Occurrence and Interactions of Mycobiomes with the Anopheles gambiae Host

Anopheles mosquitoes are colonized by diverse microorganisms that may influence hosts‟ biology and vectorial capacity. Resident Anopheles bacterial symbionts isolated from these hosts are known to influence traits such as generational fitness, survival, host seeking,and infectivity of pathogens such asPlasmodium falciparum.Eukaryotic symbionts such as fungi residing in the gut of Anopheles gambiaecould confer similar phenotypes, but are undexplored. It was hypothesized that fungi associating with Anopheles could establish a stable and generational association with these mosquitoes thereby influencing their fitness and could offer protection against malaria parasites. The objective of this study was to characterize fungalsymbionts of Anopheles gambiaeand establish their tripartite interactions with the parasite. Anopheles samples obtained from screen houses mimicking field environmental setting at icipe-Thomas Odhiambo Campus (iTOC) Mbita were used for fungal isolation, while field samples screened for the isolated fungi were collected from selected parts of Western, Central and Coastal Kenya. Using standard tissue culture techniques and rDNA-ITS sequencing, 25 fungi were isolatedand identified from developmental stages of semi-field mosquitoes. These isolates were found to belong to the generaAspergilus, Penicillium, Periconia, Epicoccum, Leptosphaerulina, Hyphopichia, Alternaria, Lichtheimia, Cladosporium, Hasegawazyma, Marasphaerium, and Dothideomycetes sps.Eight of these isolates namely Penicillium, Periconia, Epicoccum, Leptosphaerulina, Alternaria, Lichtheimia, Cladosporium, and Hasegawazymasp resided in both the midguts and ovaries, and were selected for re-introduction to the immature stages to establish symbiont-vector association. One of these isolates namely Leptosphaerulinasp. was found to stably associate with An. gambiae. This fungus undergoestransstadial transmission across developmental stagesof An. gambiae and was detected in their progeny. Field surveys indicated that it was present in the field-collected larvae and adult Anopheles sp. at moderate intensities across sampled geographical regions. The presence of Leptosphaerulina sp. in the infected host wasmanifested by deposition of a distinctive melanin in their fat tissues (melanotic phenotype).It was suggested that melanosis contributed to the establishment and persistence of Leptosphaerulina in the host tissuessince symbiont was localized in the midgut and reproductive tissues. This colonization was found to elevate host basal immunity suggesting that systemic infectioncaused mild interference to the host‟s fitness by slowinglarval development and reduction in fecundity during the first generation of infection. Protein coding genes linked to KEGG pathways suggests that the presence of this fungus is likely to modulate host nutrient metabolism. However, Plasmodium challenge experiments with laboratory and field mosquitoes showed that Leptosphaerulina exhibits subtle effects on the development of P.falciparum sporozoites (Using unpaired t-test,An. arabiensis: t(98)=1.365, p=0.1755; An. gambiae: t(121)=1.955, p=0.0529; field collected An. gambiaecomplex: t(36)=0.5885, p=0.5599). This suggested the existence of a complex host-fungus-parasite tripartite interaction. Upon establishing that cultivable fungi utilize mixed routes of transmission, it was hypothesized that a non-culturable fungus of Anopheles namelyMicrosporidia MB previously identified through high throughput sequencing and found to protect hosts ainstPlasmodiumfalciparumcould also exploit mixed modes of transmission.The rationale for developing a sustainable mode of spreading Microsporidia MBis based on the the need to increase field prevalence to a level that is sufficient in lowering malaria disease burden. Notably, it was established that the spread of Microsporidia MBwas only found to occur between adult mosquitoeswith evidence between infected individuals of opposite sexes. Thissuggested that Microsporidia MB is transmitted through venereal/sexual means.Importantly, Microsporidia MBwas observed in the male gonad and found to undergoa series of developmental stages in the ejaculatory duct and is secreted in the male seminal fluid. Observation of sperms in the spermathecae of females acquiring Microsporidia through insemination and a lack of transmission in same sex cohorts (through direct contact)signifythat this symbiont can be transmitted horizontally through sexual/venereal route. To conclude, the study identified cultivable fungi that associate with various developmental stages of Anopheles gambiae species. One of these fungi namely Leptosphaerulina sp. was found to establish a stable co-existence across developmental stages of these hosts with minimal fitness cost and occurred naturally inthe wild caught Anopheles gambiae complex. It was also found that Microsporidia MBexploitssexual transmission to spread through Anopheles populations.It is recommended that a sequencing approach that targets uncultured fungi could reveal mycobiomes that were not identified in this study. It would also be important to study mechanisms utilised by these symbionts to undergo maternal transmission. These fungi could be secreting secondary metabolites which ought to be studied in relation to host fitness and Plasmodium blocking. To utilise Leptosphaerulina in paratransgensisis it would be important to transform it with effector molecules. A large scale trial of the properties that enhance males‟ competence in the dissemination of Microsporidia MBis important for successful field trial implementation.Taken together, these findings suggest that fungal symbionts establish stable association with Anopheles hosts and could be utilized to either deliver anti-Plasmodium factors or used directly to block transmission of Plasmodium thereby contributing to the current modus operandi ofintegrated malaria control strategies