Osmotic balance in insects is regulated by the excretory system, consisting of Malpighian tubules and the gut under the control of diuretic and antidiuretic factors. Terrestrial insects must conserve water, and antidiuresis is the norm, only interrupted by brief diuretic periods. Surprisingly, little is known about antidiuresis in insects. Two antidiuretic strategies have been described. The first antidiuretic mechanism involves the reabsorption of fluid from the primary urine in the hindgut. More recently, a second antidiuretic strategy was reported, consisting of inhibition of primary urine formation by the Malpighian tubules. Recently, we isolated, characterized, and cloned the gene encoding for the antidiuretic neurohormone (the neuropeptide RhoprCAPA-2) acting on the Malpighian tubules of Rhodnius prolixus. Here we describe a third, novel mechanism central to the antidiuretic strategy of R. prolixus, the inhibition of ion and fluid transport across the anterior midgut by RhoprCAPA-2. Our results show that RhoprCAPA-2 (1 micromol/l) reduces serotonin-stimulated fluid transport from 83 +/- 11 to 12 +/- 12 nl/min and equivalent short-circuit current from 20 +/- 4 to 5 +/- 0.7 microA/cm(2) in diuretic hormone-stimulated anterior midgut. RhoprCAPA-2 appears to function independently of intracellular cGMP or Ca(2+) in the midgut. Thus, the antidiuretic neurohormone RhoprCAPA-2 has multiple target tissues, and we hypothesize that RhoprCAPA-2 functions to coordinate the transport activity of the anterior midgut and Malpighian tubules so that the rate of fluid transport into the haemolymph by the anterior midgut matches the transport rate of Malpighian tubules to maintain the volume and ion composition of haemolymph.