Transient knockdown of Anopheles stephensi LRIM1 using RNAi increases Plasmodium falciparum sporozoite salivary gland infections

Abstract

Background: Plasmodium falciparum (Pf) sporozoites (PfSPZ) can be administered as a highly protective vaccine conferring the highest protection seen to date. Sanaria® PfSPZ vaccines are produced using aseptically reared Anopheles stephensi mosquitoes. The bionomics of sporogonic development of P. falciparum in A. stephensi to fully mature salivary gland PfSPZ is thought to be modulated by several components of the mosquito innate immune system. In order to increase salivary gland PfSPZ infections in A. stephensi and thereby increase vaccine production efficiency, a gene knock down approach was used to investigate the activity of the immune deficiency (IMD) signaling pathway downstream effector leucine-rich repeat immune molecule 1 (LRIM1), an antagonist to Plasmodium development.

Methods: Expression of LRIM1 in A. stephensi was reduced following injection of double stranded (ds) RNA into mosquitoes. By combining the Gal4/UAS bipartite system with in vivo expression of short hairpin (sh) RNA coding for LRIM1 reduced expression of LRIM1 was targeted in the midgut, fat body, and salivary glands. RT-qPCR was used to demonstrate fold-changes in gene expression in three transgenic crosses and the effects on P. falciparum infections determined in mosquitoes showing the greatest reduction in LRIM1 expression.

Results: LRIM1 expression could be reduced, but not completely silenced, by expression of LRIM1 dsRNA. Infections of P. falciparum oocysts and PfSPZ were consistently and significantly higher in transgenic mosquitoes than wild type controls, with increases in PfSPZ ranging from 2.5- to tenfold.

Conclusions: Plasmodium falciparum infections in A. stephensi can be increased following reduced expression of LRIM1. These data provide the springboard for more precise knockout of LRIM1 for the eventual incorporation of immune-compromised A. stephensi into manufacturing of Sanaria's PfSPZ products.

Keywords: Anopheles stephensi; Gene silencing; Immune system; Mosquito; Oocyst; PfSPZ vaccine; Plasmodium falciparum; Sporozoite.

Fig. 1

Amino acid sequence and predicted structural organization of Anopheles stephensi LRIM1. Grey—signal peptide; Red—leucine rich repeat regions; Blue—cysteine; Green—Coiled coil domains

Fig. 2

Relative transcript levels of immune genes in adult female Anopheles stephensi. LRIM1, APL1C, TEP1 and Caspar in the midgut (a–c) and carcass (d–f) of female Anopheles stephensi 24 h (a, d), 48 h (b, e) and 72 h (c, f) after feeding on blood with (black bars) or without (gray bars) Plasmodium falciparum gametocytes. Bars represent mean of triplicate experiments ± standard error of mean. *p < 0.05; **p < 0.02; ***p < 0.01. Asterisks above each bar show comparison to baseline; asterisks above horizontal lines show comparison between treatments. Transcript levels are reported as a fold expression compared to naïve non-blood fed females

Fig. 3

Relative transcript levels of LRIM1, APL1C, TEP1 and Caspar in salivary glands of female Anopheles stephensi 14 days post Plasmodium falciparum infection. Transcript levels are reported as a fold expression compared to non- infected blood fed females. Error bars indicate standard error of the mean of three independent replicates. *p < 0.05; **p < 0.02; ***p < 0.01. Asterisks above each bar show comparison to baseline

Fig. 4

LRIM1 expression and survival of female Anopheles stephensi after dsRNA injection. a Expression of LRIM1 in whole mosquitoes 4 days after injection. Average transcript abundance is relative to control mosquitoes injected with dsEGFP. Transcript levels of ribosomal S7 gene were used as a calibrator. Bars indicate standard error of the mean of three independent replicates. **p < 0.02; ***p < 0.01. Asterisks above horizontal lines show comparison between treatments and control. b Survival of female Anopheles stephensi after injection of dsRNA. Fifty female mosquitoes were used for each treatment. Bars indicate standard error of the mean of three independent experiments

Fig. 5

Relative transcript abundance of LRIM1 in driver and silencer LRIM1 mosquito lines. LRIM1 expression in GAL4::LRIM1-silencer lines F2, M2, and M7 was determined in the midguts and carcasses 24 h post blood meal, and salivary glands 14 days post blood meal. For each genotype the midguts or carcasses of 10 females or salivary glands of 30 females were pooled and RNA extracted. Transcript abundance is shown relative to wild type mosquitoes using ribosomal S7 gene as a calibrator. Bars indicate mean and standard error of the mean of three independent experiments

Fig. 6

Survival of the progeny from crosses of LRIM1-silencer lines with the MLB24 Gal4 driver line. a F2, b M2, c M7 line. Fifty female pupae from each genotype were pooled and adults observed for 21 days post emergence. The cage was examined daily and dead individuals removed, the genotype determined. No statistical differences were observed among the genotypes examined

Fig. 7

Midgut bacterial load in a cross of LRIM1-silencer M7 with MLB24 Gal4 driver Anopheles stephensi. Serial dilutions of midgut homogenate of 10 individual females of each genotype were plated on LB agar. CFUs were calculated after 48 h incubation at 27 °C. Error bars indicate the standard error of the mean of three independent experiments

Fig. 8

Plasmodium falciparum infections in progeny from a cross of LRIM1-silencer M7 with MLB24 Gal4 driver Anopheles stephensi. Oocyst infections were determined on day 7 post blood meal and PfSPZ infections on day 21–25 post blood meal. Circles represent the number of oocysts on a single midgut; horizontal black bars represent the median oocysts in each genotype. Three independent biological replicates were pooled, and significance was determined by a Kruskal–Wallis test followed by Dunn’s post-test in the case of multiple comparisons. For PfSPZ, circles represent the mean intensity in a pool of 30 salivary glands from each of three experiments