A possible malaria control approach involves the dissemination in mosquitoes of inherited symbiotic microbes to block Plasmodium transmission. However, in the Anopheles gambiae complex, the primary African vectors of malaria, there are limited reports of inherited symbionts that impair transmission. We show that a vertically transmitted microsporidian symbiont (Microsporidia MB) in the An. gambiae complex can impair Plasmodium transmission. Microsporidia MB is present at moderate prevalence in geographically dispersed populations of An. arabiensis in Kenya, localized to the mosquito midgut and ovaries, and is not associated with significant reductions in adult host fecundity or survival. Field-collected Microsporidia MB infected An. arabiensis tested negative for P. falciparum gametocytes and, on experimental infection with P. falciparum, sporozoites aren't detected in Microsporidia MB infected mosquitoes. As a microbe that impairs Plasmodium transmission that is non-virulent and vertically transmitted, Microsporidia MB could be investigated as a strategy to limit malaria transmission.

The recently discovered Anopheles symbiont, Microsporidia MB, has a strong malaria transmission-blocking phenotype in Anopheles arabiensis, the predominant Anopheles gambiae species complex member in many active transmission areas in eastern Africa. The ability of Microsporidia MB to block Plasmodium transmission together with vertical transmission and avirulence makes it a candidate for the development of a symbiont-based malaria transmission blocking strategy. We investigate the characteristics and efficiencies of Microsporidia MB transmission between An. arabiensis mosquitoes. We show that Microsporidia MB is not transmitted between larvae but is effectively transmitted horizontally between adult mosquitoes. Notably, Microsporidia MB was only found to be transmitted between male and female An. arabiensis, suggesting sexual horizontal transmission. In addition, Microsporidia MB cells were observed infecting the An. arabiensis ejaculatory duct. Female An. arabiensis that acquire Microsporidia MB horizontally are able to transmit the symbiont vertically to their offspring. We also investigate the possibility that Microsporidia MB can infect alternate hosts that live in the same habitats as their An. arabiensis hosts, but find no other non-anopheline hosts. Notably, Microsporidia MB infections were found in another primary malaria African vector, Anopheles funestus s.s. The finding that Microsporidia MB can be transmitted horizontally is relevant for the development of dissemination strategies to control malaria that are based on the targeted release of Microsporidia MB infected Anopheles mosquitoes.

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Anopheles mosquitoes are colonized by diverse microorganisms that may impact on host biology and vectorial capacity. Eukaryotic symbionts such as fungi have been isolated from Anopheles, but whether they are stably associated with mosquitoes and transmitted transstadially across mosquito life stages or to subsequent generations remains largely unexplored. Here, we show that a Leptosphaerulina sp. fungus isolated from the midgut of An. gambiae can be stably associated with An. gambiae host and that it imposes low fitness cost when re-introduced through co-feeding. This fungus is transstadially transmitted across An. gambiae developmental stages and to their progeny. It is present in field-caught larvae and adult mosquitoes at moderate levels across geographical regions. We observed that Leptosphaerulina sp. induces a distinctive melanotic phenotype across the developmental stages of mosquito. As a eukaryotic symbiont that is stably associated with An. gambiae Leptosphaerulina sp. can be explored for paratransgenesis.

Background: The discovery of the Anopheles symbiont, Microsporidia MB in An. arabiensis and the subsequent demonstration that it blocks malaria transmission, undergoes vertical and horizontal transmission suggests that it is a promising candidate for developing a symbiont-based malaria transmission-blocking strategy. We investigated the prevalence and characteristics of Microsporidia MB in An. gambiae s.s., another primary malaria vector species in Kenya. Methods: Field collected females were confirmed Microsporidia MB positive after oviposition. The egg counting of Microsporidia MB infected and non-infected individuals used to infer the effects of Microsporidia MBon fecundity. The duration to pupation, the adult sex ratio and survival were quantified to determine if Microsporidia MBinfection has similar characteristics in An. gambiae and An. arabiensis hosts. The intensity of Microsporidia MB infections in tissues such as midgut, gonads and carcasses was determined by qPCR. To investigate horizontal transmission, virgin individuals of opposite sexes that were either Microsporidia MB infected or non-infected were placed in the standard cages for 48 hours and allowed to mate. After this period, transmission was confirmed by assessment of Microsporidia MB infection status by qPCR. Results: We observed that Microsporidia MB is naturally found at low prevalence in Anopheles gambiae s.s. collected in Western Kenya. Microsporidia MB shortened development time from larvae to pupae, but other fitness parameters such as fecundity, sex ratio, and adult survival did not differ between Microsporidia MB infected and non-infected host. Microsporidia MB intensities were high in the male gonadal tissue. Transmission experiments indicate that Microsporidia MB utilizes both maternal and horizontal transmission in An. gambiae s.s. Conclusions: The finding that Microsporidia MB naturally infects An. gambiae s.s., undergoes maternal and horizontal transmission and is avirulent signifies that many of the characteristics of Microsporidia MB infection observed in An. arabiensis hold true for Anopheles gambiae s.s. This is an early indication that Microsporidia MB could equally be developed as a tool for malaria transmission blocking across different Anopheles species.

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