (n=370), Dytiscidae 3.8% (n=313) were common. A total of 5,260 mosquito larvae were sampled, whereby Anopheles funestus group were 60.3% (n= 3,170), Culex spp. 24.3% (n= 1,279), An. gambie s.l. 8.3% (n= 438) and other anophelines 7.1% (n= 373). Permanent and aquatic habitats larger than 100m2 were positively associated with An. funestus group larvae (P<0.05) and predator abundance (P<0.05). Habitats with submerged vegetation were negative associated with An. funestus group larvae (P<0.05). Only dissolved oxygen (DO) was positively and significantly affect the abundance of An. funestus group larvae (P<0.05). While predators abundance were not impacted by all physicochemical parameters. Conclusion: Six potential predator families were common in aquatic habitats of An. funestus larvae group. Additional studies are needed to demonstrate the efficacy of different predators on larval density and adult fitness traits. Interventions leveraging the interaction between mosquitoes and predators can be established to disrupt the transmission potential and survival of the An. funestus mosquitoes. Key words: Anopheles funestus group, predators, aquatic habitats, malaria transmission, Ifakara Health Institute, and biological control.

Background Malaria transmission can be highly heterogeneous between and within localities, and is influenced by factors such as survival and biting frequencies of Anopheles mosquitoes. This study investigated the relationships between the biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes, which currently dominate malaria transmission in south-east Tanzania. The study also examined how such relationships may influence malaria transmission and control. Methods Female An. funestus were collected in houses located 50–100 m, 150–200 m or over 200 m from the nearest known aquatic habitats. The mosquitoes were exposed to 1×, 5× and 10× the diagnostic doses of deltamethrin or permethrin, or to the synergist, piperonyl butoxide (PBO) followed by the pyrethroids, then monitored for 24 h-mortality. Ovaries of exposed and non-exposed mosquitoes were dissected to assess parity as a proxy for biological age. Adults emerging from larval collections in the same villages were tested against the same insecticides at 3–5, 8–11 or 17–20 days old. Findings Mosquitoes collected nearest to the aquatic habitats (50-100 m) had the lowest mortalities compared to other distances, with a maximum of 51% mortality at 10× permethrin. For the age-synchronized mosquitoes collected as larvae, the insecticide-induced mortality assessed at both the diagnostic and multiplicative doses (1×, 5× and 10×) increased with mosquito age. The highest mortalities at 1× doses were observed among the oldest mosquitoes (17–20 days). At 10× doses, mortalities were 99% (permethrin) and 76% (deltamethrin) among 8–11 day-olds compared to 80% (permethrin) and 58% (deltamethrin) among 3–5 day-olds. Pre-exposure to PBO increased the potency of both pyrethroids. The proportion of parous females was highest among mosquitoes collected farthest from the habitats. Conclusion In this specific setting, older An. funestus and those collected farthest from the aquatic habitats (near the centre of the village) were more susceptible to pyrethroids than the younger ones and those caught nearest to the habitats. These findings suggest that pyrethroid-based interventions may remain at least moderately effective despite widespread pyrethroid-resistance, by killing the older, less-resistant and potentially-infective mosquitoes. Further studies should investigate how and whether these observations could be exploited to optimize malaria control in different settings.

Background: Variation in mosquito body size and the ability to penetrate long-lasting insecticide-treated nets (LLINs) remains unknown. This study evaluated the ability of Anopheles funestus and A. arabiensis to penetrate commercially available treated and untreated bednets and how this behaviour affects mosquito mortality. Methods: Three types of LLINs; DawaPlus 2.0, PermaNet 2.0, Olyset 2.0, and untreated (Safi Net) were tested inside a semi-field system. One hundred 3–5-day-old female A. funestus and A. arabiensis were released in a chamber with a sleeping adult volunteer under a treated or untreated bednet. Mosquitoes that penetrated inside the nets were collected every two hours using a mouth aspirator. Live mosquitoes were put in paper cups, fed on glucose ad libitum and their mortality rate was monitored for 48 h. Results: The ability of A. funestus to penetrate treated and untreated bednets was significantly higher than for A. arabiensis for all three LLIN net types (P<0.001). For both species the penetration rate was higher for untreated bednets than treated ones except for the Olyset net. Of all mosquitoes that penetrated the net, regardless of the species, >90% successfully blood-fed on the sleeping volunteer. Compared to A. arabiensis, significant mortality was recorded for A. funestus that were caught inside Olyset nets within 48 hrs of monitoring (P<0.001). Conclusions: These findings demonstrate the ability of A. funestus and A. arabiensis mosquitoes to penetrate the human-occupied treated and untreated bednets. Despite this ability, mosquitoes that penetrated the bednet succumbed to death within two days.