Overexpression of Activated AMPK in the Anopheles stephensi Midgut Impacts Mosquito Metabolism, Reproduction and Plasmodium Resistance

Abstract

Mitochondrial integrity and homeostasis in the midgut are key factors controlling mosquito fitness and anti-pathogen resistance. Targeting genes that regulate mitochondrial dynamics represents a potential strategy for limiting mosquito-borne diseases. AMP-activated protein kinase (AMPK) is a key cellular energy sensor found in nearly all eukaryotic cells. When activated, AMPK inhibits anabolic pathways that consume ATP and activates catabolic processes that synthesize ATP. In this study, we overexpressed a truncated and constitutively active α-subunit of AMPK under the control of the midgut-specific carboxypeptidase promotor in the midgut of female Anopheles stephensi. As expected, AMPK overexpression in homozygous transgenic mosquitoes was associated with changes in nutrient storage and metabolism, decreasing glycogen levels at 24 h post-blood feeding when transgene expression was maximal, and concurrently increasing circulating trehalose at the same time point. When transgenic lines were challenged with Plasmodium falciparum, we observed a significant decrease in the prevalence and intensity of infection relative to wild type controls. Surprisingly, we did not observe a significant difference in the survival of adult mosquitoes fed either sugar only or both sugar and bloodmeals throughout adult life. This may be due to the limited period that the transgene was activated before homeostasis was restored. However, we did observe a significant decrease in egg production, suggesting that manipulation of AMPK activity in the mosquito midgut resulted in the re-allocation of resources away from egg production. In summary, this work identifies midgut AMPK activity as an important regulator of metabolism, reproduction, and innate immunity in An. stephensi, a highly invasive and important malaria vector species.

Keywords: AMPK; Anopheles stephensi; Plasmodium falciparum; malaria; metabolism; midgut; reproduction.

Figure 1

Generation of the HA-AMPKαT176D transgenic An. stephensi and protein and transcript expression profile of the transgene in adult females. (A) Full length AMPKα subunit versus constitutively active AMPKα subunit. Changes include removal of the inhibitory domains, conversion of Thr176 to Asp176 and the addition of an HA epitope. (B) Schematic of the construct genetically engineered into An. stephensi mosquitoes. (C) Comparison of transgenic (TG) and non-transgenic (NTG) siblings. Top panel: NTG (1 & 2) and TG pupae (3 and 4) under white light. Middle panel: TG and NTG under fluorescence and a GFP filter. Bottom panel: merge of top and middle panels. (D) Representative immunoblot of total proteins isolated from the midguts or carcasses (whole body minus midgut) of TG and NTG mosquitoes and probed with anti-HA antibody (top) or anti-GAPDH antibody (bottom) as loading control. (E) Total RNA was isolated from the midguts or carcasses of both TG and NTG mosquitoes and converted into cDNA. Transgene specific primers were used to amplify HA-AMPKαT176D (top). Actin-specific primers were used as positive control to verify the integrity of the cDNA (bottom). No template controls served as negative control. All transcript and protein expression studies were replicated a minimum of 3 times.

Figure 2

Expression profile of HA-AMPKαT176D. (A) Representative immunoblot detailing the expression of HA-AMPKαT176D and GAPDH in the midguts of non-blood-fed (NBF) and blood-fed TG mosquitoes at 6, 12, 24, 36, 48 and 72 h post-BM. The lower graph depicts the average expression of transgene protein normalized to GAPDH loading controls. Data are represented as means ± SEMs from three replicates with independent cohorts of mosquitoes. Significant differences between the post-BM treatments and the NBF controls were determining using a one-way ANOVA followed by a Dunnett’s multiple comparisons test (* p < 0.05). (B) HA-AMPKT176D protein expression at the different mosquito developmental stages. (C) Representative immunoblot of the expression of phospho-AMPK and GAPDH in the midguts of NBF and blood-fed (3, 6, and 24 h post-BM) WT and TG mosquitoes. Average expression of endogenous p-AMPK relative to GAPDH loading controls and shown relative to levels in NBF mosquitoes and in homozygous TG females compared to wild type at 3, 6, and 24 h post-BM. Data are represented as means from five replicates with independent cohorts of mosquitoes. (D) Three independent nuclear and cytoplasmic fractions of homozygous TG mosquito midguts collected at different time points post-BM were prepared and probed with anti-HA antibody and GAPDH as loading control.

Figure 3

Effect of HA-AMPKαT176D overexpression on An. stephensi glycogen, lipids, and trehalose. (A) Glycogen (B) Trehalose, and (C) Lipids were extracted and assayed from pools of five females. Average concentrations ± SEMs are shown. Data were analyzed using Student’s t test (n = 15 biological replicates). * p < 0.05 relative to wild type controls.

Figure 4

Survivorship of sugar-fed and blood-fed AMPKαT176D hemizygous transgenic (TG) and non-transgenic (NTG) An. stephensi. (A) A representative survivorship curve comparing hemizygous TG and NTG siblings reared under identical conditions and provided with 10% sucrose ad libitum. Lifespan experiments were replicated twice with independent cohorts of mosquitoes. (B) A representative survivorship curve comparing hemizygous TG and NTG siblings reared under identical conditions and provided with a daily bloodmeal and 10% sucrose solution. Lifespan experiments were replicated five times with independent cohorts of mosquitoes. The table summarizes the sample sizes, means, and statistical analyses results (* p < 0.05) of lifespan data using the Wilcoxon test for sugar-fed and blood-fed mosquitoes.

Figure 5

Survivorship of sugar-fed and blood-fed AMPKαT176D homozygous transgenic (TG) and wild type An. stephensi. (A) A representative survivorship curve comparing homozygous AMPKαT176D TG and wild type siblings reared under identical conditions and provided with 10% sucrose ad libitum. Lifespan experiments were replicated five times with independent cohorts of mosquitoes. (B) A representative survivorship curve comparing homozygous AMPKαT176D TG and wild type siblings reared under identical conditions and provided with a daily bloodmeal and 10% sucrose solution. Lifespan experiments were replicated twice with independent cohorts of mosquitoes. The table summarizes the sample sizes, means, and statistical analyses results (* p < 0.05) of lifespan data using the Wilcoxon test for sugar-fed and blood-fed mosquitoes.

Figure 6

Impact of HA-AMPKαT176D overexpression on lifetime fecundity. Graphs represent total egg production of hemizygous (A) and homozygous (B) TG females compared with NTG or wild type control females, respectively, throughout their lifespans. No significant differences were observed between hemizygous TG and NTG or homozygous TG and wild type females (p > 0.05). (C) Egg counts for TG and wild type female mosquitoes. Results are presented for each replicate separately, including the number of females tested (N), the mean number of eggs laid ± SEMs, and p-values by Wilcoxon test.

Figure 7

HA-AMPKαT176D overexpression reduces Plasmodium falciparum infection prevalence and intensity. Transgenic (hemizygous and homozygous) mosquitoes and wild type controls were provided with an artificial bloodmeal enriched with P. falciparum NF54 gametocytes. Ten days after infection, midguts were dissected and the numbers of P. falciparum oocysts were counted. (A) Prevalence reflects the percentage of mosquitoes infected with at least one oocyst in the midgut. (B) Intensity of infection reflects the mean number of oocysts found in infected mosquitoes. The mean numbers of oocysts per midgut ± SEMs are plotted. WT n = 91; homozygous TG n = 94; hemizygous TG n = 87. Infection prevalence was analyzed by Fisher’s exact test and intensity by Mann–Whitney (** p < 0.01, *** p < 0.001).