382 INTRODUCTION Rhodnius prolixus is a blood-feeding hemipteran and is one of the species responsible for the transmission of Chagas’ disease. Rhodnius ingest enormous blood-meals and then undergo a period of rapid diuresis for 2–3·h during which they excrete approximately 40% of the volume of the blood-meal. This rapid, post-feeding diuresis, is under neurohormonal control and involves the coordination and integration of several processes and tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and muscle contractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood- meal, mixing of the haemolymph, as well as the expulsion of waste. Several factors have been suggested to play a role during this rapid diuresis, including serotonin (Lange et al., 1988; Maddrell et al., 1991; Maddrell et al., 1993), corticotropin-releasing factor (CRF)- like diuretic hormone (DH) (Te Brugge et al., 1999) and kinin-like peptides (Te Brugge et al., 2001). These factors stimulate Malpighian tubule secretion and/or muscle contractions of the gut during diuresis, while the cardioactive peptide 2b (CAP 2b )-like peptides decrease Malpighian tubule secretion in R. prolixus (Quinlan et al., 1997; Paluzzi and Orchard, 2006). Another family of peptides, the calcitonin-like DH, were first isolated and sequenced from extracts of brain and corpus cardiacum (CC) of the cockroach D. punctata (Furuya et al., 2000), and shown to stimulate secretion of Malpighian tubules from both D. punctata and Locusta migratoria (Furuya et al., 2000). These DH 31 -like peptides have now been predicted from the genome or sequenced from tissue extracts of several species including Drosophila melanogaster (Coast et al., 2001), Anopheles gambiae (Coast et al., 2005), Aedes aegypti, Bombyx mori, Apis mellifera, Tribolium castaneum and Formica polyctena (Schooley et al., 2005). These sequences display a high degree of identity to the D. punctata sequence. The T. castaneum, B. mori, D. melanogaster and A. gambiae sequences have 94, 74, 71 and 68% identity, respectively, to the amino acid sequence from D. punctata (Table·1), while the sequences from A. mellifera and F. polyctena (with the exception of the last two unknown amino acids) are identical to the cockroach sequence (Table·1). Using an affinity-purified antibody raised against Dippu-DH 31 in whole-mount immunohistochemistry, we found Dippu-DH 31 - like immunoreactivity in cell bodies and processes throughout the central nervous system (CNS) of 5th instar R. prolixus (Te Brugge et al., 2005). Specifically, immunoreactivity was observed in the medial and lateral neurosecretory cells of the brain, which send processes to the retrocerebral complex, and in dorsal unpaired median (DUM) neurons of the mesothoracic ganglionic mass (MTGM), which send processes to neurohaemal sites on the surface of the abdominal nerves. Dippu-DH 31 -like immunoreactive processes were also observed over the anterior dorsal vessel, dorsal hindgut and the salivary glands. Immunohistochemical analysis 1·h The Journal of Experimental Biology 211, 382-390 Published by The Company of Biologists 2008 doi:10.1242/jeb.013771 Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH 31 ) from Rhodnius prolixus Victoria A. Te Brugge 1, *, David A. Schooley 2 and Ian Orchard 1 1 Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada, L5L 1C6 and 2 Department of Biochemistry, University of Nevada, Reno, NV 89557, USA *Author for correspondence (e-mail: victoria.tebrugge@utoronto.ca) Accepted 30 October 2007 SUMMARY Diuresis in the blood-gorging hemipteran Rhodnius prolixus is under neurohormonal control and involves a variety of processes and tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and muscle contractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood-meal, mixing of the haemolymph, as well as the expulsion of waste. One of the neurohormones that might play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH 31 ) family of insect peptides. Previously we have demonstrated the presence of DH 31 -like peptides in the central nervous system (CNS) and gut of R. prolixus 5th instars. In the present work, a DH 31 from the CNS of 5th instar R. prolixus was isolated using reversed-phase liquid chromatography (RPLC), monitored with an enzyme-linked immunosorbent assay (ELISA) combined with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, and sequenced using tandem mass spectrometry and Edman degradation. This neuropeptide is the first to be sequenced in R. prolixus and has a sequence identical to that found previously for Dippu-DH 31 from the cockroach Diploptera punctata. In previous studies testing Rhopr/Dippu-DH 31 in Malpighian tubule secretion assays, we demonstrated increases in the rate of secretion that were small, relative to that induced by serotonin, but nevertheless 14-fold over baseline. In the present study, we investigated second messenger pathways in response to Rhopr/Dippu-DH 31 and found no increase or decrease in cyclic adenosine monophosphate (cyclic AMP) content of the Malpighian tubules. DH 31 -like immunoreactivity is present over the dorsal hindgut, anterior dorsal vessel and dorsal diaphragm, and bioassays of the R. prolixus dorsal vessel and hindgut indicate that Rhopr/Dippu-DH 31 increases the frequency of muscle contractions of both tissues. Second messenger pathways were also investigated for the dorsal vessel and hindgut. Key words: amino acid, Rhodnius prolixus, calcitonin, diuretic hormone. THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY