Abstract
BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.
Highlights
The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise
It provides a thorough description of how gametocyte cultures can be synchronized with mosquito rearing and how mosquito maintenance and feeding regimes can influence the success of infection
Dark‐feeding increases mosquito infection and standard membrane feeding assay (SMFA) sensitivity The optimized mosquito husbandry described in the Methods section and schematically presented in Fig. 1 resulted in mosquitoes with significantly larger body size as measured by the wing length compared to mosquitoes generated with standard colony rearing (Fig. 2a; P < 0.001)
Summary
The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The newest data suggest that this progress is progressively coming to a halt or, being reversed in some countries, indicating that the current malaria intervention tools and strategies may have reached their maximum capacity. This highlights the urgency of developing new complementary technologies targeting malaria transmission in order to achieve the milestones of the new WHO Global Technical Strategy for Malaria 2016–2030 [1]. The engineered effectors can be driven through the wild vector population using gene drive technologies such as CRISPR/Cas9 [7,8,9,10,11]
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