Non-Coding RNAs Potentially Involved in Pyrethroid Resistance of Anopheles funestus Population in Western Kenya.

Backgrounds: The resurgence of Anopheles funestus, a dominant vector of human malaria in western Kenya was partly attributed to insecticide resistance. However, evidence on the molecular basis of pyrethroid resistance in western Kenya is limited. Noncoding RNAs (ncRNAs) form a vast class of RNAs that do not code for proteins and are ubiquitous in the insect genome. Here, we demonstrated that multiple ncRNAs could play a potential role in An. funestusresistance to pyrethroid in western Kenya. Materials and Methods: Anopheles funestus mosquitoes were sampled by aspiration methods in Bungoma, Teso, Siaya, Port Victoria and Kombewa in western Kenya. The F1 progenies were exposed to deltamethrin (0.05%), permethrin (0.75%), DDT (4%) and pirimiphos-methyl (0.25%) following WHO test guidelines. A synergist assay using piperonyl butoxide (PBO) (4%) was conducted to determine cytochrome P450s' role in pyrethroid resistance. RNA-seq was conducted on a combined pool of specimens that were resistant and unexposed, and the results were compared with those of the FANG susceptible strain. This approach aimed to uncover the molecular mechanisms underlying pyrethroid resistance. Results: Pyrethroid resistance was observed in all the sites with an average mortality rate of 57.6%. Port Victoria had the highest level of resistance to permethrin (MR=53%) and deltamethrin (MR=11%) pyrethroids. Teso had the lowest level of resistance to permethrin (MR=70%) and deltamethrin (MR=87%). Resistance to DDT was observed only in Kombewa (MR=89%) and Port Victoria (MR=85%). A full susceptibility to P-methyl (0.25%) was observed in all the sites. PBO synergist assay revealed high susceptibility (>98%) to the pyrethroids in all the sites except for Port Victoria (MR=96%, n=100). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by imipenemase (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP-binding cassettes (2%) and carboxylesterases(1%). Conclusions: This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention development could help in insecticide resistance management.

Microsporidia MB in the primary malaria vector Anopheles gambiae sensu stricto is avirulent and undergoes maternal and horizontal transmission.

BACKGROUND: The demonstration that the recently discovered Anopheles symbiont Microsporidia MB blocks malaria transmission in Anopheles arabiensis and undergoes vertical and horizontal transmission suggests that it is a promising candidate for the development of a symbiont-based malaria transmission-blocking strategy. The infection prevalence and characteristics of Microsporidia MB in Anopheles gambiae sensu stricto (s.s.), another primary vector species of malaria in Kenya, were investigated. METHODS: Field-collected females were confirmed to be Microsporidia MB-positive after oviposition. Egg counts of Microsporidia MB-infected and non-infected individuals were used to infer the effects of Microsporidia MB on fecundity. The time to pupation, adult sex ratio and survival were used to determine if Microsporidia MB infection has similar characteristics in the host mosquitoes An. gambiae and An. arabiensis. The intensity of Microsporidia MB infection in tissues of the midgut and gonads, and in carcasses, was determined by quantitative polymerase chain reaction. To investigate horizontal transmission, virgin males and females that were either Microsporidia MB-infected or non-infected were placed in standard cages for 48 h and allowed to mate; transmission was confirmed by quantitative polymerase chain reaction targeting Microsporidia MB genes. RESULTS: Microsporidia MB was found to naturally occur at a low prevalence in An. gambiae s.s. collected in western Kenya. Microsporidia MB shortened the development time from larva to pupa, but other fitness parameters such as fecundity, sex ratio, and adult survival did not differ between Microsporidia MB-infected and non-infected hosts. Microsporidia MB intensities were high in the male gonadal tissues. Transmission experiments indicated that Microsporidia MB undergoes both maternal and horizontal transmission in An. gambiae s.s. CONCLUSIONS: The findings that Microsporidia MB naturally infects, undergoes maternal and horizontal transmission, and is avirulent in An. gambiae s.s. indicate that many of the characteristics of its infection in An. arabiensis hold true for the former. The results of the present study indicate that Microsporidia MB could be developed as a tool for the transmission-blocking of malaria across different Anopheles species.

Horizontal Transmission of the Symbiont Microsporidia MB in Anopheles arabiensis.

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.

The fungus Leptosphaerulina persists in Anopheles gambiae and induces melanization.

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.

A microsporidian impairs Plasmodium falciparum transmission in Anopheles arabiensis mosquitoes.

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.