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.

Malaria imposes an enormous burden on sub-Saharan Africa, and evidence that incidence 22could be starting to increase again 1 suggests the limits of currently applied control strategies 23 have now been reached. A possible novel control approach involves the dissemination in 24 mosquitoes of inherited symbiotic microbes to block transmission. This strategy is 25 exemplified by the use of transmission-blocking Wolbachia in Aedes aegypti against dengue 26 virus 2-7 . However, in the Anopheles gambiae complex, the primary African vectors of 27 malaria, there limited reports of inherited symbionts with transmission-blocking capacity 8-28 10 . Here we show that a newly discovered vertically transmitted species of Microsporidia 29 symbiont in the An. gambiae complex blocks Plasmodium transmission. Microsporidia MB is 30 present at moderate prevalence in geographically dispersed populations of An. arabienesis 31 in Kenya, localized to the mosquito midgut and ovaries, and is not associated with significant 32 reductions in adult host fecundity or survival. Field collected Microsporidia MB-infected An. 33 arabiensis were never found to harbor P. falciparum gametocytes and on experimental 34 infection with P. falciparum no sporozoites could be detected in Microsporidia MB-infected 35 mosquitos. As a Plasmodium transmission-blocking microbe that is non-virulent and 36 vertically transmitted, Microsporidia MB could be exploited as a novel malaria control tool. 37Microsporidia are a group of obligately intracellular simple eukaryotes, classified within or as a 38 sister group to fungi, and found in a wide range of hosts, but most commonly invertebrates. Their 39 lifecycles include a meront phase during proliferation, and spores with chitinous cell walls 40 involved in host to host transmission through spore ingestion. Species with solely horizontal 41 transmission usually show greater virulence and lower host specificity, but where a combination 42 of horizontal and vertical transovarial transmission occurs, lower virulence is advantageous, and 43 is normally associated with a higher degree of host specificity 11-12 . Sex ratio distortion toward 44 females has been reported (a manipulation characteristic of transovarially transmitted symbionts), 45 for example Dictyocoela microsporidia in Amphipod crustaceans 13 . Various microsporidia species 46 have been reported in mosquitoes 14-25 , with simple or complex lifecycles 14 but all of which are 47