Progress in molecular genetics makes possible the development of alternative disease control strategies that target the competence of mosquitoes to transmit pathogens. We tested the regulatory region of the vitellogenin (Vg) gene of Aedes aegypti for its ability to express potential antipathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg-promoter fragment driving the expression of the Defensin A (DefA) coding region, one of the major insect immune factors. PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation, showed that the Vg-DefA transgene insertion was stable. The Vg-DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24-h postblood meal, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin were accumulated in the hemolymph of bloodfed female mosquitoes, persisting for 20 -22 days after a single blood feeding. Purified transgenic defensin showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes. Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood meal-triggered cascade rather than by infection. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in pathogen transmission.
The Rel͞NF-B transcription factor Relish performs a central role in the acute-phase response to microbial challenge by activating immune antibacterial peptides. We cloned and molecularly characterized the gene homologous to Drosophila Relish from the mosquito Aedes aegypti. Unlike Drosophila Relish, Aedes Relish has three alternatively spliced transcripts encoding different proteins. First, the predominant Aedes Relish transcript of 3.9 kb contains both the Rel-homology domains and the inhibitor B (I B)-like domain, which is similar to Drosophila Relish and to the mammalian p105 and p100 Rel͞NF-B transcription factors. Second, Aedes Relish transcript contains Rel-homology domains identical to those of the major transcript but it completely lacks the I B-like domain-coding region, which has been replaced by a unique 3 -untranslated region sequence. In the third transcript, a deletion replaces most of the N-terminal sequence and Relhomology domains; however, the I B-like domain is intact. All three Aedes Relish transcripts were induced by bacterial injection but not by blood feeding. In vitro-translated protein from the Rel-only construct specifically binds to the B motif from Drosophila cecropin A1 and Aedes defensin genes. PCR and Southern blot hybridization analyses show that these three transcripts originated from the same large inducible mRNA encoded by a single Relish gene.
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