Phenylalanine metabolism regulates reproduction and parasite melanization in the malaria mosquito

Abstract

The blood meal of the female malaria mosquito is a pre-requisite to egg production and also represents the transmission route for the malaria parasite. The proper and rapid assimilation of proteins and nutrients in the blood meal creates a significant metabolic challenge for the mosquito. To better understand this process we generated a global profile of metabolite changes in response to blood meal of Anopheles gambiae, using Gas Chromatography-Mass Spectrometry (GC-MS). To disrupt a key pathway of amino acid metabolism we silenced the gene phenylalanine hydroxylase (PAH) involved in the conversion of the amino acid phenylalanine into tyrosine. We observed increased levels of phenylalanine and the potentially toxic metabolites phenylpyruvate and phenyllactate as well as a reduction in the amount of tyrosine available for melanin synthesis. This in turn resulted in a significant impairment of the melanotic encapsulation response against the rodent malaria parasite Plasmodium berghei. Furthermore silencing of PAH resulted in a significant impairment of mosquito fertility associated with reduction of laid eggs, retarded vitellogenesis and impaired melanisation of the chorion. Carbidopa, an inhibitor of the downstream enzyme DOPA decarboxylase that coverts DOPA into dopamine, produced similar effects on egg melanization and hatching rate suggesting that egg chorion maturation is mainly regulated via dopamine. This study sheds new light on the role of amino acid metabolism in regulating reproduction and immunity.

Figure 1. Transcriptional analysis of PAH in various tissues and following dsRNA injection.

A) Q-PCR determination of PAH mRNA levels in the head, midgut, ovaries and remaining carcass of A. gambiae females in response to blood meal. Pools of 3 females were dissected and their total RNA extracted at a non-blood-fed (NBF) stage as well as 3 h, 24 h and 48 h post-blood meal (PBM). PAH transcript abundance is represented as mean proportion ± SD of the expression of the ribosomal protein gene RPL19 of 3 independent biological repeats (t-test, *p<0.05, **p<0.01). B) 24 h PBM PAH expression was down-regulated in dsPAH injected females compared to dsLacZ injected controls. Transcript abundance was standardized to RPL19 and represented as the mean proportion ± SD of the expression recorded in the LacZ control of 3 independent biological repeats (t-test,*p<0.05).

Figure 2. GC-MS mosquito metabolome in response to PAH knockdown.

A) Metabolites were extracted from 2 females and pooled for GC-MS analysis. The green bars represent the mean percentage of total metabolite signal ± SD in fed and non-fed dsLacZ injected controls from 4 independent biological repeats. The heatmap represents the mean fold change in metabolite signal in non-fed and fed dsPAH injected mosquitoes compared to the respective non-fed and fed dsLacZ injected controls. ^aputative metabolite identity (poor signal to noise), ^blabile metabolite, therefore only approximate quantification. B) Targeted analysis of GC-MS detectable metabolites affected by PAH knockdown within the phenylalanine metabolism pathway. 24 h post-blood meal aqueous metabolites were extracted from 2 blood-fed females injected with dsPAH or dsLacZ and pooled for analysis. The circles represent the mean metabolite levels (thick line) ± SD (dashed line) of dsPAH females relative to the metabolite levels of the dsLacZ controls.

Figure 3. Reduced PAH activity does not decrease the survival of adult blood-fed mosquitoes.

Mean survival ± SEM of dsPAH females and dsLacZ injected controls A) after a single blood meal of 6 independent experiments (PBM- time post-blood meal) (log-rank test, p>0.05) B) in response to multiple blood meals (time of blood meal is indicated by circles) of 2 independent experiments (log-rank test, p>0.05).

Figure 4. Large amounts of ingested phenylpyruvate are required to decrease the survival of adult A. gambiae mosquitoes.

Females were fed on naive blood or blood supplemented with phenylpyruvate (PP) or phenylalanine (Phe) at a concentration of 10–50 mM and their survival was recorded daily until 7 days post blood meal (PBM). Combined survival data from three independent experiments are displayed as mean ± SEM (log-rank test, ***p<0.001).

Figure 5. Knockdown of PAH causes a reduced melanization of P. berghei ookinetes.

In 3 independent biological repeats dsPAH- and dsLacZ-injected (control) females were fed on a mouse infected with P. berghei parasites. Mosquito midguts were dissected and examined for oocysts 8 days after infection. A) Displayed is the proportion of females which harboured at least 1 oocyst (Likelihood of Infection: Fisher’s exact test, p>0.05) B) Oocyst load of dsPAH and dsLacZ injected females. The bars represent the mean ± SEM (Mann-Whitney U test, p>0.05). Only females with at least 1 oocyst and/or melanized ookinete were included in the analysis. C) Represented is the proportion of melanized ookinetes to the total number of oocysts per dsPAH or dsLacZ injected female. The bars indicate the mean ± SEM (t-test of arc-sine square root transformed proportion, **p<0.01).

Figure 6. PAH knockdown leads to reduced fertility of A. gambiae mosquitoes.

A) The mean proportion ±SEM of dsPAH and dsLacZ (control) injected females that oviposited (Likelihood of oviposition: Fisher’s exact test, ***p<0.001). B) Mean number ± SEM of eggs per ovipositing female injected with either dsLacZ or dsPAH. Only females that oviposited 1 egg or more were included in the analysis (t-test, **p<0.01). C) Mean ± SEM hatching rate of dsPAH and dsLacZ control injected females (t-test of arc-sine square root transformed proportion, *p<0.05). D) Upon dissection (N = 6 per time point) we observed that ovaries of dsPAH injected females were smaller. Females which did not oviposit contained a large fraction of undeveloped eggs in their ovaries 5 days post blood meal. Scale bar: 400 µm. E) When females were placed into water-filled oviposition cups to lay eggs, we observed unmelanized eggs 24 h post-oviposition laid by dsPAH injected females, indicating a malfunctioning melanin biosynthesis. Scale bar: 400 µm.

Figure 7. Injection of carbidopa caused reduced egg viability and melanization in A. gambiae.

A) The mean proportion ±SEM of PBS (control) and carbidopa injected females that oviposited (Likelihood of oviposition: Fisher’s exact test, *p<0.05). B) Mean number ± SEM of eggs per ovipositing female injected with either PBS or carbidopa. (t-test, p>0.05). C) Mean ± SEM hatching rate of PBS and carbidopa injected females (t-test of arc-sine transformed proportion, ***p<0.001). Data were combined from 4 independent experiments. D) In response to carbidopa injection we observed a large proportion of light and unmelanized eggs. Displayed are the mean ± SEM melanization rate of eggs laid by PBS or carbidopa injected females from 2 independent experiments (t-test of arc-sine square root transformed proportion, ***p<0.001).