Engineered anopheles immunity to Plasmodium infection

Abstract

A causative agent of human malaria, Plasmodium falciparum, is transmitted by Anopheles mosquitoes. The malaria parasite is under intensive attack from the mosquito's innate immune system during its sporogonic development. We have used genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes through blood meal-inducible expression of a transgene encoding the IMD pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and microbial infection as a result of timely concerted tissue-specific immune attacks involving multiple effectors. The relatively weak impact of this genetic modification on mosquito fitness under laboratory conditions encourages further investigation of this approach for malaria control.

Figure 1. Resistance of genetically engineered Rel2-expressing immune-enhanced

A. stephensi to P. falciparum . (A) Fluorescent images of larvae and adults of the vitellogenin- (Vg, red) and carboxypeptidase- (Cp, green) driven Rel2 transgenic lines and hybrid (Hyb, merged into yellow) transgenic line, along with the wt control (WT, non-fluorescent) strain. (B) qRT-PCR analysis of -fold changes in the expression of the Rel2 transgene and effector genes at 0 to 48 h post-blood-meal (PBM), with standard errors shown. Dots represent fold change in expression of the genes of interest (GOI) of the transgenic mosquito tissues compared to that of wt mosquito tissues at the corresponding time PBM. The ribosomal protein S7 gene was used for normalization of the cDNA templates. (C) Green fluorescent P. falciparum (3D7(GFP)) oocysts in the midguts of WT control, Cp (Cp11), Vg (Vg1), and Hyb mosquitoes at 8 days post-infection (dpi). (D, E) P. falciparum (NF54) oocyst loads (including zeros) of WT control, homozygous Cp11 and Vg1 and heterozygotes hybrid transgenic mosquitoes at 8 dpi when fed on blood with a standard 0.3% (D) and 0.05% (E) gametocytemia. (F) P. falciparum (NF54) ookinete loads in the midgut lumen at 24 h post-infection (hpi). (G) P. falciparum (NF54) sporozoite loads in the salivary glands at 14 dpi. Assays were performed with at least three biological replicates, and the numbers of parasites from equal numbers of samples (midguts, midgut lumen, or salivary glands) from different replicates were pooled for the dot-plot. Each dot represents the number of oocysts, ookinetes, or sporozoites in an individual midgut, midgut lumen, or salivary gland respectively, and the horizontal lines (red) indicate the median values. Mann-Whitney test determined p-values (shown here) and Kruskal-Wallis test was used to calculate p-values and determine the significance of oocysts, ookinetes, or sporozoites numbers. Detailed statistical information of infection assays with N, range, prevalence, p-values of prevalence determined by Chi-square test, median (with or without zeros), p-values of infection intensity (with or without zeros) calculated through Kruskal-Wallis and Mann-Whitney tests, and % decrease of oocysts, ookinetes, or sporozoites loads are presented in Table S3.

Figure 2. Implication of anti-Plasmodium effector genes in refractoriness of immune-enhanced transgenic mosquitoes.

Depletion of TEP1, APL1, LRRD7, and PGRP-LC through RNAi-mediated gene silencing resulted in changes in the P. falciparum oocyst intensity at 8 dpi in the non-transgenic wild type (WT), Cp, Vg, and hybrid transgenic mosquitoes. GFP dsRNA-injected mosquitoes served as control. Points indicate the absolute value of oocyst counts in individual mosquitoes, and horizontal red bars in each column represent the median value of oocysts from three replicates. P-values were calculated by Mann-Whitney test. (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001). Detailed statistical information of infection assays with n, range, prevalence, median, p-values, and % increased oocysts load are presented in the table beneath the figure as well as in Table S3.

Figure 3. Antibacterial resistance of immune-enhanced transgenic mosquitoes.

(A) The midgut microbial flora (total bacterial load) of female transgenic and wt control (WT) mosquitoes at 24, 48, and 72 h PBM (mean±SEM). A Student's t-test was used to calculate p-values and determine significance. (B) Survival rates of transgenic mosquitoes upon challenge with either Gram-negative (E. coli: 350,000 CFU) or Gram-positive (S. aureus: 210,000 CFU) bacteria at 4 dpi. Kaplan-Meier survival analysis with log-rank test was used to determine the p-values, and p<0.05 indicates significance (detailed Kaplan-Meier survival curves with three biological replicas are presented in Figure S5). (C) P. falciparum oocyst infection intensities in the septic (non-antibiotic treated) and aseptic (antibiotic-treated) transgenic and WT mosquitoes at 8 dpi. Antibiotic-treated wt mosquitoes became more susceptible to P. falciparum infection than did non-antibiotic-treated wt mosquitoes, while oocyst intensities in transgenic hybrid mosquitoes showed no change. At least three biological replicates are included, with each dot representing the number of oocysts in an individual midgut, and the horizontal lines (red) indicating the median values. P-values were calculated by a Mann-Whitney test.

Figure 4. Fitness assessment of immune-enhanced transgenic mosquitoes.

(A–D) Life spans of transgenic and wt mosquitoes maintained on 10% sucrose solution (A); or mosquitoes fed on a single naïve human blood 5 d post emergence and then maintained on sugar solution (B); or mosquitoes that were provided 3 naïve blood meals (days 0, 4, 8) and then maintained on sugar solution (C); or mosquitoes that were provided a Pf-infected blood meal (0.3% gametocytemia) and then maintained on sugar solution (D). The mean values from three replicates are shown, with the standard error bars. Survival rates were analyzed by Kaplan-Meier survival analysis with Wilcoxon test to determine the significance, and detailed statistical information is shown in Table S4. (E) Eggs laid by female transgenic and wt mosquitoes from three biological replicates: Each dot represents the eggs laid by an individual female after a single blood meal. The median values (red horizontal bars) are shown. P-values were calculated with a Mann-Whitney test. (F) -Fold change in the expression of the A. stephensi endogenous vitellogenin 1 (AsVg1) gene, at 6, 12, 24, and 48 h PBM (mean±SEM) compared to that of the non-fed mosquitoes. The cDNA templates were normalized using the AsS7 gene. (G) Hatch rates indicate the average percentage of eggs giving rise to 1st instar larvae, as determined by three biological replicates. Mean values for hatch rates and standard errors (SE) of replicates are indicated.