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First published online August 3, 2006
Journal of Experimental Biology 209, 3071-3078 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02349

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Identification of two cationic amino acid transporters required for nutritional signaling during mosquito reproduction

Geoffrey M. Attardo^*,, Immo A. Hansen^*, Shin-Hong Shiao and Alexander S. Raikhel

Center for Disease-Vector Research, Department of Entomology and the Institute for Integrative Genome Biology, University of California Riverside, 3401 Watkins Drive, Riverside, CA-92521, USA

View larger version (23K): [in a new window]   Fig. 1. Specific amino acids (AAs) are essential for 20-hydroxyecdysone (20E)-activation of the vitellogenin (vg) gene. Fat bodies from 3- to 5-day-old mosquitoes were cultured for 3 h at 27°C in media lacking individual AAs in the presence of 20E (10-6 mol l-1). Total RNA was isolated from three groups of six fat bodies per treatment. cDNA was synthesized from equal amounts of DNase I-treated total RNA. Real-time PCR was used to quantify levels of vg mRNA. Data was normalized by real-time PCR analysis of actin levels in the cDNA samples. vg production in responses to 20E stimulation in the `withdrawal' medias are presented as the mean percentage (± s.e.m. of triplicate samples) relative to the response observed in the control medium containing complete AAs.

View larger version (11K): [in a new window]   Fig. 2. Amino acid stimulation of the vg gene depends on Na+/K+-ATPase and V-ATPase activity. Treatment with 10-6 mol l-1 bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase, and 10-5 mol l-1 ouabain, an inhibitor of Na+/K+-ATPase, results in a significant reduction of the vg gene expression. (A) Fat bodies of 3- to 5-day-old mosquitoes were dissected and incubated in Aedes physiological saline (APS) with different treatments as indicated. After 1 h the APS was replaced with media containing different combinations of amino acids (AAs), bafilomycin A1 and ouabain, and the fat bodies were incubated at room temperature for 6 h. Total RNA was isolated from three groups of three fat bodies per treatment. cDNA was synthesized from equal amounts of DNase I-treated total RNA. Gene expression was analyzed using vg-specific real-time PCR primers. Values are means ± s.e.m. of triplicate samples. (B) Treatment with bafilomycin A1 or ouabain does not inhibit 20-hydroxyecdysone (20E) activation of the E74 early gene (E74B isoform). As a control, fat bodies were dissected and pre-treated in APS as described above. After 1 h the APS was replaced with media containing different combinations of 10-6 mol l-1 20E, 10-6 mol l-1 bafilomycin A1 and 10-5 mol l-1 ouabain. The fat bodies were incubated at room temperature for 6 h and processed as described above. Gene expression was analyzed using E74-specific real-time PCR primers. Values are means ± s.e.m. of triplicate samples.

View larger version (39K): [in a new window]   Fig. 3. Dendrogram/bootstrap analysis of insect and vertebrate cationic amino acid transporters. Sequence similarity was assessed using amino acid alignments in ClustalW and a rooted tree was calculated by the neighbor-joining method. Confidence values were derived by bootstrapping the dataset, using 1000 replicates. The alignment was visualized using Treeview 1.6.6 (Page, 1996).

View larger version (22K): [in a new window]   Fig. 4. Expression of Aaslif and AaiCAT2 mRNA. Relative mRNA levels in fat body tissue of female mosquitoes at different points of the first reproductive cycle were determined by real-time PCR. (A) Schematic diagram of the different phases of female mosquito fat bodies during the first reproductive cycle. The previtellogenic period starts with a 3 day preparation period during which the mosquito fat body gains competence to respond to 20-hydroxyecdysone (20E) and produce yolk protein precursor (YPPs). This is followed by a state-ofarrest that lasts until a blood meal (BM) is taken. The vitellogenic period is divided into a synthesis phase during which the YPPs are produced, and a termination phase, during which the fat body undergoes remodeling and returns to its previtellogenic state of being a store for lipid, protein and glycogen reserves (Raikhel and Dhadialla, 1992). (B) Aaslif mRNA expression profile. (C) AaiCAT2 mRNA expression profile. PBM, post blood meal.

View larger version (31K): [in a new window]   Fig. 5. RNAi-mediated knockdown of Aaslif and AaiCAT2 inhibits amino acid stimulation of the vg gene. 1 day-old mosquitoes were injected with 0.6-1.0 µg of the following dsRNAs: the non-coding region of a control bacterial gene (MAL), the coding region of neutral amino acid transporter (NAT), the coding region of the Aaslif gene (Slif), the coding region of the AaiCAT2 gene (iCAT2), or a 1:1 mixture of both Aaslif and AaiCAT2 dsRNAs. Mosquitoes were allowed to recover for 5 days. Fat bodies from these mosquitoes were then dissected and cultured in either the presence or absence of amino acids (AAs) for 6 h. Total RNA was isolated from three groups of six fat bodies per treatment. cDNA was synthesized from equal amounts of DNase I-treated total RNA. (A) Gene expression was analyzed using vg-specific real-time PCR primers. Data were normalized by real-time PCR analysis of actin levels in the cDNA samples. Values are means ± s.e.m. of triplicate samples. (B) Knockdown of transporter genes was confirmed by RT-PCR analysis of the same cDNA used for the analysis in A.