Host range of Microsporidia MB in aquatic macrofauna inhabiting Anopheles breeding sites.

Microsporidia MB, a recently discovered endosymbiont Anopheles arabiensis mosquitoes, shows great potential as a malaria-blocking tool due to its Plasmodium-blocking phenotype. The symbiont utilizes vertical (mother-to-offspring) and horizontal (sexual) transmission routes to propagate across anopheline populations. Microsporidia MB occurs throughout the Anopheles life cycle, with no evidence to date of the existence of a secondary host. In this study, we conducted a comprehensive molecular screening of Microsporidia MB in aquatic macrofauna associated with Anopheles larval habitats to determine whether they serve as secondary hosts for Microsporidia MB. We collected aquatic macrofauna from anopheline breeding sites along the Ahero irrigation scheme, including insects from the orders Hemiptera (families Naucoridae, Micronectidae, Nepidae, and Notonectidae), Coleoptera (families Staphylinidae, Noteridae, Hydrophilidae, and Dytiscidae), Diptera (anopheline and non-anopheline larvae and pupae), Orthoptera, and Ephemeroptera; snails (Hygrophila); crustaceans (Podocopida); and frogs (Anura). We detected Microsporidia MB in 15.04% of anopheline larvae, but not in non-anopheline mosquito larvae, pupae, or any other aquatic macrofauna, despite their continuous interaction with anopheline mosquitoes in the larval habitats. The absence of Microsporidia MB detection across the diverse assemblage of aquatic macrofauna surveyed in this study suggests that these taxa are unlikely to constitute major secondary hosts or reservoirs of the symbiont. While broader surveys across additional taxa are needed to fully exclude alternative hosts, the consistent detection of Microsporidia MB at high infection intensities in Anopheles mosquitoes supports a strong host association. This reinforces its potential as a targeted, species-specific, and environmentally safe symbiont-based intervention for malaria control.IMPORTANCEMalaria continues to pose a significant global health burden, particularly in sub-Saharan Africa, and causes thousands of deaths annually. Microsporidia MB is an anopheline-associated symbiont that is vertically and horizontally transmitted in Anopheles mosquitoes and shows great promise as a complementary malaria control tool due to its Plasmodium blocking phenotype. However, its lifecycle is not fully understood. Understanding its lifecycle and host range is important for assessing the symbiont's ecological safety and feasibility for field release. This study found no evidence of Microsporidia MB in a wide range of aquatic macrofauna using sensitive molecular screening, despite their frequent contact with infected mosquito larvae. While non-detection does not rule out rare or transient infections, the results suggest that these organisms are unlikely to serve as major hosts of the symbiont. This apparent host specificity reduces concerns about spreading to non-target species and supports the development of Microsporidia MB as a targeted malaria-control intervention.