TOR signaling is required for amino acid stimulation of early trypsin protein synthesis in the midgut of Aedes aegypti mosquitoes

Abstract

Blood meal digestion in mosquitoes occurs in two phases, an early phase that is translationally regulated, and a late phase that is transcriptionally regulated. Early trypsin is a well-characterized serine endoprotease that is representative of other early phase proteases in the midgut that are only synthesized after feeding. Since the kinase Target of Rapamycin (TOR) has been implicated as a nutrient sensor in other systems, including the mosquito fat body, we tested if TOR signaling is involved in early trypsin protein synthesis in the mosquito midgut in response to feeding. We found that ingestion of an amino acid meal by female mosquitoes induces early trypsin protein synthesis, coincident with phosphorylation of two known TOR target proteins, p70S6 kinase (S6K) and the translational repressor 4E-Binding Protein (4E-BP). Moreover, in vitro culturing of midguts from unfed mosquitoes led to amino acid-dependent phosphorylation of S6K and 4E-BP which could be blocked by treatment with rapamycin, a TOR-specific inhibitor. Lastly, by injecting mosquitoes with TOR double stranded RNA (dsRNA) or rapamycin, we demonstrated that TOR signaling was required in vivo for both phosphorylation of S6K and 4E-BP in the midgut, and for translation of early trypsin mRNA in response to amino acid feeding. It may be possible to target the TOR signaling pathway in the midgut to inhibit blood meal digestion, and thereby, decrease fecundity and the spread of mosquito borne diseases.

Figure 1

Differential expression of early trypsin RNA and protein levels in Ae. aegypti mosquitoes in response to feeding. A) Time course of early trypsin RNA levels in the midgut of unfed (UF) and blood fed mosquitoes as determined by quantitative real time RT-PCR. Each bar represents the mean and standard deviation of replica PCR results taken from three independent cohorts of mosquitoes. Early trypsin RNA levels are expressed as relative values compared to endogenous transcript levels of the ribosomal S7 protein gene using 120 hours as the maximum amount. Data was analyzed by ANOVA with ** P < 0.01 comparing early trypsin transcript levels in fed versus unfed mosquitoes. B) Early trypsin transcript levels in unfed and amino acid fed mosquitoes measured over a 48 hour period using QRT-PCR and the same relative transcript scale based on S7 transcripts as in A. Data is presented as mean and standard deviation of three mosquito cohorts fed and dissected in different experiments. C) Midgut early trypsin protein levels in amino acid fed mosquitoes based on Western blotting using an anti-early trypsin rabbit polyclonal antibody. The loading control for the Western blot was an anti-tubulin antibody. This is a representative Western blot from multiple independent experiments. Note that this particular early trypsin antibody recognized numerous non-specific ~25–35 kDa proteins in the midguts of blood fed mosquitoes that precluded its use in Western blotting using protein extracts from these mosquitoes (data not shown).

Figure 2

Amino acid feeding induces phosphorylation of 4E-BP and S6K in the mosquito midgut. The antibodies used in this Western blot were ph-4E-BP, a phospho-specific 4E-BP antibody that detects phosphorylation at Thr37 and Thr 46; d4E-BP, a Drosophila melanogaster anti-4E-BP antibody that detects both phosphorylated and unphosphorylated 4E-BP; ph-S6K, a phospho-specific antibody that detects phosphorylated S6K at Thr412; and S6K which detects both phosphorylated and unphosphorylated S6K. This is a representative Western blot from multiple independent experiments.

Figure 3

Rapamycin inhibits 4E-BP and S6K phosphorylation in isolated midguts cultured in amino acid rich media. Isolated midguts from unfed mosquitoes were cultured in amino acid free M199 media in the presence or absence of various concentrations of rapamycin for 60 minutes and then transferred to fresh M199 media containing amino acids and the same concentrations of rapamycin for an additional 90 minutes. Protein extracts were prepared and analyzed by Western blotting as described in figure 2. This is a representative Western blot from multiple independent experiments.

Figure 4

The TOR signaling pathway accounts for half of the amino acid induced translation in isolated midguts from unfed mosquitoes. Midguts were cultured for 60 mins in amino acid free M199 media in the absence of any inhibitors, or in the presence of 20 µM rapamycin or 10 µg/ml puromycin, a general translational inhibitor. The midguts were then transferred to fresh M199 media containing amino acids and the same concentrations of inhibitors. This media also contained [³⁵S] methionine/cysteine as a trace label to follow protein synthesis. Incubation continued for 90 minutes after which the pooled midguts were homogenized and TCA precipitable protein was analyzed by scintillation counting. Data is expressed as the mean and standard deviation of disintegrations per minute per midgut from three independent experiments. Asterisks (*) indicate that the data point is significantly different (p<0.05) from the control sample.

Figure 5

TOR signaling is required for 4E-BP phosphorylation in the midguts of amino acid fed mosquitoes. A) TOR RNA levels were determined by quantitative RT-PCR in the midgets of different cohorts of unfed mosquitoes six days post-injection. Mean early trypsin RNA levels relative to S7 ribosomal protein gene transcripts were determined using midgut RNA from control (uninjected), and TOR or LUC injected mosquitoes. Asterisk (*) indicates that TOR RNA levels were significantly different (p<0.05) from the non-injected or LUC-injected control mosquitoes. B) Effect of TOR dsRNA injection on egg development as determined by egg size. Egg size was determined using a microscopic micrometer. Data is expressed as the average size of eggs from 10 different mosquitoes per sample point using more than one cohort of mosquitoes. The mean egg sizes were significantly different (*) between the LUC and TOR injected mosquitoes, as well as, between the TOR injected and unfed controls (p<0.05). C) Effect of TOR dsRNA injection on the phosphorylation of 4E-BP and S6K as determined by Western blotting. Protein extracts were prepared 90 minutes after amino acid feeding and analyzed with the same antibodies described in figure 2. Representative Western blots from multiple experiments are shown.

Figure 6

TOR signaling is required for early trypsin protein synthesis in amino acid fed mosquitoes. A) Injection of TOR dsRNA had no effect on early trypsin transcript levels in unfed mosquitoes based on quantitative RT-PCR. B) Injection of TOR dsRNA or rapamycin decreased early trypsin protein levels in amino acid fed mosquitoes. Western blotting was performed the same as in figure 1. The doublet band seen here likely reflects the presence of unprocessed zymogen. This is a representative Western blot from multiple independent experiments.