Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) have resulted in a major decrease in malaria transmission. However, it has become apparent that malaria can be effectively transmitted despite high coverage of LLINs/IRS. Residual transmission can occur due toPlasmodium-carryingAnophelesmosquitoes that are insecticide resistant and have feeding and resting behavior that reduces their chance of encountering the currently deployed indoor malaria control tools. Residual malaria transmission is likely to be the most significant hurdle to achieving the goal of malaria eradication and research and development towards new tools and strategies that can control residual malaria transmission is therefore critical. One of the most promising strategies involves biological agents that are part of the mosquito microbiome and influence the ability ofAnophelesto transmitPlasmodium.These differ from biological agents previously used for vector control in that their primary effect is on vectoral capacity rather than the longevity and fitness ofAnopheles(which may or may not be affected). An example of this type of biological agent isMicrosporidia MB, which was identified in field collectedAnopheles arabiensisand caused complete inhibition ofPlasmodium falciparumtransmission without effecting the longevity and fitness of the host.Microsporidia MBbelongs to a unique group of rapidly adapting and evolving intracellular parasites and symbionts called microsporidia. In this review we discuss the general biology of microsporidians and the inherent characteristics that make some of them particularly suitable for malaria control. We then discuss the research priorities for developing a transmission blocking strategy for the currently leading microsporidian candidateMicrosporidia MBfor malaria control.

Background: Microsporidia MB (MB) is a naturally occurring symbiont of Anopheles and has recently been identified as having a potential to inhibit the transmission of Plasmodium in mosquitoes. MB intensity is high in mosquito gonads, with no fitness consequences for the mosquito, and is linked to horizontal (sexual) and vertical (transovarial) transmission from one mosquito to another. Maximising MB intensity and transmission is important for maintaining heavily infected mosquito colonies for experiments and ultimately for mosquito releases. We have investigated how diet affects the MB-Anopheles arabiensis symbiosis phenotypes, such as larval development and mortality, adult size and survival, as well as MB intensity in both larvae and adults. Methods: F₁ larvae of G₀ females confirmed to be An. arabiensis and infected with MB were either combined (group lines [GLs]) or reared separately (isofemale lines [IMLs]) depending on the specific experiment. Four diet regimes (all mg/larva/day) were tested on F₁ GLs: Tetramin 0.07, Tetramin 0.3, Gocat 0.3 and Cerelac 0.3. GLs reared on Tetramin 0.3 mg/larva/day were then fed either a 1% or 6% glucose diet to determine adult survival. Larvae of IMLs were fed Tetramin 0.07 mg and Tetramin 0.3 mg for larval experiments. The mosquitoes in the adult experiments with IMLs were reared on 1% or 6% glucose. Results: Amongst the four larval diet regimes tested on An.arabiensis development in the presence of MB, the fastest larval development highest adult emergence, largest body size of mosquitoes, highest prevalence and highest density of MB occurred in those fed Tetramin 0.3 mg/larva/day. Although adult MB-positive mosquitoes fed on 6% glucose survived longer than MB-negative mosquitoes, there was no such effect for those fed on the 1% glucose diet. Development time, wing length and adult survival were not significantly different between MB-infected and uninfected An. arabiensis fed on the Tetramin 0.07 mg/larva/day diet, demonstrating that the MB-conferred fitness advantage was diet-dependent. Conclusions: Microsporidia MB does not adversely impact the development and fitness of An. arabiensis, even under limited dietary conditions. The diet regime of Tetramin 0.3 mg/larva/day + 6% glucose for adults is the superior diet for the mass rearing of MB-infected An. arabiensis mosquitoes. These results are important for rearing MB-infected An. arabiensis in the laboratory for experiments and the mass rearing required for field releases. Graphical

Background: Understanding the ecology and behaviour of disease vectors, including the olfactory cues used to orient and select hosts and egg-laying sites, are essential for the development of novel, insecticide-free control tools. Selected graminoid plants have been shown to release volatile chemicals attracting malaria vectors; however, whether the attraction is selective to individual plants or more general across genera and families is still unclear.Methods: To contribute to the current evidence, we implemented bioassays in two-port airflow olfactometers and in large field cages with four live graminoid plant species commonly found associated with malaria vector breeding sites in western Kenya:Cyperus rotundusandC. exaltatusof the Cyperaceae family, andPanicum repensandCynodon dactylonof the Poaceae family. Additionally, we tested one Poaceae species,Cenchrus setaceus, not usually associated with water. The volatile compounds released in the headspace of the plants were identified using gas chromatography/mass spectrometry.Results: All five plants attracted gravid vectors, with the odds of a mosquito orienting towards the choice-chamber with the plant in an olfactometer being 2–5 times higher than when no plant was present. This attraction was maintained when tested with free-flying mosquitoes over a longer distance in large field cages, though at lower strength, with the odds of attracting a female 1.5–2.5 times higher when live plants were present than when only water was present in the trap.Cyperus rotundus,previously implicated in connection with an oviposition attractant, consistently elicited the strongest response from gravid vectors. Volatiles regularly detected were limonene, β-pinene, β-elemene and β-caryophyllene, among other common plant compounds previously described in association with odour-orientation of gravid and unfed malaria vectors.Conclusions: The present study confirms that gravidAnopheles gambiaesensu stricto use chemical cues released from graminoid plants to orientate. These cues are released from a variety of graminoid plant species in both the Cyperaceae and Poaceae family. Given the general nature of these cues, it appears unlikely that they are exclusively used for the location of suitable oviposition sites. The utilization of these chemical cues for attract-and-kill trapping strategies must be explored under natural conditions to investigate their efficiency when in competition with complex interacting natural cues.Graphical

Background: Microsporidia MB is a naturally occurring symbiont in Anopheles arabiensis mosquitoes that inhibits the development of Plasmodium. It is transmitted both vertically and horizontally, enabling its spread within mosquito populations. Currently, mating is the only known mechanism for horizontal transmission. Understanding the factors that influence Microsporidia MB transmission during mating is crucial for developing a malaria transmission-blocking strategy based on this symbiont. Methods: The effect of mosquito age on Microsporidia MB transmission was determined through mating bioassays between infected and uninfected Anopheles arabiensis males and females in three age groups: 3–4 days, 7–8 days, and 10–11 days. Mating bioassays were also conducted to determine if Microsporidia MB infection affects the individual male mating frequencies and mating competitiveness of male mosquitoes. To assess the effect of Microsporidia MB-infection on swarming under field conditions, swarming and non-swarming An. arabiensis male mosquitoes were collected and compared for Microsporidia MB prevalence. Results: The age of mosquitoes does not affect the transmission of Microsporidia MB from males to females (χ² = 11.6, df = 12, p = 0.47). However, transmission of the Microsporidia MB from female mosquitoes to males was not observed in the 3–4 days old age group. Although heterogeneous, there is higher overall transmission from male to female (41.5%) compared to female to male (22.4%). When individual males (Microsporidia MB infected or uninfected) were mated with females, Microsporidia MB-infected males on average mated two times more than the Microsporidia MB-uninfected F1 male mates from the age of 3–4 days to death (t = 2.2, df = 56.8, p = 0.03). Also, Microsporidia MB-infected males when combined in a cage with Microsporidia MB uninfected males were twice as competitive (χ² = 4.74, df = 1, p = 0.02) to the uninfected males in mating with uninfected females. In natural swarms, the proportion of Microsporidia MB-infected males was significantly higher compared to the non-swarming male mosquitoes (χ² = 685.5, df = 1, p < 0.0001). Conclusion: There is a moderate, although heterogenous, horizontal transmission of Microsporidia MB across all age groups, except from 3–4 days old, infected females to males. Microsporidia MB-infected male mosquitoes were almost twice as competitive in mating as their uninfected counterparts. Therefore, Microsporidia MB infected males can potentially disseminate Microsporidia MB in the natural mosquito populations, thus, contributing to malaria control. However, semi-field studies are required to validate these results in a natural environment.

Background: Microsporidia MB, an endosymbiont naturally found in Anopheles mosquitoes inhibits transmission of Plasmodium and is a promising candidate for a transmission-blocking strategy that may involve mosquito release. A rapid assessment was carried out to develop insight into sociodemographic factors, public health concerns, and malaria awareness, management, and prevention practices with the willingness to accept and participate in Microsporidia MB-based transmission-blocking strategy to develop an informed stakeholder engagement process. Methods: The assessment consisted of a survey conducted in two communities in western Kenya that involved administering a questionnaire consisting of structured, semi-structured, and open questions to 8108 household heads. Results: There was an overall high level of willingness to accept (81%) and participate in the implementation of the strategy (96%). Although the willingness to accept was similar in both communities, Ombeyi community was more willing to participate (OR 22, 95% CI 13–36). Women were less willing to accept (OR 0.8, 95% CI 0.7–0.9) compared to men due to fear of increased mosquito bites near homes. Household heads with incomplete primary education were more willing to accept (OR 1.6, 95% CI 01.2–2.2) compared to those educated to primary level or higher. Perceiving malaria as a moderate or low public health issue was also associated with a lower willingness to accept and participate. Experience of > 3 malaria cases in the family over the last six months and knowledge that malaria is transmitted by only mosquito bites, increased the willingness to accept but reduced the willingness to participate. Awareness of malaria control methods based on mosquitoes that cannot transmit malaria increases the willingness to participate. Conclusion: The study showed a high level of willingness to accept and participate in a Microsporidia MB-based strategy in the community, which is influenced by several factors such as community, disease risk perception, gender, education level, knowledge, and experience of malaria. Further research will need to focus on understanding the concerns of women, educated, and employed community members, and factors that contribute to the lower disease risk perception. This improved understanding will lead to the development of an effective communication strategy.

Microsporidia MB is a natural symbiont of Anopheles arabiensis Patton, 1905 that blocks Plasmodium transmission without compromising mosquito fitness. Developing this symbiont for malaria control requires a thorough understanding of its transmission dynamics across generations under environmentally relevant conditions. This study aimed to evaluate the transgenerational spread of Microsporidia MB in semi-field settings. From field-collected infected females (G0), several independent replicates of Microsporidia MB-infected An. arabiensis were established and maintained under semi-field conditions for six generations (F1–F6). All the mosquitoes were screened for Microsporidia MB prevalence and infection intensity using qPCR after death. Temperature and humidity and wing size were recorded. Two replicates sustained through F6. Microsporidia MB prevalence and intensity increased from F1 to F3–F4 before declining. A weak positive correlation was observed between prevalence and temperature, but not humidity. Female mosquitoes exhibited higher infection intensities. Female wing size varied across generations, while male size remained consistent. This first study on Microsporidia MB transgenerational transmission under semi-field conditions revealed a pattern of initial increase followed by decline in prevalence and intensity. The stability of male size supports the potential use of infected males in release programs. These findings underscore additional investigations into environmental drivers for mosquito symbiont prevalence.