A protocol for membrane feeding assays to determine the infectiousness of P. falciparum naturally infected individuals to Anopheles gambiae

Abstract

Mosquito feeding assays play an important role in quantifying malaria transmission potential in epidemiological and clinical studies. At present, membrane feeding assays are incompletely standardised. This affects our understanding of the precision of the assay and its suitability for evaluating transmission-blocking interventions. Here, we present a detailed protocol for membrane feeding using Anopheles gambiae mosquitoes and naturally P. falciparum infected individuals.

Figure 1.

Outline of the procedure for detecting and counting Plasmodium oocysts in the mosquito midgut. (Steps are described clockwise). First, an anesthetised mosquito is placed in a drop of PBS and placed under a dissecting microscope (top left). The posterior tip of the abdomen is then slowly pulled from the remainder of the body with a pair of forceps, while a second pair of forceps is used to grasp and stabilise the thorax. In cases when the midgut does not pull out with the abdominal tip, the midgut and foregut may be separated by cutting through the mosquito with a surgical knife or razor at the thoraco-abdominal junction (top left, dotted red line) and the viscera can then be pulled out of the resulting hole in the anterior abdomen. During this process the visceral organs are normally pulled from the body along with the last abdominal segments (top right). The Malpighian tubules as well as any debris are then cut and removed from the midgut using either a knife or forceps (top right). The midguts are then soaked in a convex glass well containing mercurochrome or individually in a drop of mercurochrome on the slide for the desired time (bottom middle; between 5- 15 minutes, depending on the concentration of mercurochrome made up in distilled water). After staining the midguts are mounted in PBS on a glass slide, a cover slip is added, and then samples are visualised under a compound microscope (bottom right). Either phase contrast or bright field can be used depending on the model of microscope. (Inset) Close-up of the oocysts on the mosquito midgut.