Peptides 26 (2005) 29–42 Presence and activity of a Dippu-DH 31 -like peptide in the blood-feeding bug, Rhodnius prolixus V.A. Te Brugge a,∗ , V.C. Lombardi b , D.A. Schooley b , I. Orchard a a Department of Biology, University of Toronto, Mississauga, Ont., Canada L5L 1C6 b Department of Biochemistry, University of Nevada, Reno, NV 89557 USA Received 28 July 2004; accepted 31 August 2004 Abstract The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a period of rapid diuresis which is under neurohormonal control. In both cockroach (Diploptera punctata) and fruit fly (Drosophila melanogaster) a calcitonin-like DH 31 neuropeptide has been identified [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795–804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469–74] and demonstrated to be active on Malpighian tubule secretion [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795–804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469–74]. Using an antibody raised against D. punctata (Dippu) DH 31 , we demonstrate the presence of Dippu-DH 31 -like immunoreactivity in the CNS, salivary glands, hindgut and neurohemal sites of 5th instar Rhodnius. Double-label immunohistochemistry for Dippu-DH 31 -like and serotonin-like immunoreactivity demonstrates some co-localization of these factors in cells of the mesothoracic ganglionic mass (MTGM) and in neurohemal sites on the abdominal nerves. When tested on Rhodnius 5th instar Malpighian tubules, Dippu-DH 31 stimulated minor increases in rate of secretion. Dippu-DH 31 tested in combination with serotonin resulted in increases in the rate of secretion which were at least additive. © 2004 Elsevier Inc. All rights reserved. Keywords: Calcitonin-like; DH 31 ; Diuretic hormone; Immunohistochemistry; Neurohemal; Diploptera punctata 1. Introduction The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a profound diuresis which is un- der neurohormonal control. We are interested in determining the identity of the neurohormones controlling diuresis, the timing of their release, their effect on the Malpighian tubules (and other tissues involved in diuresis) and their potential interactions. Previously we have demonstrated the presence of serotonin [6], corticotropin-releasing factor (CRF)-like [13] and kinin-like immunoreactivity [14] in the central ner- ∗ Corresponding author. Tel.: +1 905 569 4752; fax: +1 905 828 3792. E-mail address: vtebrugg@utm.utoronto.ca (V.A. Te Brugge). vous system (CNS) of 5th instar Rhodnius. These factors are present in neurohemal sites in the corpus cardiacum (CC) and in the abdominal nerves (ABN) and are released into the hemolymph after the start of feeding [7,16]. However, only serotonin [8] and CRF-like peptide(s) [15] have been shown to stimulate Rhodnius Malpighian tubules to increase their rate of secretion. Interestingly, serotonin is synergistic with extracts of the mesothoracic ganglionic mass (MTGM) [9,15] but not with non-native CRF-like peptides [15], suggesting that there may be another factor(s) present in the CNS that is active on the Malpighian tubules. In the cockroach Diploptera punctata, a calcitonin-like diuretic hormone, Dippu-DH 31 , has been isolated and se- quenced from extracts of brain and CC [3], and demonstrated 0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.08.025