Activation of Akt signaling reduces the prevalence and intensity of malaria parasite infection and lifespan in Anopheles stephensi mosquitoes

Abstract

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60-99%. Of those mosquitoes that were infected, we observed a 75-99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18-20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.

Figure 1. Generation of the CP-myr-AsteAkt-HA transgenic mosquito line and protein and transcript expression profile of the transgene in adult females.

A. Schematic of the construct genetically engineered into A. stephensi mosquitoes. See text for a description of the construct. B. Comparison of transgenic (TG) and non-transgenic (NTG) siblings. Top panel: non-transgenic (left) and transgenic fourth instar larvae (right) under white light. Middle panel: non-transgenic and transgenic mosquitoes under fluorescence and a DsRed filter. Bottom panel: merge of top and middle panels. C. Two transgene-specific primer sets were used to amplify the transgene from the genomic DNA of transgenic and non-transgenic siblings. Primers to AsteActin were used to verify the integrity of the DNA. D. Total RNA was isolated from the midguts or carcasses (i.e., entire body minus midgut) of both transgenic (TG) and non-transgenic (NTG) mosquitoes and converted into cDNA. Transgene specific primers were used to amplify myr-AsteAkt. Primers to AsteActin were used to verify the integrity of the cDNA. E. Total protein was isolated from the midguts or carcasses of transgenic and non-transgenic mosquitoes, separated electrophoretically on a 12% SDS-PAGE gel. Proteins were blotted and then probed with anti-HA antibody or anti-GAPDH antibody to assess protein loading.

Figure 2. Expression profile of the transgene during a reproductive cycle.

A. Total RNA was isolated from the midguts and carcasses of ten non-bloodfed (NBF) and ten bloodfed transgenic mosquitoes at 2 h, 24 h, 48 h and 72 h post-bloodfeeding and converted into cDNA. The 72 h sample was collected post-oviposition. Transgene specific qRT-PCR primers were used to amplify myr-AsteAkt from the cDNA; amplification of ribosomal protein S7 was used for normalization. The qRT-PCR experiments were performed in triplicate and replicated twice with separate cohorts of mosquitoes. Data are represented as means ± SEMs. B. Total protein was isolated from the midguts of transgenic mosquitoes at various time points during a reproductive cycle. Proteins were blotted and probed with anti-HA antibody or anti-GAPDH antibody to assess protein loading. C. Average expression of transgenic protein normalized to GAPDH loading controls and shown relative to levels in non-bloodfed mosquitoes (NBF). Data are represented as means ± SEMs from four replicates with separate cohorts of mosquitoes.

Figure 3. Membrane localization of the transgene.

A. Immunocytochemistry of myr-AsteAkt-HA transgenic or non-transgenic paraffin embedded midgut sections using an anti-HA-fluorescein antibody. Images were acquired under brightfield illumination to visualize the midgut epithelial architecture or a FITC filter to visualize the overexpressed protein. To determine localization the images were merged. Arrows indicate the cell membrane of the midgut epithelial cells. Five midguts from transgenic and non-transgenic mosquitoes were analyzed. B. Immunocytochemistry of myr-AsteAkt-HA transgenic or non-transgenic midgut sheets using an anti-HA-fluorescein antibody. At least 10 midgut sheets from three separate cohorts of mosquitoes were analyzed by confocal microscopy. Three representative myr-AsteAkt-HA transgenic and non-transgenic midguts are shown. C. Membrane (M), cytoplasmic (C), and nuclear (N) fractions of transgenic mosquito midguts were prepared and these fractions, and an intact midgut sample (MG), were probed with anti-HA antibody. This immunoblot is representative of three experiments.

Figure 4. Increased FOXO phosphorylation due to dietary insulin and myr-AsteAkt-HA.

A. Total protein from the midguts of non-transgenic (NTG) mosquitoes fed a bloodmeal either containing buffer or 1.7×10⁻⁴ µM human insulin were immunoblotted and probed with anti-phospho-FOXO1 (p-FOXO) antibody and anti-GAPDH to assess protein loading. B. A representative immunoblot of increased FOXO phosphorylation in myr-AsteAkt-HA transgenic mosquitoes compared to non-transgenic controls. Total protein from the midguts of transgenic (TG) and non-transgenic (NTG) mosquitoes maintained under identical conditions and fed only sucrose. The proteins were immunoblotted and probed with anti-phospho-FOXO1 (p-FOXO) antibody. The blot was then stripped and re-probed with and anti-GAPDH antibody to assess protein loading. This experiment was replicated three times.

Figure 5. Resistance of heterozygous transgenic and non-transgenic mosquitoes to P. falciparum infection.

Heterozygous transgenic (TG) and non-transgenic (NTG) sibling mosquitoes were provided with an artificial bloodmeal containing P. falciparum NF54 gametocytes. Ten days after infection, the midguts were dissected and the number of P. falciparum oocysts counted. The experiment was replicated three times with separate cohorts of mosquitoes. A. Infection prevalence was defined as the percentage of mosquitoes that had at least one oocyst on the midgut. Parasite prevalence was significantly lower in transgenic mosquitoes compared to the nontransgenic control (** indicates p<0.0001, * indicates p = 0.0008). B. Summary statistics of the parasite data from all three experiments including zeros or excluding zeros (mosquitoes without oocysts). Analyses were performed for each replicate separately and for all three replicates combined. When zeros were omitted, the small number of transgenic mosquitoes infected with at least one parasite (n = 1–14) obviated statistical analysis.

Figure 6. Resistance of homozygous transgenic and non-transgenic mosquitoes to P. falciparum infection.

Homozygous transgenic (TG) mosquitoes and non-transgenic (NTG) mosquitoes were provided with an artificial bloodmeal containing P. falciparum NF54 gametocytes. Ten days after infection, the midguts were dissected and the numbers of P. falciparum oocysts were counted. A. Infection prevalence was defined as the percentage of mosquitoes that had at least one oocyst on the midgut. Homozygous transgenic mosquitoes were significantly less prone to infection (** indicates p<0.0001). B. Summary statistics of parasite data from all three experiments. Analyses were performed for each replicate separately and for the three replicates combined.

Figure 7. Lifespan experiments of sugarfed or bloodfed transgenic and non-transgenic mosquitoes.

A. A representative survivorship curve comparing transgenic (TG) and non-transgenic (NTG) siblings reared under identical conditions and provided with only a 10% sucrose solution. Lifespan experiments were replicated three times with separate cohorts of mosquitoes. B. A representative survivorship curve comparing transgenic (TG) and non-transgenic (NTG) siblings reared under identical conditions and provided with weekly bloodmeals and a 10% sucrose solution. Lifespan experiments were replicated three times with separate cohorts of mosquitoes. C. Summary of the samples sizes, medians, means, and statistical significance for sugarfed and bloodfed mosquitoes.