Abstract
The saliva of hematophagous arthropods is enriched with a complex mixture of antihemostatic molecules, the biological functions of which are largely unknown. Anopheline antiplatelet protein (AAPP) from malaria vector mosquito exhibits strong antiplatelet activity when bound directly to host collagen by its C-terminus and through its N-terminus with Ca2+-binding activity. To investigate the biological functions of AAPP in blood feeding behavior and malaria transmission, we generated transgenic Anopheles stephensi mosquito lines expressing anti-AAPP antibody single-chain fragment (scFv) in their salivary glands. The AAPP-specific collagen-binding activity was completely abolished by AAPP-scFv complex formation in the saliva. Probing and prediuresis time, feeding success, blood meal size, and fecundity, which are all fitness characteristics, were significantly reduced in the transgenic mosquitoes. However, oocysts number in these mosquitoes were not significantly reduced following blood meal intake from Plasmodium berghei-infected mice. These results show that although AAPP plays an important role in mosquito blood feeding, its neutralizing activity did not affect sporogonic development in our laboratory model, but its high fitness cost would pose a survival risk for parasite-infected mosquitoes in nature.
Highlights
In mosquitoes, transgenesis-based gene silencing and saliva protein inactivation are potentially useful methods for analyzing the effects of saliva proteins on mosquito physiology
We have established a female salivary gland-specific transgene expression system in transgenic Anopheles stephensi mosquitoes using the promoter region of the anopheline antiplatelet protein (AAPP) gene, which encodes an inhibitor of collagen-induced platelet aggregation in the salivary glands[9,10,11]
The 45Ca2+ overlay assay directly evidences the Ca2+ binding property of rAAPPex[1,2,3,4], which is www.nature.com/scientificreports estimated to be eight times lower than that of calmodulin when equimolar amounts were tested (Fig. 1D)
Summary
Transgenesis-based gene silencing and saliva protein inactivation are potentially useful methods for analyzing the effects of saliva proteins on mosquito physiology. We have established a female salivary gland-specific transgene expression system in transgenic Anopheles stephensi mosquitoes using the promoter region of the anopheline antiplatelet protein (AAPP) gene, which encodes an inhibitor of collagen-induced platelet aggregation in the salivary glands[9,10,11]. Protein of the Phlebotomus papatasi sand fly[12], the repeat region of the Plasmodium berghei circumsporozoite protein (CSP)[13,14] and the anti-P. falciparum circumsporozoite protein (PfCSP) single-chain Ab (scFv)[15] have been functionally expressed in the salivary glands as a component of saliva This system could be used for gene silencing or protein inactivation for studying the effects of saliva proteins on the ecological attributes of mosquitoes. These results indicate that AAPP plays an important role in facilitating blood feeding, but impairment of blood feeding behavior did not affect the malaria vectorial capacity (sporogonic development) in our laboratory model
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