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Figure S1. Percent retention of the first and second intron of CtVg genes during combined non-reproductive stages (larvae and pupae) in Culex tarsalis. Data were quantified from reverse transcriptase-PCR products using intron-spanning primers, CtVg1intron1 and intron2 (left graph) and CtVg2intron1 and intron2 (right graph). The bar graphs represent the mean ± sem. **P < 0.0001, Student's t-test.

Figure S2. Translation of intron-retained vitellogenin transcripts from Culex tarsalis, Anopheles gambiae and Aedes aegypti reveals premature termination codons. The translation consists of the first exon (not underlined) followed by the retained first intron (underlined) and in some, partial sequences from the second exon (not underlined). ‘Stop’ indicates a premature termination codon present due to the retention of the first intron in the sequence.

Figure S3. A. Partial amino acid alignment between a cloned Anopheles gambiae Upf1 (AgUpf1), predicted nonsense-mediated mRNA decay protein 1 from Culex quinquefasciatus (CqNMDprotein1, accession # XP_001862543), Drosophila melanogaster Upf1 (DmUpf1, accession # NP_572767) and Homo sapiens Upf1 (HsUpf1, accession # AAC51140). Shaded residues match AgUPF1. B. Partial amino acid alignment between a cloned sequence of An. gambiae Upf2 (AgUpf2), predicted Upf2 from Aedes aegypti and C. quinquefasciatus (AaUpf2 and CqUpf2, accession # XP_001660932 and XP_001851054), D. melanogaster Upf2 (DmUpf2, accession # NP_572434) and H.sapiens Upf2 (HsUpf1, accession # EAW86331). Shaded residues match AgUPF2.

Figure S4. Expression of AgVg measured by quantitative PCR in newly emerged fat bodies treated with 20-hydroxyecdysone (20E), actinomycin D (ACD) and rapamycin as compared with previtellogenic fat bodies treated with 20E. AgVg expression is shown relative to AgS7. The bar graphs represent the mean ± sem. **P < 0.0001. NE, newly emerged adult female; PV, previtellogenic adult female.

Figure S5. Mosquito fat body culture. The mosquito abdomen is dissected and cut on the ventral side down the anteroposterior axis. Abdomens are opened such that the cuticle is exposed to air and the fat body cells (which adhere to the wall of the abdomen) are exposed to the media. These are then referred to as ‘fat bodies’. Fat bodies are cultured five to a well of a 96-well plate as shown in the picture above. Arrow points to a single fat body.

Table S1. Primer sequence information. T7 promoter sequence is underlined.

Table S2. Vitellogenin intron 1 and 2 lengths in base pairs (bp).

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