Adipokinetic hormone signaling in the malaria vector Anopheles gambiae facilitates Plasmodium falciparum sporogony

Abstract

An obligatory step in the complex life cycle of the malaria parasite is sporogony, which occurs during the oocyst stage in adult female Anopheles mosquitoes. Sporogony is metabolically demanding, and successful oocyst maturation is dependent on host lipids. In insects, lipid energy reserves are mobilized by adipokinetic hormones (AKHs). We hypothesized that Plasmodium falciparum infection activates Anopheles gambiae AKH signaling and lipid mobilization. We profiled the expression patterns of AKH pathway genes and AgAkh1 peptide levels in An. gambiae during starvation, after blood feeding, and following infection and observed a significant time-dependent up-regulation of AKH pathway genes and peptide levels during infection. Depletion of AgAkh1 and AgAkhR by RNAi reduced salivary gland sporozoite production, while synthetic AgAkh1 peptide supplementation rescued sporozoite numbers. Inoculation of uninfected female mosquitoes with supernatant from P. falciparum-infected midguts activated AKH signaling. Clearly, identifying the parasite molecules mediating AKH signaling in P. falciparum sporogony is paramount.

Fig. 1. Dissecting the adipokinetic hormone (AKH) signaling pathway in An. gambiae.

a Simplified diagrammatic representation of the proposed AKH signaling and insulin/insulin-like growth factor signaling (IIS) pathway in in An. gambiae. Color-coded genes were targeted for transcript quantification using RT-qPCR. AgAkhR = An. gambiae adipokinetic hormone receptor, AgLsdp1 = An. gambiae Lipid storage droplets surface-binding protein 1, AgLp = An. gambiae lipophorin, AgILP = An. gambiae insulin-like peptide, AgInR = An. gambiae ILP receptor. Other ILPs in the IIS pathway that were quantified include AgILP1, AgILP4, AgILP5, AgILP6, and AgILP7. b Schematic representation of study design depicting starvation (i), blood feeding (ii), P. falciparum infection (iii), and AKH perturbation assays (iv). The time points represent the hours of starvation, hours post bloodmeal and days postinfectious bloodmeal when mosquitoes were sampled for analysis. RT-qPCR = reverse transcription quantitative PCR, ELISA enzyme-linked immunosorbent assay. The figures were created with BioRender.com.

Fig. 2. Induction of AKH signaling and insulin/insulin-like signaling (IIS) pathways in An. gambiae.

a Experimental approach for measuring AKH and IIS signaling. b Representative replicate showing time-dependent fold change in expression of AKH (AgAkh1, AgAkh2, AgAkhR, AgLsdp1, and AgLp) and IIS (AgILP2, AgILP3 and AgInR) pathways genes during starvation relative to age-matched sugar-fed females. All expression levels were normalized against the control gene 60 S ribosomal protein L32 (AgRPL32). Sugar-fed expression levels were standardized to 1 (dotted line). N = 3 pools of 3 mosquitoes for each treatment at each time point. Bars not capped with an asterisk were not statistically different from sugar-fed controls. c Representative data showing AgAkh1 peptide titers in sugar-fed and starved female An. gambiae at different time points detected by competitive peptide ELISA. N = 3 pools of 3 mosquitoes for each treatment at each time point. d Representative data showing time-dependent fold change in expression of AKH and IIS pathways genes following blood feeding relative to age-matched sugar-fed females. All expression levels were normalized against the control gene AgRPL32. Sugar-fed expression levels were standardized to 1 (dotted line). N = 3 pools of 3 mosquitoes for each treatment at each time point. Bars not capped with an asterisk were not statistically different from sugar-fed controls. e Representative replicate showing AgAkh1 peptide titers in sugar-fed and blood-fed female An. gambiae at different time points post bloodmeal. N = 3 pools of 3 mosquitoes for each treatment at each time point. f Representative data showing time-dependent fold change in expression of AKH and IIS pathways genes in P. falciparum-infected An. gambiae relative to uninfected mosquitoes at different time points. All expression levels were normalized against the control gene AgRPL32. Uninfected blood-fed expression levels were standardized to 1 (dotted line). N = 3 pools of 3 mosquitoes for each treatment at each time point. Bars not capped with an asterisk were not statistically different from uninfected blood-fed controls. g Representative data showing AgAkh1 peptide titers in uninfected and P. falciparum-infected An. gambiae at different time points. N = 3 pools of 3 mosquitoes for each treatment at each time point. Error bars represent standard error of the mean. Differences in gene expression between sugar-fed and starved, sugar-fed and blood-fed mosquitoes, and P. falciparum-infected and uninfected mosquitoes were detected by independent samples t-test or two-sample Wilcoxon test at 95% confidence interval. * < 0.05, ** < 0.01, *** <0.001, ns = not significant (P > 0.05). All statistical analyses were performed at a 95% confidence interval. A total of 9 mosquitoes pooled in groups of three were analyzed for each time point for each feeding status. The assays were repeated three times (Supplementary Fig. S1). A subset of infected mosquitoes (N = 20-21) from each replicate were dissected 8 dpi to confirm infection prevalence and intensity using midgut oocyst count as a proxy for infection. P. falciparum oocyst prevalence and intensities were 85% and 35 oocysts per midgut, respectively. AgAkhR An. gambiae adipokinetic hormone receptor, AgLsdp1 Lipid storage droplets surface-binding protein 1, AgLp lipophorin, AgILP insulin-like peptide, AgInR ILP receptor, uBM non-infectious (uninfected) bloodmeal, iBM infectious bloodmeal, dpib days postinfectious bloodmeal.

Fig. 3. RNAi-silencing of An. gambiae AKH signaling reduces P. falciparum salivary gland sporozoite infection intensity.

a Experimental approach to reduce AgAkh1 levels. b Efficiency of double strand RNA (dsRNA) treatment on knockdown of AgAkh1, AgAkh2, and AgAkhR transcripts. N = 8 pools of 3 mosquitoes for each treatment. c Duration of the knockdown effect of dsAgAkh1 and dsAgAkh1 + dsAgAkhR treatment on AgAkh1 transcript levels. N = 6 pools of 3 mosquitoes for each treatment at each time point. d Duration of the knockdown effect of dsAgAkh1, dsAkhR, and dsAgAkh1 + dsAgAkhR treatment on AgAkhR transcript levels. N = 6 pools of 3 mosquitoes for each treatment at each time point. e Confirmation of AgAkh1 and AgAkhR transcript knockdown in P. falciparum-infected An. gambiae. N = 9 pools of 3 mosquitoes for each treatment. f Reduction of AgAkh1 peptide levels in P. falciparum-infected An. gambiae following AgAkh1 transcript knockdown. N = 9 pools of 3 mosquitoes for each treatment at each time point. Error bars represent standard error of the mean. g P. falciparum oocyst diameters in the midguts of An. gambiae treated with dsRNA. Numbers shown in brackets are the total number of oocysts whose diameters were measured. h Representative brightfield images of stained oocyst-infected midguts of An. gambiae treated with dsRNA. Inserts show single oocyst from midguts of dsGFP (i and ii) and dsAgAkh1 (iii and iv) treated female An. gambiae. The scale bars in the large images are 50 µm and the scale bars in the inserts are 25 µm. i P. falciparum sporozoite intensity from salivary glands of dsRNA treated An. gambiae detected by qPCR. dsAgAkh1 + R = dsAgAkh1 + dsAgAkhR. dpib = days post infectious bloodmeal, dpi = days post injection. The fold change in gene expression is relative to controls. Figures represent pooled data from three biological replicates. Error bars represent standard error of the mean. Differences in gene expression were detected by independent samples t-test or two-sample Wilcoxon test at 95% confidence interval. * < 0.05, ***< 0.001, ns = not significant (P > 0.05).

Fig. 4. Direct modulation of AgAkh1 peptide levels enhances oocyst maturation and salivary gland sporozoite infection intensity.

a Experimental approaches to increase Akh1 levels. b Effect of synthetic AgAkh1 and AgAkh2 peptide injection on AKH signaling pathway downstream genes. All expression levels were normalized against the control gene 60 S ribosomal protein L32 (AgRPL32). Gene expression levels of PBS-injected females were standardized to 1 (dotted line). N = 3 pools of 3 mosquitoes for each treatment at each time point. Bars not capped with an asterisk were not statistically different from sugar-fed controls. c P. falciparum oocyst diameter from the midguts of An. gambiae treated with synthetic AgAkh1 (SAgAkh1) and AgAkh2 (SAgAkh2) peptides at 3 and 5 dpi. Numbers shown in brackets are the total number of oocysts whose diameters were measured. d P. falciparum salivary gland sporozoite intensity from An. gambiae treated with synthetic peptides at 3 and 5 dpi detected by qPCR. Differences in oocyst diameters and sporozoite genome copy number were detected using linear mixed-effects models. PBS = phosphate buffered saline, * < 0.05, ** < 0.01, *** < 0.001, ns = not significant (P > 0.05). All statistical analyses were performed at a 95% confidence interval. e Relative expression of AKH pathway genes following treatment with supernatant collected from P. falciparum-infected midguts at 3-, 4- and 5-dpi (days postinjection) following overnight incubation in serum-free RPMI. N = 9 pools of 3 mosquitoes for each treatment at each time point. Supernatant from midguts of mosquitoes fed on noninfectious blood meal or RPMI media were used as controls. f Relative expression of AKH pathway genes following treatment with supernatant from P. falciparum-infected midguts incubated in RPMI or RPMI + FBS at 4-dpi. Supernatant from midguts of mosquitoes fed on noninfectious blood meal or media only were used as controls. N = 9 pools of 3 mosquitoes for each treatment at each time point. g AgAkh1 peptide titers in female An. gambiae treated with supernatant from P. falciparum-infected midguts 4-dpi. N = 9 pools of 3 mosquitoes for each treatment at each time point. Gene expressions were normalized to 60 S ribosomal protein L32 (AgRPL32). The fold change in gene expression is relative to RPMI or RPMI + FBS media-treated females (standardized to 1). Error bars represent standard error of the mean. Differences in gene expression between mosquitoes treated with supernatant from uninfected and infected midguts were detected by a two-sample Wilcoxon test. Differences in peptide concentration were detected by a Kruskal-Wallis test and a two-sample Wilcoxon test. * < 0.05, ** < 0.01, *** < 0.001, ns = not significant (P > 0.05). Figures represent pooled data from three biological replicates. All statistical analyses were performed at a 95% confidence interval. uMS uninfected midgut supernatant, iMS infected midgut supernatant, dpib days postinfectious bloodmeal, dpi days postinjection.

Fig. 5. Graphical representation of measured AKH/IIS pathway gene transcript levels across time and experimental conditions.

AKH/IIS pathway gene transcript levels increase in starved (light gray) conditions between 3 and 6 hours, as verified by AgAkh1 peptide titers (light gray asterisks), in comparison to the sugar-fed condition (light blue). Between 6 and 48 hours, the starved then blood-fed treatment (light red) increases in AKH/IIS pathway gene transcript levels, as verified by peptide titers at these timepoints (light red asterisks), in comparison to the starved then sugar-fed group (dark blue). AKH/IIS pathway gene transcript levels increase from 3–8 days in the Plasmodium-infected blood-fed condition (purple), with spikes coinciding with oocyst development and sporozoite release (pictorially represented under the purple Plasmodium development arrow) and verified by peptide titers (purple asterisks), in comparison to the blood-fed only samples (dark red). Proposed but not measured trends for each condition are displayed as color-matched dashed lines.