P3.92 Friday, Jan. 6 Scaling of Manduca sexta midgut tissue and amino acid transporter expression YEOH, Aaron/J; VELA-MENDOZA, Allison/V; GILLEN, Chris/M*; Kenyon College; Kenyon College; Kenyon College firstname.lastname@example.org
We assessed mass and gene expression in 3rd through 5th instar Manduca sexta midgut. Wet mass of gut tissue increased in comparison to total body mass with a scaling exponent of 0.85, while gut content mass scaled at a 1.33 exponent, indicating that surface area may become increasingly limiting in larger larvae. Scaling exponents of anterior and posterior midgut were 0.96 and 0.85, above the 0.67 value predicted for isometric scaling, while the exponent for middle midgut was 0.70. Dry masses showed the same pattern. KAAT1 is a potassium-amino acid transporter expressed on the apical membrane of Manduca sexta midgut. We tested the hypothesis that increased expression of membrane proteins helps compensate for the decreased relative surface area of the midgut in later instars. We compared mRNA expression between 4th and 5th instar larvae. Expression of the KAAT1 gene was 2.3 to 3.1 fold higher in 5th compared to 4th instar larvae, supporting the hypothesis of increased membrane protein expression in larger larvae. KAAT1 was expressed 300 to 1500 fold higher in middle and posterior midgut compared to anterior midgut. We are optimizing RNA interference to characterize the consequences of decreased KAAT1 expression. A plasmid construct with a 453bp KAAT1 PCR fragment and flanking T7 promoters was developed for the production of KAAT1 dsRNA. We are testing the effects of different delivery methods, timing and dosage with assessment of phenotype and gene expression with qPCR.