The mosquito is an obligatory vector for transmission of malaria, a disease that kills 2 million people every year. Plasmodium, the causative agent of malaria, must physically traverse the mosquito peritrophic matrix (PM) in order to complete its life cycle in the mosquito. In both larvae and adults, the PM completely surrounds the food bolus. Enzymes secreted by the midgut epithelium must cross the PM to reach the ingested food and the products of digestion must cross the PM in the opposite direction to be absorbed by the midgut epithelial cells. We devised a new non-invasive in vivo assay for estimating larval PM permeability. The assay also allows for assessment of permeability of the caecal membrane, a structure that separates each caecum from the gut lumen. Permeability was estimated by appearance of fluorescently-labeled dextrans (size range 4.4 - 2,000 kDa) within the gastric caecae of mosquito larvae. The larval PM was permeable to dextrans of 148 kDa and smaller, while the caecal membrane was permeable to dextrans 19.5 kDa and smaller. The assay was also used to devise a treatment that disrupts the PM sufficiently to allow the passage of virus-sized particles. Dithiothreitol was effective and chitinase only partially effective in disrupting the PM. Both agents did not have any toxic effects on the larvae. Cycloheximide, Polyoxin D, Pronase and Calcofluor had no effect on permeability. The results will be discussed in terms of the physiology of digestion and the ability of rendering the larval midgut epithelium accessible to viruses and other large particles. To clone Anopheles gambiae larval PM genes we prepared a [cardia-minus-midgut] subtraction library enriched for cardia sequences. One clone from this library - Ag-Lper1 - was further characterized. Anti-Ag-Lper1 antibodies react strongly with cytoplasmic vesicles of cells in the outer cell layer of the cardia. Northern blots detects a ~750 nucleotide-long RNA in cardia but not in gut or carcass RNA samples. The predicted 183 amino acid protein contains a signal sequence and a glutamine-rich (11%) amino acid sequence with similarity to involucrin, which is a major structural protein in the mammalian epidermis. Both the PM and the epidermis form barriers separating two compartments. A cDNA encoding a putative PM protein that is also rich in glutamine was isolated from an ADULT library. To isolate Aedes aegypti PM gene counterparts, we are currently immunoscreening an adult midgut cDNA expression library with an anti-PM serum.

Index terms: Anopheles gambiae, Aedes aegypti, permeability, cardia, caecal membrane, peritrophic matrix genes

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