Plasmodium gallinaceum ookinetes adhere specifically to the midgut epithelium of Aedes aegypti by interaction with a carbohydrate ligand

During the course of its development in the mosquito and transmission to a new vertebrate host, the malaria parasite must interact with the mosquito m idgut and invade the gut epithelium. To investigate how the parasite recogn izes the midgut before invasion, we have developed an in vitro adhesion ass ay based on combining fluorescently labelled ookinetes with isolated midgut epithelia from blood-fed mosquitoes, Using this assay, we found that Plasm odium gallinaceum ookinetes readily adhered to midguts of Aedes aegypti, mi micking the natural recognition of the epithelium by the parasite, This int eraction is specific: the ookinetes preferentially adhered to the lumen (mi crovillar) side of the gut epithelium and did not bind to other mosquito ti ssues, Conversely, the binding was not due to a non-specific adhesive prope rty of the midguts, because a variety of other cell types, including untran sformed P. gallinaceum zygotes or macrogametes, did not show similar bindin g to the midguts, High concentrations of glycosylated (fetuin, orosomucoid, ovalbumin) or non-glycosylated (bovine serum albumin) proteins, added as n on-specific competitors, failed to compete with the ookinetes in binding as says. We also found that the adhesion of ookinetes to the midgut surface is necessary for sporogonic development of the parasite in the mosquito. Anti bodies and other reagents that blocked adhesion in vitro also reduced oocys t formation when these reagents were combined with mature ookinetes and fed to mosquitoes. Chemical modification of the midguts with sodium periodate at pH5.5 destroyed adhesion, indicating that the ookinete binds to a carboh ydrate ligand on the surface of the midgut, The ligand is sensitive to peri odate concentrations of less than 1 mmol l(-1), suggesting that it may cont ain sialic-acid-like sugars. Furthermore, free N-acetylneuraminic acid comp eted with the ookinetes in binding assays, while other monosaccharides had no effect. However, in agreement with the current belief that adult insects do not contain sialic acids, we were unable to detect any sialic acids in mosquito midguts using the most sensitive HPLC-based fluorometric assay cur rently available, We postulate that a specific carbohydrate group is used b y the ookinete to recognize the midgut epithelium and to attach to its surf ace. This is the first receptor-ligand interaction demonstrated for the ook inete stage of a malaria parasite. Further characterization of the midgut l igand and its parasite counterpart may lead to novel strategies of blocking oocyst development in the mosquito.