Identification of kinin-related peptides in the disease vector, Rhodnius prolixus.

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX(1)X(2)WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX(1)X(2)WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.

The antidiuretic neurohormone RhoprCAPA-2 downregulates fluid transport across the anterior midgut in the blood-feeding insect Rhodnius prolixus.

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.

Biological activity of diuretic factors on the anterior midgut of the blood-feeding bug, Rhodnius prolixus.

Probing of a host and ingestion of a blood-meal in a fifth instar Rhodnius prolixus results in a cascade of tightly integrated events, including salivary gland secretion, plasticization of the abdominal cuticle, increased ion and water movement across the anterior midgut (crop) and Malpighian tubules (which rapidly produce urine) and the regular expulsion of urine from the hindgut. In this study we have focussed on the role of the anterior midgut during the rapid postprandial diuresis. The huge blood-meal is pumped into the anterior midgut, during feeding, then modified by diuresis and stored until it is digested. Changes in the anterior midgut activity are rapid. Within minutes of the commencement of feeding there is an increase in the frequency of anterior midgut contractions and diuresis begins with the movement of salt and water across the epithelium of the anterior midgut into the haemolymph. While serotonin, a diuretic hormone in R. prolixus, is known to play a role in the physiological activity of the anterior midgut, we were interested in exploring further the role of serotonin, and other diuretic peptides. We have tested the activity of several peptides, including R. prolixus calcitonin-like diuretic hormone (Rhopr-DH 31), corticotropin-releasing factor (CRF)-like peptide from Zootermopsis nevadensis DH (Zoone-DH) and a kinin from Leucophaea maderae, Leucokinin 1 (LK1). These peptides families are known to be present in the central nervous system of R. prolixus, are putative neurohormones released into the haemolymph after the start of feeding, and have been shown to have activity on a variety of tissues involved in post-feeding diuresis. We show here that both serotonin and Zoone-DH increase the cAMP content of the anterior midgut and that serotonin, Zoone-DH and cAMP analogues increase absorption of water from the anterior midgut, increase the short circuit current and voltage, while decreasing the resistance across the epithelium. While LK1 and Rhopr-DH 31 do not significantly increase absorption, or short circuit current, LK1 does significantly decrease the resistance and transepithelial voltage of the anterior midgut epithelium. All of the factors studied increase the frequency of contractions of the anterior midgut.