IP Excretiony

Neuropeptide control of insect hindgut reabsorption: Recent molecular advances

J. Phillips 1 , J. Meredith 1 , Y. Wang 1 , Y. Zhao 1 & H. Brock 1

1 Dept. of Zoology, Univ. of British Columbia, Vancouver, BC V6T 1Z4, Canada

We previously have described epithelial transport mechanisms in locust (Schistocerca gregaria) hindgut responsible for water/ionic homeostasis, pH regulation, and nitrogen excretion, leading to the first models for insect reabsorptive epithelia. The dominant process is an electrogenic Cl - pump that drives K + and fluid reabsorption. Chloride transport can be continuously monitored in vitro as a short-circuit current, and this has served as a very specific bioassay to identify several neuropeptide stimulants of hindgut transport processes, including the major one in nervous corpora cardiaca acting on the ileum, Ion Transport Peptide (ITP). ITP is 72 amino acids long, terminally amidated, and has 3 disulphide bridges which are conserved relative to a large family of crustacean neurohormones to which ITP belongs. ITP is the first member of this protein family to be synthesized and its cDNA expressed using several insect cell/viral vector systems. Both transient and stably transformed Drosophila Kc1 cells secrete a very active form of ITP (KcITP) that is cleaved from its prepropeptide correctly at the N-terminus (unlike baculovirus-infected Sf9 cells). This system has allowed us to use site-directed mutagenesis to identify specific amino acids at both the N and C termini that are essential for activation of the ITP receptor (ITP-R). Similar studies show that each of the disulphide bridges is required for full activity of KcITP. Inactive mutant forms of ITP were also tested for antagonistic activity on ITP-R stimulated by native ITP with potential pest control strategies in mind. We are using molecular techniques and bioassays to study evolutionary divergence of ITP homologues, including those in the most important agricultural pests (Lepidoptera, Coleoptera) and disease vectors (Diptera).

Index terms: Schistocerca gregaria, neuropeptide, expression, mutagenesis

Copyright: The copyrights of this abstract belong to the author (see right-most box of title table). This document also appears in Session 13 – INSECT PHISIOLOGY, NEUROSCIENCES, IMMUNITY AND CELL BIOLOGY Symposium and Poster Session, ABSTRACT BOOK II – XXI-International Congress of Entomology, Brazil, August 20-26, 2000.