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Depletion of AGAP Bioavailability of complement-like protein TEP1 in both hemocoelFIGURE 4. Depletion of AGAP001476 specifically increases Plasmodium survival, which is accompanied by a decline in TEP1 and an increase in vitellogenin level. Each mosquito was injected with 0.4 g of dsluc, dsAGAP001476, or dsAGAP006241. The oocyst number and the percentage of infected mosquitoes are shown in A. Mosquitoes receiving dsAGAP001476 had significantly more oocysts in their midguts than those receiving dsluc or dsAGAP006241. The expression levels of selected genes involved in innate immune responses and the two innexin genes of interest were assessed by qPCR and https://britishrestaurantawards.org/members/pansy07africa/activity/282734/ normalized against actin levels in B. Apart from a reduction in AGAP001476, mosquitoes receiving dsAGAP001476 had less TEP1 at 1 and 2 dpi and more vitellogenin at 2 dpi. Values in A represent the median from a representative experiment that was repeated three times. Values in B represent means S.E. of the mean compiled from four independent experiments. *, p 0.05; **, p 0.01; ***, p 0.001 by Kruskal-Wallis test and Dunn's multiple comparisons test in A, and two-way ANOVA and Bonferroni's multiple comparisons test in B.SEPTEMBER 5, 2014 ?VOLUME 289 ?NUMBERJOURNAL OF BIOLOGICAL CHEMISTRYAnopheles Innexins and Malaria PathogenesisAGAP140 120 100 80 60 40 20 0150dsluc dsAGAPAGAP250 200 150 100 50 0dsluc dsAGAP*0 1150TEPAPL1C200LRIM150 100 100 100 50dsluc dsAGAP50dsluc dsAGAP50dsluc dsAGAP0 0 800 700 600 500 400 300 200 100 0 0 1 2 10 0 250 200 150 10 0 1Vitellogenindsluc dsAGAPLipophorin200Cactus150100100 50 0 0 1dsluc dsAGAP50dsluc dsAGAP0 0 200 1Caspar250 200 150 100 100 50 0 0 1 2 50dsluc dsAGAPRelish200dsluc dsAGAPRelishdsluc dsAGAP150150100500 0 10 0 1Time post-infection (dpi)FIGURE 5. Expression levels of innexins and selected genes involved in innate immunity in mosquitoes receiving dsluc or dsAGAP006241. Whole mosquitoes that received dsRNA injection were collected at 0, 1, and 2 dpi for mRNA analysis. The expression levels of selected genes involved in innate immune responses and the two innexin genes of interest were assessed by qPCR and normalized against actin level. Only AGAP006241 level showed a significant reduction in dsAGAP006241-treated mosquitoes, whereas other selected immune markers remained unchanged. Values represent means S.E. of the mean compiled from at least four independent experiments. *, p 0.05 by two-way ANOVA and Bonferroni's multiple comparisons test.24892 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 289 ?NUMBER 36 ?SEPTEMBER 5,Anopheles Innexins and Malaria PathogenesisTime (hpi) Silver StainRelative vitellogenin levelAdsAGAP001476 dsluc 18 24 48 0 18 24 48 VitellogeninTEP1-Fdsluc dsAGAP*1.IBTEP1cut PPO0.0 0 dsluc dsAGAP001476 18Relative TEP1cut levelRelative TEP1-F level14 12 10 8 6 4 2dsluc dsAGAP6 5 4 3 2 1* *0 18 24B1 DPITEP1 TOPRO3 dsluc dsdsAGAPCIP: APL1CBound140 120 100nsTEP1cut LRIM1/APL1CIgG ctrl80 60 40 20*dslucTEP1cutBound0 dsluc dsAGAPLRIM1/APL1C TEP1-FSupernatantSupernatant120 100 80 60 TEP1-FnsTEP1cut** **LRIM1/APL1CTEP1cut LRIM1/APL1C PPO40 20 0 dsluc dsAGAPFIGURE 6. The TEP1-LRIM1-APL1C complex was reduced in dsAGAP001476 mosquitoes at 1 dpi. A, Western blot (IB) analysis and silver staining of hemolymph collected from mosquitoes treated with dsluc or dsAGAP001476 at 0, 18, 24, and 48 hpi. More TEP1 (both TEP1-F and TEP1cut) was detected in the hemolymph during Plasmodium infection, but TEP1cut was induced to a lesse.