Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity

Abstract

Background: 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 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.

Methods: The 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.

Results: A 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.

Conclusion: A 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.

Keywords: Anopheles coluzzii; Anopheles gambiae; Gametocyte; Gene drive; Malaria; Mosquito population replacement; Plasmodium falciparum; Standard membrane feeding assay.

Fig. 1

Overview of the protocol for synchronized mosquito and gametocyte culture coupled with the dark-feeding and resting regime

Fig. 2

Mosquito infection with Plasmodium falciparum using the streamlined SMFA and mosquito dark-feeding and resting regime. a Comparison of wing length as a proxy for body size between mosquitoes reared as reported in this manuscript (optimal; opt) and mosquitoes generated under standard colony maintaining regime (std). b Comparison of RBC count in the mosquito gut bolus, used as a proxy to bloodmeal volume, between mosquitoes maintained in dark conditions for 3 h prior to, during and several hours after blood feeding (dark-feeding regime) compared mosquitoes fed using the standard SMFA protocol. c Pie-charts showing P. falciparum oocyst infection prevalence in mosquitoes under dark-feeding regime compared to mosquitoes fed using the standard SMFA protocol. Numbers in brackets show the number of mosquitoes used for each replicate as well as the total number of mosquitoes. d Overall P. falciparum infection intensities between mosquitoes under dark-feeding regime and mosquitoes fed using the standard SMFA protocol. e Forest plot showing estimate of odds ratio (± 95% CI) of oocyst intensities between mosquitoes under a dark-feeding regime and control. Squares and diamond shows the sample size for each replicate and the total, respectively

Fig. 3

Summary of infections obtained using synchronized mosquito and Plasmodium falciparum gametocyte culture and the dark-feeding regime. a Scatter plot of mean infection intensities versus prevalence (%) for 123 feeds that used a total of 2703 mosquitoes. The fitting line was generate using Sigmoidal 4PL analysis, X is log (oocyst count) and Y is the prevalence: Y = 86.65/(1 + 10^{(− 2.73 − 0.03X)}). b Parasite infection intensity for 63 control feeds (no further treatment). Dots and lines represent median and range, respectively

Fig. 4

Effect of AMPs on Anopheles coluzzii infections with Plasmodium falciparum. a Infection intensity in mosquitoes fed on cultured P. falciparum gametocytes spiked with water (control), and AMPs at a 50 μM dose. b, c Infection intensity in mosquitoes fed on cultured gametocytes spiked with modified magainin and melittin, respectively. d Infection intensity in mosquitoes fed on cultured gametocytes spiked with different concentration of melittin-EENPG. Dots shows oocyst counts for individual midguts and red lines show median infection intensities