Research ArticleMOLECULAR BIOLOGY

Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles

See allHide authors and affiliations

Science Advances  13 May 2020:
Vol. 6, no. 20, eaay5898
DOI: 10.1126/sciadv.aay5898
  • Fig. 1 Generation of transgenic mosquitoes (MultiEff) using an AgCp-driven transgene array with five anti-Plasmodium effectors targeting the malaria parasite in the midgut, and P. falciparum infection phenotypes at both oocyst and sporozoite stages.

    (A) Schematic illustration of transgenic targeting of parasite midgut infection stages and the design of the transgenes to target the parasites at this stage. (B) Five known anti-Plasmodium effectors (Melittin, TP10, EPIP, Shiva1, and Scorpine) were selected. The gene array with these five anti-Plasmodium effector genes was synthesized through GenScript Inc. and cloned under the AgCp promoter, followed by the trypsin terminator (TrypT) in the pBac[3xP3-EGFPafm] vector. These five antiparasitic effector genes were transcribed on one cassette separated by viral P2A sequences, translated into one polypeptide, and self-spliced into five individual peptides with an extra viral P2A amino acid tag on the first four peptides. (C) Fluorescent images of a positive larva and an adult transgenic mosquito. (D) Polymerase chain reaction (PCR) validation of the partial [500 base pairs (bp)] transgene cassette of MultiEff in the transgenic mosquitoes. (E) Transcript abundance of the transgene in the gut of MultiEff transgenic mosquitoes at various time points post-blood meal (PBM). Each bar represents the relative fold change in the transgene as compared to the control at time 0 hour. The S7 ribosomal gene was used to normalize the complementary DNA (cDNA) templates. Error bars indicate SEM. (F to H) P. falciparum (NF54) oocyst and sporozoite infection intensities and prevalence at 8 days post-infection (dpi) in the gut or 14 dpi in the salivary glands (SG) when fed on blood with a medium (0.05%) (F) or low (0.01%) (G and H) gametocytemia. At least three biological replicates were pooled for the dot plots. Each dot represents the number of parasites in an individual gut or a pair of salivary glands, with the median values indicated by red bars. P values were calculated by a Mann-Whitney test. Detailed statistical analysis is presented in table S2. (I) Midgut microbial flora of female transgenic MultiEff and WT control (AsWT) mosquitoes at 0-, 24-, 48-, and 72-hour PBM (mean ±SEM). (J and K) P. falciparum oocyst infection intensities and prevalence in the aseptic (antibiotic-treated) and septic (non–antibiotic-treated) transgenic and AsWT mosquitoes at 8 dpi. (L) Expression of AMP and anti-Plasmodium effector as fold change in expression through quantitative reverse transcription PCR (qRT-PCR). Error bars indicate SEM. CEC1, Cecropin 1; DEF1, Defensin 1; GAM1, Gambicin 1; FBN9, Fibrinogen-related protein 9.

  • Fig. 2 Generation of transgenic mosquitoes targeting the sporozoite-stage malaria parasite in the hemolymph with single-chain antibodies fused to antiparasitic effectors and P. falciparum infection phenotypes at both the oocyst and sporozoite stages.

    (A) Schematic illustration of transgenic targeting of parasite sporozoite infection stages and the design of AsVg-driven transgenes to be expressed in the fatbody after the blood meal, to specifically target the parasites at this stage. (B) Schematic representation of single-chain antibody (ScFv) targeting the CSP protein fused to AMP. The single-chain antibodies consist of variable regions of the VH heavy and VL light chains. Each transgene encodes a short 5–amino acid polypeptide linker between VH and VL and a long 15–amino acid (aa) sequence linking the VH to the AMP peptides (CecC, PLA2, and Scorpine), including the CecA signal peptide sequence (SP). Three individual transformation plasmids, pBAC-AsVg-CecC (or PLA2, Scorpine)–ScFv (with the red fluorescent eye reporter gene 3xP3 dsRed), were used for the germline transformation. AsVg promoter with the same AsVg endogenous terminator (AsVg 3′-UTR) was used. (C) Fluorescent images of a positive larva and an adult transgenic ScorpScFv mosquito. (D) PCR validation of the partial transgene cassette (~500 bp) of CecScFv, PLAScFv, and ScorpScFv in the transgenic mosquitoes. (E) Transcript abundance of the transgene in the fatbody of ScorpScFv transgenic line at various time points PBM. Each bar represents the relative fold change in the transgene as compared to the control at time 0 hour. The S7 ribosomal gene was used to normalize the cDNA templates. Error bars indicate SEM. (F to I) P. falciparum (NF54) oocyst and sporozoite infection intensities and prevalence of the three transgenic ScFv lines (PLAScFv, CecScFv, and ScorpScFv) at 8 dpi in the gut or 14 dpi in the salivary glands (SG) without (F and G) or with (H and I) additional naïve blood meals at days 5 and 9 post-infectious blood meal (PIBM). Each dot represents the number of parasites in an individual midgut or salivary glands, and the horizontal lines (red) indicate the median values. Detailed statistical analysis is presented in table S2.

  • Fig. 3 The hybrid lines ScorpScFv with CpRel2 or with MultiEff targeting the malaria parasite at multiple infection stages result in near-complete refractoriness.

    (A and B) P. falciparum (NF54) oocyst and sporozoite infection intensities and prevalence of two transgenic lines (ScorpScFv and CpRel2) and the hybrid line of the two transgene cassettes (Rel2ScFv) at 8 dpi in the gut (A) or 14 dpi in the salivary glands (SG) (B) with additional blood meals at days 5 and 9 PIBM. (C and D) P. falciparum (NF54) oocyst and sporozoite infection intensities of two transgenic lines (ScorpScFv and MultiEff) and the MultiEffScFv hybrid transgenic mosquitoes at 8 dpi in the gut (C) or 14 dpi in the salivary glands (SG) (D), with additional blood meals on days 5 and 9 PIBM. Assays were performed with at least four biological replicates, and the horizontal lines (red) indicate the median values. Mann-Whitney test was used to calculate P values and determine the significance of parasite numbers. A χ2 test was used to compare infection prevalence values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed statistical analysis is presented in table S3. (E) Schematic illustration of transgenic targeting of parasite midgut and sporozoite infection stages using carboxypeptidase and vitellogenin promoter-driven transgenes.

  • Fig. 4 Fitness effects of three transgenic lines with anti-Plasmodium activity.

    (A and B) Life spans of the female mosquitoes of the three transgenic A. stephensi lines maintained on 10% sucrose solution (A) or after one blood meal on mice (B) with WT control (AsWT). The life spans of ScorpScFv and MultiEff mosquitoes were significantly shorter than those of the controls when the mosquitoes were fed once on the naïve mice (P < 0.01 and P < 0.001, respectively). The pooled values from three replicates are shown, with SE. Survival rates were analyzed by Kaplan-Meier survival analysis with Wilcoxon test to determine the significance. (C and D) The wing lengths of the three anti-Plasmodium transgenic mosquito (CecScFv, ScorpScFv, and MultiEff) females or males did not differ from those of the control mosquitoes (AsWT). (E) Numbers of eggs laid by female homozygous transgenic ScorpScFv and MultiEff were significantly lower than those of the control AsWT mosquitoes. Each dot represents the eggs laid by an individual female after a single blood meal on mice. The median values (black horizontal bars) are shown. The P values were calculated with a Mann-Whitney test. (F) Hatch rates indicate the average percentage of eggs giving rise to first- and second-instar larvae, as shown with the bars indicating the mean values and SE. Each dot represents the hatch rate of the eggs laid by an individual female after blood meal. (G) Life spans of male mosquitoes of the three transgenic lines and AsWT controls maintained on 10% sucrose solution. (H and I) Comparison of life spans of the female hybrid transgenic line mosquitoes (MultiEffScFv) to the parental single transgenic lines (MultiEff and ScorpScFv) and the AsWT controls maintained on 10% sucrose solution (H) or after one naïve blood meal on mice (I).

  • Table 1 Selected AMPs and anti-Plasmodium peptides for transgenic expression in the genetically modified mosquitoes.

    EffectorsCharacterizationParasite stage(s)Function or
    mechanism
    Expression
    method
    Inhibition (%)Reference
    MelittinBee antimicrobial
    peptides
    OokineteLysis of parasitesSynthesized100.0%(27)
    TP10 dimerWasp antimicrobial
    peptides
    OokineteLysis of parasitesSynthesized100.0%(2527)
    Shiva1Cecropin-like
    synthetic peptide
    Gametes OokineteLysis of parasitesPara-transgenesis94.3%(27)
    EPIP four repeatsEnolase-
    plasminogen
    interaction peptide
    OokineteBlock midgut
    invasion
    Para-transgenesis97.7%(20)
    ScorpineScorpion venomGametogenesis
    Ookinete
    Cecropin and
    defensin-like lytic
    peptide
    Para-transgenesis97.8%(23)

Supplementary Materials

  • Supplementary Materials

    Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles

    Yuemei Dong, Maria L. Simões, George Dimopoulos

    Download Supplement

    This PDF file includes:

    • Fig. S1
    • Tables S1 to S3
    • Supporting text

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article