SLC7 amino acid transporters of the yellow fever mosquito Aedes aegypti and their role in fat body TOR signaling and reproduction

Abstract

Background: An important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.

Methodology/principal findings: We identified 11 putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All 11 A. aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these 'knockdowns' reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal.

Conclusions/significance: Our analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.

Fig. 1. Phylogenetic tree of SLC7 transporters

The evolutionary history was inferred using the neighbor-joining method. The percentage of bootstrap test (1,000 replicates) is shown next to the nodes. The tree was drawn using Fig Tree software. The analysis involved 13 human (black letters) and 11 mosquito AA sequences (white letters in grey boxes). Predicted members of the CAT subfamily are surrounded by a black line, members of the HAT subfamily are surrounded by two grey lines.

Fig. 2. Expression levels of SLC7 transporters in whole mosquitoes during development and vitellogenesis

A. Overall expression of AaCATs during larval stages, adult male, and adult female mosquitoes during vitellogenesis. Expression was assayed with q-RT-PCR. RNA was isolated from three groups of 150 1st instar larvae, 130 2nd instar larvae, 75 3rd instar larvae, 30 4th instar larvae, 15 pupae, and 3 adult mosquitoes. PE- Post Eclosion, PBM- Post Blood Meal. Five replicates were analyzed, and their means separated by Tukey-Kramer HSD (p,0.05). Means which share the same letter are not significantly different. B. Expression of HATs during larval stages and in adult mosquitoes. Overall expression of AaHATs in during larval stages, adult male, and adult female mosquitoes during vitellogenesis. Expression was assayed as described above.

Fig. 2. Expression levels of SLC7 transporters in whole mosquitoes during development and vitellogenesis

A. Overall expression of AaCATs during larval stages, adult male, and adult female mosquitoes during vitellogenesis. Expression was assayed with q-RT-PCR. RNA was isolated from three groups of 150 1st instar larvae, 130 2nd instar larvae, 75 3rd instar larvae, 30 4th instar larvae, 15 pupae, and 3 adult mosquitoes. PE- Post Eclosion, PBM- Post Blood Meal. Five replicates were analyzed, and their means separated by Tukey-Kramer HSD (p,0.05). Means which share the same letter are not significantly different. B. Expression of HATs during larval stages and in adult mosquitoes. Overall expression of AaHATs in during larval stages, adult male, and adult female mosquitoes during vitellogenesis. Expression was assayed as described above.

Fig. 3. SLC7 knockodown reduces TOR signaling in mosquito fat bodies

A: AA induced S6K phosphorylation in fat body culture. Upper panel: Western Blot transfer membranes showing the increase of P-S6K in fat bodies of mosquitoes at different time points after placement in FBCM. Second panel: A second blot was probed with anti p70-S6K antibodies as control. Third panel: A third blot was probed with anti actin antibodies as loading control. Lower panel: Densitometric analysis of expression levels. P-S6K levels were normalized against actin. B: Effect of RNAi-mediated SLC7 knockdowns on P-S6K levels. Upper panel: Western Blot showing the levels of P-S6K after knockdown of the transporter shown in the column. Second panel: A second blot was probed with anti p70-S6K antibodies as control. Third panel: A third blot was probed with anti actin antibodies as loading control. Lower panel: Densitometric analysis of P-S6K expression levels. P-S6K levels were normalized against actin. Three biological replicates were analyzed, and their means separated by Tukey-Kramer HSD (p,0.05). Means which share the same letter are not significantly different. C: The effect of L-arginine withdrawal on S6K phosphorylation. Upper panel: Western Blot showing phospho-S6K levels of fat bodies that were treated with PBS, balanced AA media, and AA media without L-arginine. Lower panel: A second blot was probed with anti p70-S6K antibodies as control.

Fig. 4. RNAi-mediate knockdown causes a decrease in average clutch size per female

A: Average number of eggs laid by each female. The total number of eggs was take from 45 females and measured by egg counting though a stereo microscope. B: Comparison of the egg distributions of AaCAT3 injected mosquitoes (white bars) and eGFP injected mosquitoes (grey bars).

Fig. 5. Working Model: SLC7 regulation of TOR signaling in the Mosquito Fat Body

A. Transfer of fat bodies onto FBCM results in a balanced rise of intracellular essential AAs and subsequent de-repression of TOR via the tuberous sclerosis complex (TSC1 & 2) which activates yolk protein precursor gene expression. B. RNAi-mediated knockdown of individual SLC7 transporters resulted in unbalanced intracellular AA concentrations. Under these conditions TOR signaling is reduced and yolk protein precursor gene expression is repressed.