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First published online February 15, 2006
Journal of Experimental Biology 209, 907-915 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02083

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Distribution, activity and evidence for the release of an anti-diuretic peptide in the kissing bug Rhodnius prolixus

Jean-Paul Paluzzi^* and Ian Orchard

Department of Biology, University of Toronto at Mississauga, Mississauga, ON, L5L 1C6, Canada

View larger version (36K): [in a new window]   Fig. 1. Composite camera lucida drawing of PRXamide-like immunoreactive cells and processes in the central nervous system of R. prolixus. Left, dorsal view; right, ventral view. Filled cells indicate strong PRXamide-like immunoreactivity (PRXa-LI) and open cells indicate weak PRXa-LI. Within the mesothoracic ganglionic mass (MTGM), the ventral paired median cells give rise to immunoreactive processes which project dorsally and then exit the CNS via the second, third and fourth abdominal nerves (ABN) where they develop neurohaemal sites. PRO, prothoracic ganglion; SOG, sub-oesophageal ganglion. Scale bar, 200 µm.

View larger version (145K): [in a new window]   Fig. 2. PRXamide-like immunoreactivity (PRXa-LI) in fifth-instar R. prolixus. Dorsal views of (A) brain, (B) sub-oesophageal ganglion (SOG), (C) prothoracic ganglion and (D) the mesothoracic ganglionic mass (MTGM). In A, putative lateral neurosecretory cells and medial neurosecretory cells are indicated by open and filled arrows, respectively. In B, note the strong immunoreactivity in putative neurohaemal sites on the dorsal vessel (open arrows) and light staining over the corpus cardiacum (filled arrow). In C, note the numerous medial processes with PRXa-LI that originate in the SOG and project into the MTGM. In D, note the lateral paired cells (open arrows) and the processes originating from the ventral paired neurosecretory cells (filled arrows). Scale bar, 100 µm.

View larger version (139K): [in a new window]   Fig. 3. PRXamide-like immunoreactivity (PRXa-LI) in fifth-instar R. prolixus. Ventral views of (A) brain, (B) sub-oesophageal ganglion, (C) prothoracic ganglion (PRO) and (D) the mesothoracic ganglionic mass. In A, note the lateral immunoreactive cells (open arrows). In B, note the numerous bilaterally paired cell bodies (open arrows) lying medially. In C, note the lightly staining immunoreactive varicosities over the ventral PRO. In D, note the immunoreactive cell bodies (open arrows) and extensive neurohaemal-like immunoreactivity on the abdominal nerves (closed arrows). The intensity of staining of these cells is greatly reduced 3–4 h following feeding (see later). Scale bar, 100 µm.

View larger version (69K): [in a new window]   Fig. 4. PRXamide-like immunoreactivity (PRXa-LI) in fifth-instar R. prolixus. (A) Frontal ganglion (FG) with numerous immunoreactive cell bodies; (B) immunoreactive neurohaemal sites on the second (ABN2), third (ABN3) and fourth (ABN4) abdominal nerves. In B, note that abdominal nerves two and three contain more elaborate immunoreactive neurohaemal sites. Scale bar, 100 µm.

View larger version (63K): [in a new window]   Fig. 5. Time-course immunohistochemical analysis of the ventral paired medial neurosecretory cells in the MTGM of fifth-instar R. prolixus. Immunohistochemical analysis was conducted on a group of animals that were either fed for 20 min on rabbit's blood (hatched bars) or not fed (white bars). (A–G) PRXamide-like immunoreactivity (PRXa-LI) was examined at 1, 2, 3, 4, 5, 24 and 48 h post feeding, respectively. (H) Confocal image of the ventral paired median neurosecretory cells showing the labelling scheme utilized in A–G. Scale bar, 50 µm. *PRXa-LI that differs significantly from controls (unfed) (P<0.05, ANOVA and Tukey post-test).

View larger version (17K): [in a new window]   Fig. 6. Dose–response curve demonstrating Mas-CAPA-1 inhibition of secretion by Malpighian tubules stimulated with 50 nmol l–1 5-HT. Control tubules received 50 nmol l–1 5-HT. Values are mean ± s.e.m., N=8 or more tubules.

View larger version (22K): [in a new window]   Fig. 7. Inhibition of secretion (stimulated with 50 nmol l–1 5-HT) with increasing doses of Fraction 25 from RP-HPLC. Values are expressed as a percentage of control (normal secretion of tubules stimulated with 50 nmol l–1 5-HT). Values are mean ± s.e.m., N=8 or more tubules. *Statistically significant inhibition (P<0.001).

View larger version (29K): [in a new window]   Fig. 8. Change in levels of intracellular cGMP in tubules stimulated with 5-HT alone or in combination with Mas-CAPA-1 (500 nmol l–1) or Fraction 25 (F25; 10 CNS equivalents) vs saline alone. 5-HT lowers cGMP levels, and these levels can be restored to those with saline alone or above by Mas-CAPA-1 or Fraction 25 (*significantly different from 5-HT alone at P<0.05). In addition, levels of intracellular cGMP in tubules stimulated with Fraction 25 were also significantly higher () than tubules stimulated with Mas-CAPA-1 or saline alone (P<0.05).