The malaria disease burden remains a major impediment to good health and economic development in many regions of sub-Saharan Africa. We have recently reported that a microsporidian symbiont (Microsporidia MB) naturally blocks Plasmodium transmission in Anopheles arabiensis, a major vector of malaria in Africa. Microsporidia MB could form the basis of a novel transmission blocking intervention for malaria control. However, the development of Microsporidia MB as an intervention a strategy will require a better understanding of the symbiont’s biology. Of particular relevance are the natural mosquito to mosquito transmission routes that enable Microsporidia MB to spread within Anopheles mosquito populations and which could potentially be used to disseminate Microsporidia MB as part of a malaria transmission blocking strategy. We investigate the natural routes of Microsporidia MB’s mosquito to mosquito transmission and find that it can be transmitted horizontally between adult An. arabiensis of opposite sexes. This finding will aid the development of a Microsporidia MB dissemination strategy, potentially involving targeted release of Microsporidia MB infected Anopheles mosquitoes.

Malaria continues to be a major health threat across sub-Saharan Africa, with this region accounting for 93% of the global malaria deaths (World Health Organization, 2020). The major preventive strategies for malaria control remain the use of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). In conjunction with improvements in case detection and management, these strategies have reduced malaria cases by up to 40% between 2000 and 2015 (Bhatt et al., 2015). However, progress has plateaued and possibly reversed, with case levels remaining the same between 2014 and 2016 and increasing between 2016 and 2017 (D’Alessandro, 2018; World Health Organization, 2020). It is apparent that current malaria control strategies have their limitations and there is a vital need for complementary tools (Huijben and Paaijmans, 2018).

The malaria transmission cycle relies on female Anopheles mosquitoes becoming infected by feeding on human blood that contains the Plasmodium gametocyte stage. Plasmodium gametocytes undergo a series of developmental changes before traversing the mosquito midgut to form a sporogonic oocyst, which produces sporozoites that are released into the mosquito hemocoel. Sporozoites in the hemocoel travel to the mosquito salivary glands to enter the mosquito’s saliva, which results in an infected mosquito, usually 8–14 days after the bloodmeal (Baton and Ranford-Cartwright, 2005). This transmission cycle can be impeded by inhibitory interactions with mosquito-associated microbes (Romoli and Gendrin, 2018). One of the most promising new management strategies involves the use of vertically (mother to offspring) transmitted symbiotic microbes that prevent the establishment of disease-causing viruses in mosquito vectors. This strategy is currently used as a control mechanism against the arboviral disease, Dengue, through the bacterial symbiont, Wolbachia (Moreira et al., 2009; Bian et al., 2010; Hoffmann et al., 2011; Walker et al., 2011; Frentiu et al., 2014; Ant et al., 2018; Nazni et al., 2019).

The Anopheles-associated symbiont Microsporidia MB colonizes mosquito ovaries and is vertically transmitted. This microsporidian can also block the transmission of malaria by Anopheles mosquitoes (Herren et al., 2020), and therefore could potentially contribute to the control of malaria. The successful deployment of symbiont-based vector-borne disease control strategies requires the ability to spread symbionts through host insect populations and the maintenance of a high prevalence of infection. In Wolbachia-based strategies, cytoplasmic incompatibility can effectively drive symbionts through mosquito populations. In the absence of cytoplasmic incompatibility, other driving mechanisms would be required to spread Microsporidia MB through Anopheles populations. Microsporidia MB is naturally found in populations of Anopheles mosquitoes in Kenya, ranging in prevalence from 0 to 25% (Herren et al., 2020). From the standpoint of symbiont-based control strategies, the different Microsporidia MB transmission routes could be relevant for interventions that could generate a higher prevalence of the transmission-blocking symbiont in Anopheles mosquito populations, leading to reductions in malaria transmission.

Microsporidia are a diverse clade of obligate, intracellular organisms that infect an array of hosts, including vertebrates and invertebrates and are found in both terrestrial and aquatic environments (Vossbrinck and Debrunner-Vossbrinck, 2005). The morphology of Microsporidia can be simplified into the meront phase, which is present during proliferation, and the spore, which is resistant to environmental degradation and transmission-specialized. Microsporidian spores are characterized by a chitinous wall and a polar filament involved in host cell penetration (Stentiford et al., 2013). In arthropods, Microsporidian transmission can occur vertically (mother to offspring) and horizontally (from one individual to another of the same generation, Stentiford et al., 2013). There are also many reported incidences of microsporidians using a combination of vertical and horizontal transmission. Vertical transmission generally occurs via the transovarial route with spores germinating on the periphery or inside of ovaries to colonize developing eggs. Vertical transmission is associated with greater host specificity and lower Microsporidia burden and virulence (Vávra and Lukeš, 2013). There are different forms of horizontal transmission in arthropod-associated Microsporidia, however the most widespread is oral and involves the ingestion of spores, which subsequently germinate and inject their sporoplasm into the host intestinal cells through a polar filament. Microsporidia that predominately rely on oral horizontal transmission tend to be associated with lower levels of host specificity and high virulence as microsporidian spores will usually be released en masse from deceased hosts to infect other hosts (Han and Weiss, 2017). Other forms of horizontal transmission that are not associated with high virulence, for example sexual transmission, have also been demonstrated in several microsporidian species. Nosema plodiae is a microsporidian pathogen of the Indian meal moth, Plodia interpunctella, which invades the reproductive organs of its host and is transmitted from male to female moths during mating (Kellen and Lindegren, 1971).

The Microsporidia transmission mode influences host specificity and life-cycle complexity (Stentiford et al., 2013). Microsporidians can be generalists, infecting a variety of different hosts or exhibit high levels of host specialization. Microsporidians can have specialization toward a single (simple lifecycle) or several intermediate hosts (complex lifecycle). Vertical and sexual transmission result in limited opportunities for Microsporidia to infect hosts of a different species and are therefore likely to lead to higher levels of host specificity. In contrast, horizontal transmission by spore ingestion is likely to be associated with lower levels of host specificity. Microsporidians with simple and complex lifecycles can use both vertical and horizontal transmission. In most cases, different spores types become specialized for different transmission routes (Stentiford et al., 2013).

We investigated a number of possible horizontal transmission routes for Microsporidia MB in An. arabiensis. We established that transmission was only found to occur between adult mosquitoes. In addition, transmission was only observed between different sexes, which indicates that Microsporidia MB is sexually transmitted in An. arabiensis.

The results clearly demonstrate that Microsporidia MB is transmitted horizontally between adult An. arabiensis. Transmission was only observed in cages that had opposite sexes of Microsporidia MB infected and uninfected adult An. arabiensis suggesting that Microsporidia MB is transmitted sexually. Anopheles gambiae s.l. males package seminal fluid that is produced in the male accessory glands into a coagulated mating plug that is digested in the female atrium several days after mating (Giglioli and Mason, 1966). Sperm received by mated female Anopheles are stored in a dedicated organ called the spermatheca, which is relied upon by Anopheles gambiae s.l. females for a lifetime of offspring production (Tripet et al., 2003). We observed that Microsporidia MB intensity was much higher in the midgut and male gonads than in the carcass of male An. arabiensis. This suggests that Microsporidia MB either migrates to or proliferates in the male gonad. We observed multinucleate Microsporidia cells only in specimens that were infected with Microsporidia MB, indicating that these cells are developmental stages of Microsporidia MB. Microsporidia MB cells were specifically localized to the An. arabiensis male ejaculatory duct. The Microsporidia MB cells observed had either 4 or 8 nuclei, which likely indicates that Microsporidia MB sporogenesis is occurring in the An. arabiensis male ejaculatory duct as 4-nuclei sporogonial plasmodia develop into an 8-nuclei stage and finally to become sporophorous vesicles. This developmental sequence has been reported in greater detail in Microsporidians associated with fire ants (Sokolova and Fuxa, 2008) and Daphnia (Refardt et al., 2008). It is therefore likely that the sporogenesis of Microsporidia MB in the male ejaculatory duct produces infectious spores that are released with seminal secretions and therefore transferred to females upon mating. Transmission from female to male An. arabiensis was also observed, but further investigation will be required to establish the basis of this transmission route.

Two findings indicate that mating is required for Microsporidia MB transmission. Firstly, the absence of Microsporidia MB transmission in same sex transmission cage experiments, and secondly the finding that in the three cage experiments where female An. arabiensis spermatheca were checked for the presence of sperm, only inseminated females acquired Microsporidia MB. The experimental design precluded the quantification of precise transmission rates, since in the majority of experiments female insemination events were not confirmed. However, in light of the low rate of female insemination in cages where spermatheca were checked, it can be expected that the rate of Microsporidia MB transmission from males to female An. arabiensis per successful mating is likely to be high.

The number of females infected and the intensity of Microsporidia MB infections in recipient females was not dependent on the intensity of Microsporidia MB in donor males. A possible explanation for this finding is that Microsporidia MB are localized to midguts and gonads. It is possible that localization to the male gonad is a pre-requisite for sexual transmission and that only the intensity of Microsporidia MB infection in gonads is correlated with transmission capacity. The finding that Microsporidia MB intensity was high in the midgut and that some male An. arabiensis had high intensity of Microsporidia MB only in the midgut suggests that this organ may play a yet to be determined role in transmission or alternatively that the midgut is a reservoir of Microsporidia MB. Notably, since the majority of gonadal tissue development occurs during metamorphosis, localization to the midgut could be required for maintenance of Microsporidia MB infection in An. arabiensis larval stages.

From the perspective of symbionts that are strictly maternally inherited, males are a dead end. In many cases, including for maternally inherited microsporidians, this can lead to the evolution of feminization or male-killing (Ironside and Alexander, 2015). Another possible outcome is that maternally inherited infections evolve to become sexually transmitted. The sexual transmission of beneficial heritable microbes has been reported in aphids (Moran and Dunbar, 2006). It is probable that in aphids sexual transmission enabled decreased pathogenicity of symbionts and co-evolution toward obligate mutualism.

Sexual horizontal transmission has been reported in a variety of insect-associated microsporidians (Knell and Mary Webberley, 2004) and in most cases it is associated with other complementary forms of transmission. Sexual transmission is likely to be more effective in insect species that have overlapping generations and higher levels of promiscuity. In Anopheles, the bacterial symbionts Asaia (Favia et al., 2007) and Serratia AS1 (Wang et al., 2017) have been shown to be sexually transmitted. It is notable that Anopheles gambiae s.l. is largely monandrous and therefore it is unlikely that symbionts could rely solely on sexual horizontal transmission. Indeed, both Asaia and Serratia AS1 are also transmitted vertically and by other horizontal transmission routes. It is notable that sexually transmitted infections of insects tend to reach much higher prevalence levels than infections with other forms of horizontal transmission (Knell and Mary Webberley, 2004). An example is the Microsporidian Nosema calcarati, which is sexually and vertically transmitted in its host Pitogenes calcaratus and found at a prevalence of 50% (Purrini and Halperin, 1982). High prevalence may be in part due to the fact that sexual transmission selects for lower levels of virulence toward the hosts. Sexually transmitted infections can manipulate insect host physiology or behavior to favor higher levels of transmission, for example sexually transmitted mites were shown to increase the mating success of male midge hosts (McLachlan, 1999). Whether any sexually transmitted pathogens of Anopheles affect mating behavior has not been established.

Microsporidia MB was not found in non-anopheline arthropods that are found in the same habitats as An. arabiensis larvae. Since Microsporidia MB is transmitted vertically and by sexual horizontal transmission, a high level of host specificity could be expected since neither vertical (Herren et al., 2020), nor sexual horizontal transmission would be effective across species. It is noteworthy that Microsporidia MB was found in another species of anopheline mosquito, An. funestus s.s., which is a primary vector of malaria in Sub-Saharan Africa. If the Microsporidia MB found in An. funestus s.s have similar characteristics to Microsporidia MB found in An. arabiensis, including Plasmodium transmission blocking, then Microsporidia MB could be developed as a tool for malaria control in several primary vector species.

To be successfully developed into a strategy to control malaria, an effective method of disseminating Microsporidia MB into Anopheles populations will need to be established. Our results show that Microsporidia MB infected male mosquitoes can infect their female counterparts and that horizontally infected females can transmit Microsporidia MB to their offspring. We previously showed that Microsporidia MB is vertically transmitted in An. arabiensis (Herren et al., 2020) and therefore Microsporidia MB infected males for releases could be produced by sorting the offspring of Microsporidia MB infected An. arabiensis colonies. These findings could be the basis for a dissemination strategy that involves targeted release of Microsporidia MB infected male Anopheles mosquitoes, potentially avoiding the need to release biting females, which would be advantageous in terms of community engagement and acceptance of the intervention. In principle, such a strategy would be similar to the mass-release of sterile males (Bouyer et al., 2020), except that instead of sterilizing females Microsporidia MB infected males would decrease the capacity of infected females and their offspring to transmit malaria for multiple generations. The capacity of Microsporidia MB to be vertically transmitted after infecting females would potentially make this approach more sustainable and cost-effective than SIT.

All the datasets presented in this study can be found an online repository: https://doi.org/10.6084/m9.figshare.14846925.v2.

JH and GN conceived and designed the majority of the experiments. GN, TM, TB, and EEM performed the majority of the experiments. GN, EEM, TM, DM, and EO collected mosquitoes and screened them for symbionts. JH, SS, EM, EEM, LM, ET, JP, JB, GN, TB, TO, FO, and GM analyzed the data. JH, TO, and FO carried out the microscopy. JH and GN wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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