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

Horizontal transmission of the symbiont Microsporidia MB in Anopheles arabiensis

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

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

Abstract 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. Graphical Abstrac

Additional file 4 of Microsporidia MB in the primary malaria vector Anopheles gambiae sensu stricto is avirulent and undergoes maternal and horizontal transmission

Additional file 4: Table S2. Horizontal transmission of Microsporidia MB between Anopheles gambiae s.s. adults reared together in cages. Values indicate intensities of Microsporidia MB infection in donor male/female and recipient male/female An. gambiae s.s. individuals reared together in a cage

Additional file 3 of Microsporidia MB in the primary malaria vector Anopheles gambiae sensu stricto is avirulent and undergoes maternal and horizontal transmission

Additional file 3: Table S1. Annual prevalence of Microsporidia MB in Anopheles gambiae s.l. Values indicate yearly (2021–2022) abundance (%) of Microsporidia MB assessed from the DNA of individual female mosquitoes sampled from Bungoma and Busia

Additional file 2 of Microsporidia MB in the primary malaria vector Anopheles gambiae sensu stricto is avirulent and undergoes maternal and horizontal transmission

Additional file 2: Figure S2. Microsporidia MB infection intensities in An. gambiae s.s. influence the rate of transmission to offspring. A Correlation of Go Microsporidia MB intensities with average G1 Microsporidia MB densities [r2 = 0.1276, P = 0.268, number of broods = 18]. B Correlation between G0 Microsporidia MB intensities and Microsporidia MB transmission to offspring (r2 = 0.625, P = 0.0056, n = 18)

Additional file 1 of Microsporidia MB in the primary malaria vector Anopheles gambiae sensu stricto is avirulent and undergoes maternal and horizontal transmission

Additional file 1: Figure S1. Sex ratio of Microsporidia MB-infected Anopheles gambiae s.s. progeny reared in pools. A Bar plots representing the sex ratio of Microsporidia MB-infected offspring and non-infected counterparts reared in pools (n = 6, where a pool consists of approximately seven individual females). B Bar plots representing the mean prevalence of Microsporidia MB transmitted to An. gambiae s.s. offspring, reared in pools, by females. Error bars represent the SEM percentage (* P < 0.05)