Electrophoretic separation and identification of phenoloxidases in hemolymph and midgut of adult Anopheles stephensi mosquitoes.

Anopheles mosquitoes frequently respond to invading malaria parasites with a rejection mechanism consisting of phenoloxidase-mediated melanization of ookinetes in the mosquito midgut epithelium. The relative roles of hemolymph vs. midgut phenoloxidase in this rejection mechanism is unclear. We have separated and identified phenoloxidase isozymes from midgut and hemolymph of Anopheles stephensi by native gel electrophoresis followed by zymography. The isozymes from the 2 sites had distinctively different electrophoretic characteristics. Hemolymph possessed 2 phenoloxidase-positive bands, both of which were bifunctional molecules that oxidized monophenol as well as o-diphenol substrates. Midgut extract possessed 3 bands that migrated more rapidly than those of the hemolymph. None of these midgut bands had detectable monophenoloxidase activity; they possessed, however, a broad spectrum of diphenoloxidase activity in their ability to oxidize both o- and p-diphenol substrates, as well as the laccase substrate syringaldazine. The 2 most rapidly migrating midgut PO bands could be distinguished from the more slowly migrating band through their insensitivity to inhibition by the chelating agent tropolone. A question to be resolved in the future relates to the relative roles of hemolymph vs. midgut phenoloxidase in mosquito defense against invasive parasites.

Inhibition of mosquito salivary gland apyrase activity by antibodies produced in mice immunized by bites of Anopheles stephensi mosquitoes.

Mice (BALB/c) were immunized to mosquito saliva by repeated bites of Anopheles stephensi mosquitoes. Studies were conducted on the ability of these mice to develop antibodies against the apyrase component of the saliva. By means of immunoprecipitation procedures and Western blot analysis, we demonstrated the presence of antiapyrase antibodies to the mosquito saliva. Furthermore, these antibodies were able to inhibit apyrase activity. Serum titers of 1:20 were able to inhibit approximately 90% of salivary gland apyrase activity, while titers of 1:160 retained the ability to inhibit more than 50% of apyrase activity. Parallel inhibition assays with immunoglobulin G (IgG) from immunized versus nonimmunized mice showed that the inhibitory activity of serum from immunized mice could be accounted for by its IgG component. Mosquito salivary gland apyrase has previously been shown to facilitate mosquito feeding by inhibiting aggregation of platelets at the mosquito bite site. However, our studies have shown that mosquitoes feeding on immunized mice had no deficiency in probing these mice for a blood meal, even in the face of high titers of anti-apyrase antibodies.

Plasmodium sporozoites possess both positively and negatively charged sites accessible on their surface.

Studies were conducted on the distribution of anionic and cationic sites on the surface of intact Plasmodium berghei and P. yoelii sporozoites. Anionic and cationic ferritins were used as probes for electron microscope studies and fluorescein conjugates of the same charged ferritins were used for correlative studies by fluorescence light microscopy. We found that the surfaces of mature sporozoites from mosquito salivary glands possess both negatively and positively charged sites that are accessible to charged ferritin molecules. Sporozoites obtained from oocysts expressed negatively charged sites but were inconsistent in their expression of positively charged sites. The charged nature of the ferritin probes was confirmed by their binding to cellulose particles or cellular components with known negative or positive charges. The electrostatic nature of the binding of ferritin to sporozoites was established by showing that binding could be blocked by competition with high concentrations of NaCl. The most likely source of these surface charges is circumsporozoite (CS) protein, the major protein covering the surface of sporozoites. Circumsporozoite proteins contain a cluster of positively charged amino acids, which may play a role as components of a surface ligand capable of binding to a liver receptor.