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. 2005 Sep 20;102(38):13568-73.
doi: 10.1073/pnas.0502815102. Epub 2005 Sep 12.

Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti

Guowu Bian  1 , Sang Woon Shin, Hyang-Mi Cheon, Vladimir Kokoza, Alexander S Raikhel
Affiliations

Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti

Guowu Bian et al. Proc Natl Acad Sci U S A. .

Abstract

Reverse genetics is a powerful tool for understanding gene functions and their interactions in the mosquito innate immunity. We took the transgenic approach, in combination with the RNA interference (RNAi) technique, to elucidate the role of mosquito REL1, a homolog of Drosophila Dorsal, in regulation of Toll immune pathway in the mosquito Aedes aegypti. By transforming the mosquitoes with DeltaREL1-A or a double-stranded RNA construct of REL1 driven by the female fat body-specific vitellogenin (Vg) promoter with the pBac[3xP3-EGFP, afm] vector, we generated two different transgenic mosquito strains, one with overexpressed AaREL1 and the second with AaREL1 knockdown. Both strains had a single copy of the respective transgene, and the expression in both transgenic mosquitoes was highly activated by blood feeding. Vg-DeltaREL1-A transgenic mosquitoes activate Toll immune pathway in the fat body by blood feeding. The overexpression of both isoforms, AaREL1-A and AaREL1-B, in Vg-DeltaREL1-A transgenic mosquitoes resulted in the concomitant activation of Aedes Spätzle1A and Serpin-27A, independent of septic injury. The same phenotype was observed in the mosquitoes with RNAi knockdown of an Aedes homolog to Drosophila cactus, an IkappaB inhibitor of Drosophila Toll pathway. The effect of the transgenic RNAi knockdown of AaREL1 on mosquito innate immunity was revealed by increased susceptibility to the entomopathogenic fungus Beauveria bassiana and the reduced induction of Spz1A and Serpin-27A gene expression after fungal challenge. These results have proven that AaREL1 is a key downstream regulator of Toll immune pathway in the mosquito A. aegypti.

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Figures

Fig. 1.
Fig. 1.
Construction of two different transgenic mosquitoes with altered REL1 activity. (A and B) Schematic diagrams of the pBac[3xp3-EGFP, afm, Vg-ΔREL1-A] and the pBac[3xp3-EGFP, afm, Vg-iREL1] transformation vector that were respectively transformed into the A. aegypti germ line. (C and D) Southern blot analyses of genomic DNA extracted from the Vg-ΔREL1-A transgenic mosquitoes, the Vg-iREL1 transgenic mosquitoes, and the parental UGAL strain, digested with BamHI (BHI), EcoRV (EV), SalI (SI), BglII (BII), ApaI (AI), and PstI (PI). The additional bands, indicated as arrows, were detected in the transgenic mosquitoes. The probe region and the restriction sites of the transformation vector are indicated in A and B.
Fig. 2.
Fig. 2.
The expression profiles of Vg-ΔREL1-A and Vg-iREL1 transgene and transgenic effects on the endogenous REL1 gene expression. (A) Northern blot analysis showing the Vg promoter-mediated activation of the transgenes after blood feeding. Endogenous REL1 transcripts, REL1-A and REL1-B, were detected in the parental mosquitoes at any stage. The additional bands, which appeared after blood feeding, were detected in the Vg-ΔREL1-A and Vg-iREL1 transgenic mosquitoes, respectively. (B and C) Real-time PCR analysis showing the relative level of endogenous REL1 transcripts in transgenic and parental mosquitoes. After blood feeding, the Vg-ΔREL1-A transgenic mosquitoes showed the overexpression of REL1 transcripts, whereas the Vg-iREL1 transgenic mosquitoes showed the knockdown of both REL1 transcripts, REL1-A (B) and REL1-B (C). PV, previtellogenic.
Fig. 3.
Fig. 3.
The effects of the transgenic Vg-ΔREL1-A and Cactus dsRNA treatments on the expression of Aedes Spz1A and Serpin-27A. (A) Northern blot analysis showing the activation of Aedes Spz1A and Serpin-27A after blood feeding in Vg-ΔREL1-A transgenic mosquitoes. (B) The expression profiles of the Aedes Spz1A and Serpin-27A 5 h and 24 h after septic injury in the Vg-ΔREL1-A transgenic mosquitoes and the parental UGAL strain 24 h PBM. (C) The increased expression of the Aedes Spz1A and Serpin-27A genes in Cactus dsRNA-treated mosquitoes (iCactus). GFP dsRNA-treated mosquitoes (iGFP) were used as a negative control. PBM (h), hours after blood meal; ASI (h), hours after septic injury.
Fig. 4.
Fig. 4.
The increased susceptibility to B. bassiana (A) and the reduced immune activation of Spz1A and Serpin-27A genes in the Vg-iREL1 transgenic mosquitoes (B). (A) The Vg-ΔREL1-A and Vg-iREL1 transgenic and wild-type UGAL mosquitoes were blood fed at 2-3 days after emerging, forced to lay eggs at 3 days PBM, and blood fed again the next day. At 24 h PBM, B. bassiana spores were challenged. The result is representative of three independent experiments. (B) The Vg-iREL1 transgenic and wild-type UGAL mosquitoes were forced to lay eggs at 3 days after the second blood feeding and then challenged with B. bassiana spores the next day. The immune activation of Spz1A and Serpin-27A were impaired in fat bodies of the Vg-iREL1 transgenic mosquitoes. ASI (h), hours after septic injury.
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