Amino acid sequence and biological activity of a calcitonin-like … · Forpo-DH 31 GLDLGLSRGFSGSQAAKHLMGLAAANY-AGXX Sequenced (Laenen, 1999) Trica-DH 31 GLDLGLGRGFSGSQAAKHLMGLAAANF-AGGP-NH

382

INTRODUCTIONRhodnius prolixus is a blood-feeding hemipteran and is one of thespecies responsible for the transmission of Chagas’ disease.Rhodnius ingest enormous blood-meals and then undergo a periodof rapid diuresis for 2–3·h during which they excrete approximately40% of the volume of the blood-meal. This rapid, post-feedingdiuresis, is under neurohormonal control and involves thecoordination and integration of several processes and tissues. Theseinclude ion and water movement across the epithelium of the cropand 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 rapiddiuresis, 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-likepeptides (Te Brugge et al., 2001). These factors stimulateMalpighian tubule secretion and/or muscle contractions of the gutduring diuresis, while the cardioactive peptide 2b (CAP2b)-likepeptides decrease Malpighian tubule secretion in R. prolixus(Quinlan et al., 1997; Paluzzi and Orchard, 2006).

Another family of peptides, the calcitonin-like DH, were firstisolated and sequenced from extracts of brain and corpus cardiacum(CC) of the cockroach D. punctata (Furuya et al., 2000), and shownto stimulate secretion of Malpighian tubules from both D. punctata

and Locusta migratoria (Furuya et al., 2000). These DH31-likepeptides have now been predicted from the genome or sequencedfrom tissue extracts of several species including Drosophilamelanogaster (Coast et al., 2001), Anopheles gambiae (Coast et al.,2005), Aedes aegypti, Bombyx mori, Apis mellifera, Triboliumcastaneum and Formica polyctena (Schooley et al., 2005). Thesesequences display a high degree of identity to the D. punctatasequence. 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 thesequences from A. mellifera and F. polyctena (with the exceptionof the last two unknown amino acids) are identical to the cockroachsequence (Table·1).

Using an affinity-purified antibody raised against Dippu-DH31

in whole-mount immunohistochemistry, we found Dippu-DH31-like immunoreactivity in cell bodies and processes throughout thecentral nervous system (CNS) of 5th instar R. prolixus (Te Bruggeet al., 2005). Specifically, immunoreactivity was observed in themedial and lateral neurosecretory cells of the brain, which sendprocesses to the retrocerebral complex, and in dorsal unpairedmedian (DUM) neurons of the mesothoracic ganglionic mass(MTGM), which send processes to neurohaemal sites on the surfaceof the abdominal nerves. Dippu-DH31-like immunoreactiveprocesses were also observed over the anterior dorsal vessel, dorsalhindgut and the salivary glands. Immunohistochemical analysis 1·h

The Journal of Experimental Biology 211, 382-390Published by The Company of Biologists 2008doi:10.1242/jeb.013771

Amino acid sequence and biological activity of a calcitonin-like diuretic hormone(DH31) from Rhodnius prolixus

Victoria A. Te Brugge1,*, David A. Schooley2 and Ian Orchard1

1Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada, L5L 1C6 and2Department of Biochemistry, University of Nevada, Reno, NV 89557, USA

*Author for correspondence (e-mail: [email protected])

Accepted 30 October 2007

SUMMARYDiuresis in the blood-gorging hemipteran Rhodnius prolixus is under neurohormonal control and involves a variety of processesand tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and musclecontractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood-meal, mixing of the haemolymph, as wellas the expulsion of waste. One of the neurohormones that might play a role in this rapid diuresis belongs to the calcitonin-likediuretic hormone (DH31) family of insect peptides. Previously we have demonstrated the presence of DH31-like peptides in thecentral nervous system (CNS) and gut of R. prolixus 5th instars. In the present work, a DH31 from the CNS of 5th instar R. prolixuswas 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 sequencedusing tandem mass spectrometry and Edman degradation. This neuropeptide is the first to be sequenced in R. prolixus and hasa sequence identical to that found previously for Dippu-DH31 from the cockroach Diploptera punctata. In previous studies testingRhopr/Dippu-DH31 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 secondmessenger pathways in response to Rhopr/Dippu-DH31 and found no increase or decrease in cyclic adenosine monophosphate(cyclic AMP) content of the Malpighian tubules. DH31-like immunoreactivity is present over the dorsal hindgut, anterior dorsalvessel and dorsal diaphragm, and bioassays of the R. prolixus dorsal vessel and hindgut indicate that Rhopr/Dippu-DH31

increases the frequency of muscle contractions of both tissues. Second messenger pathways were also investigated for thedorsal vessel and hindgut.

Key words: amino acid, Rhodnius prolixus, calcitonin, diuretic hormone.

THE JOURNAL OF EXPERIMENTAL BIOLOGY

383Rhodnius DH31 sequence and biological activity

after feeding demonstrated a reduction in the staining intensity ofprocesses on both the abdominal nerves and dorsal hindgut,suggesting that the DH31-like material may be released during therapid phase of diuresis (Te Brugge et al., 2005).

When tested on R. prolixus Malpighian tubules, Dippu-DH31

causes only small (relative to serotonin) but significant increases(14-fold) in the rate of secretion (Te Brugge et al., 2005). In otherinsects, such as D. punctata, L. migratoria and D. melanogaster,DH31 peptides stimulate larger increases in the rates of secretion(Furuya et al., 2000; Coast et al., 2001). In some insects thisincrease in secretion by DH31-like peptides is through a cyclicAMP-dependent pathway (Coast et al., 2001; Coast et al., 2005). Itis possible that the native R. prolixus DH31 is sufficiently differentfrom the other peptides such that the non-native peptides could berelatively inactive in R. prolixus. It is also possible that stimulationof Malpighian tubule secretion is not the main role for a DH31-likepeptide in R. prolixus, and that it plays other roles during diuresis,such as modifying the ionic composition of the urine, gutcontraction or heart contraction. The presence of Dippu-DH31-likeimmunoreactivity on the hindgut and the anterior dorsal vessel andits potential release as a neurohormone suggest a role for DH31-likepeptides on R. prolixus hindgut as well as other peripheral tissues,such as the dorsal vessel and heart.

In order to further our understanding of the overall feeding-related neuroendocrinological events in Rhodnius, and to appreciatethe cocktail of hormones (neuropeptides and amines) involved, wehave quantified, analysed the distribution of, isolated andsequenced a native DH31, Rhopr/Dippu-DH31. We have tested thispeptide for its possible activation of cyclic AMP pathways inMalpighian tubules, and also shown that Rhopr/Dippu-DH31 haspotential roles in other tissues involved in rapid post-feedingdiuresis.

MATERIALS AND METHODSAnimals

Fifth instars of R. prolixus (Stål 1859) were taken from a long-standing colony maintained at 25°C under 60% humidity. Theinsects were unfed, 6–8 weeks post-emergence, and previously fedon rabbit’s blood as 4th instars.

Tissue collectionCNS, whole or parts, from 5th instar male and female R. prolixuswere dissected under physiological saline (Lane et al., 1975).Tissues were extracted in 1·ml of ice-cold acidified methanol(methanol:acetic acid:water; 90:9:1). The tissues in acidifiedmethanol were frozen at –20°C, and later thawed, sonicated and

centrifuged at 8800·g for 10·min. The supernatant was decanted anddried in a SpeedVac (Savant, Farmingdale, NY, USA) and frozenat –20°C until use.

DH31 enzyme-linked immunosorbant assayThe competitive DH31 enzyme-linked immunosorbant assay(ELISA) employed the same affinity-purified antibody as was usedfor immunohistochemistry and the direct ELISA describedpreviously (Te Brugge et al., 2005). In brief, the procedure used100·�l of primary antibody solution (1:500 anti-[Cys-32]Dippu-DH31 in Hepes-buffered saline (HBS; 10·mmol·l–1 Hepes,150·mmol·l–1 NaCl, 1·mmol·l–1 MgCl2, pH 7.4), which was added toeach well of a 96-well ELISA plate (Corning easy wash®, Corning,NY, USA). The plate was covered and incubated overnight at 4°C.The antibody was then discarded and the plate washed three timeswith 200·�l per well of washing buffer (0.05% Tween 20 in HBS,pH 7.4). The contents of the plate were then discarded and the plateblocked by adding 100·�l of 5% normal goat serum in HBS to eachwell and incubating for 1·h at room temperature on a flatbed shaker.The contents were again discarded and the plate blotted. Blank, zero,standards (5 to 10·000·fmol·50·�l–1 Dippu-DH31), samples andalkaline phosphatase [Cys-32]Dippu-DH31 conjugate (1:2000) weremade up in HBS with 0.1% bovine serum albumin (BSA). Standardsand samples, run in duplicate, were added in a volume of 50·�lfollowed by the addition of an equal volume of the DH31 alkalinephosphatase conjugate to each well. The plate was covered andincubated at room temperature for 3·h on the flatbed shaker.Subsequently, the contents of the plate were discarded and the platewas washed three times with 200·�l of washing buffer and thenblotted. To each well, 100·�l of p-nitrophenyl phosphate (pNPP)liquid substrate system (Sigma-Aldrich, St Louis, MO, USA) wasadded, and the plate covered and then placed on a flatbed shaker for1.5·h at room temperature. The plate was read in a Molecular DevicesSpectramax microplate reader at 405·nm (Molecular Devices,Sunnyvale, CA, USA).

Tissue collection for DH31 purificationFour-hundred 5th instar R. prolixus CNS were dissected underphysiological saline. The tissues were extracted in batches of 50 in1·ml of ice-cold acidified methanol. The tissues in acidified methanolwere frozen at –20°C, then later thawed, sonicated and centrifugedat 8800·g (10·000·r.p.m.) for 10·min. The supernatant was decantedand dried in a SpeedVac. These tissue extracts were brought up in0.1% trifluoroacetic acid (TFA; BDH, Toronto, ON, Canada) inwater and applied to a C18 Sep-Pak cartridge (Waters Associates,Mississauga, ON, Canada) that had previously been equilibrated with

Table 1. Amino acid sequences (known or predicted) of DH31-like peptides from various insect species

Sequenced/Insect Peptide sequence predicted Reference

Dippu-DH31 GLDLGLSRGFSGSQAAKHLMGLAAANY-AGGP-NH2 Sequenced (Furuya et al., 2000)Apime-DH31

Rhopr-DH31

Forpo-DH31 GLDLGLSRGFSGSQAAKHLMGLAAANY-AGXX Sequenced (Laenen, 1999)Trica-DH31 GLDLGLGRGFSGSQAAKHLMGLAAANF-AGGP-NH2 PredictedBommo-DH31 AFDLGLGRGYSGALQAKHLMGLAAANF-AGGP-NH2 PredictedDrome-DH31 TVDFGLARGYSGTQEAKHRMGLAAANF-AGGP-NH2 Predicted (Coast et al., 2001)Anoga-DH31 TVDFGLSRGYSGAQEAKHRMAMAVANF-AGGP-NH2 Predicted (Coast et al., 2005)Aedae-DH31

Amino acids in bold are identical to those in the sequence of the cockroach Dippu-DH31.The sequences of A. mellifera DH31 (Apime-DH31) and R. prolixus DH31

(Rhopr-DH31) are identical with Dippu-DH31, and the sequence of A. aegypti DH31(Aedae-DH31) is identical with that of Anopheles gambiae (Anoga-DH31).Forpo, F. polyctena; Trica, T. castaneum; Bommo, B. mori; Drome, D. melanogaster.


384

sequential applications of 8·ml methanol, then 8·ml water, then 8·mlwater containing 0.1% TFA, and finally 2·ml of 0.1% TFA in waterwith 10·�g of protease-free BSA (Sigma-Aldrich). The cartridge wasthen washed sequentially with 8.0·ml each of water with 0.1% TFAthen 99.9% methanol (Burdick and Jackson, Muskegon, MI, USA)with 0.1% TFA, and the eluents collected. We chose to use 100%methanol in order to preserve all of the possible neuropeptidefamilies, since our interests extend beyond DH31 alone. The collectedextracts were dried in the SpeedVac and frozen at –20°C until use.

Reversed-phase high pressure liquid chromatography (RPLC)purification

The dried Sep-Pak eluents were further purified by RPLC in twobatches containing 150 and 250 CNS equivalents. Two RPLCpurification steps were used and are described below.

System AThe dried eluents were brought into solution in 9% acetonitrile(Burdick and Jackson) with 0.1% TFA and filtered using a 0.22·�mSpin-X® filter (Corning). The filtrate was then applied to an RPLCsystem that utilised a Brownlee C18 column (Mandel/Alltech,Guelph, ON, Canada). The RPLC gradient was from 9% to 60%acetonitrile with 0.1% TFA over 34·min (1.5% acetonitrile per min)at a flow rate of 1·ml·min–1. Fractions were collected, aliquoted,dried and stored at –20°C until use.

System BActive fractions, as determined by DH31 ELISA, were furtherpurified through an RPLC system that utilised a Brownlee Spheri5 phenyl column (Mandel/Alltech). The fractions were brought intosolution in 1·ml of 18% acetonitrile with 0.1% TFA. The RPLCgradient was from 18% to 60% acetonitrile over 60·min (0.7%acetonitrile per min) collected in 1·ml fractions. The fractions werealiquoted, dried and stored at –20°C until use.

Mass spectrometry and Edman degradation sequencingThe isolated sample with DH31-like immunoreactivity from 150CNS was dried by SpeedVac and analysed by the Centre ofAdvanced Protein Technology (Hospital for Sick Children,Toronto, ON, Canada). Samples were analysed using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF)mass spectrometry (Q-Star, Applied Biosystems Inc. Sciex,Concord, ON, Canada). Peaks observed in the MALDI-TOFanalysis of specific molecular weights were subjected to furtheranalysis using tandem mass spectrometry (MALDI-TOF MS/MS)and trypsin digestion.

The second batch of 250 CNS were dried and processed onRPLC in a similar manner, with the DH31-like immunoreactivefraction further subjected to molecular mass determination andsequencing. This sample was dissolved in 1% aqueous TFA withsonication and then injected onto a 3.5·�m Zorbax 300SB C8

column, 2.1·mm·� 100·mm, using a 5·ml loop (Rheodyne Model7125, Rohnert Park, CA, USA) fitted to a Hewlett Packard Model1050 chromatograph. The sample was eluted with a gradientstarting at 5% acetonitrile/0.1% TFA, increasing over 5·min to 20%acetonitrile/0.1% TFA, then increasing to 45% acetonitrile/0.1%TFA at 65·min. Peaks were collected based on their absorbance at280·nm, and aliquots analysed by MALDI-TOF MS using anApplied Biosystems Model 4700 spectrometer (Framingham, MA,USA). Aliquots of peaks of interest were then digested with trypsinand fragments were analysed by tandem MS (Applied BiosystemsModel 4700 spectrometer). Approximately half of the intact peptide

V. A. Te Brugge, D. A. Schooley and I. Orchard

was submitted for sequence analysis by Edman degradation usingan Applied Biosystems Procise Model 494 sequencer (Foster City,CA, USA).

Cyclic AMP assaysThe cyclic AMP content of the R. prolixus 5th instar Malpighiantubules, hindgut and dorsal vessel was assayed either with orwithout 3-isobutyl-1-methylxanthine (IBMX). Tissues from 5thinstars were dissected and then transferred to a microfuge tube. Theincubations with IBMX (0.5·mmol·l–1) were conducted in a totalvolume of 50·�l in physiological saline, or physiological salinecontaining serotonin (10–6·mol·l–1) or Rhopr/Dippu-DH31

(10–8·mol·l–1, 10–6·mol·l–1). Tissues were incubated for 10·min atroom temperature and then the reaction was stopped with 500·�lof boiling 0.05·mol·l–1 sodium acetate (pH 6.2). In assays where noIBMX was used the tissues were incubated in a volume of 50·�l atroom temperature for either 1 or 10·min. The assays were thenstopped with 250·�l of boiling sodium acetate. After the additionof the sodium acetate, samples were placed in a boiling water bathfor a further 5·min, then frozen at –20°C until assayed. The sampleswere thawed, sonicated and centrifuged at 8800·g for 10·min andthe supernatant decanted.

The cyclic AMP content of the supernatant was measured usingan RIA kit (Perkin Elmer, Woodbridge, ON, Canada) with

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Fig.·1. (A) DH31 ELISA standard curve (�) and the dilutions of the R.prolixus CNS (�). Note the log scale for Rhopr/Dippu-DH31.(B) Quantification of DH31-like immunoreactive material in the CNS of 5thinstar R. prolixus (male and female). Abbreviations: CC, corpus cardiacum;SOG, suboesophageal ganglion; PRO, prothoracic ganglion; MTGM,mesothoracic ganglionic mass; and ABN, abdominal nerves.



modifications as previously described (Lange and Orchard, 1986).The means ± s.e.m. were calculated and compared using Student’sunpaired t-test (N=5–10).

DH31-like immunoreactivity of the R. prolixus 5th instar dorsaldiaphragm and heart

Fifth instar R. prolixus were placed dorsal surface down intoperiphery wax and dissected under physiological saline from theventral surface. Cuticle from ventral abdominal segments 1–7 wasremoved, as was the ventral diaphragm, gut and Malpighiantubules. This exposed the dorsal vessel and heart without damagingthe alary muscles or the dorsal diaphragm. Dorsal abdominalsegments posterior to segment 5 were transferred to a solution of2% paraformaldehyde in Millonig’s buffer. Immunohistochemistrywas performed as described previously (Tsang and Orchard, 1991;Te Brugge et al., 2005), with only minor modifications. Theprimary antiserum solution utilised the affinity-purified rabbit anti-DH31 at 1:500 in 0.4% Trition X-100 with 10% normal sheep serumand incubation was carried out at 4°C for 48·h. Secondary antibodywas Cy3-labelled sheep anti-rabbit immunoglobulin at 1:200dilution (Sigma-Aldrich). In order to show more clearly the alarymuscles, heart and dorsal vessel, some fixed preparations wereexposed to a solution containing phalloidin conjugated to TRITC(Sigma-Aldrich) at a concentration of 1:1000 in PBS for 0.5·h atroom temperature, followed by a wash in PBS for 18·h at 4°C. Allpreparations were mounted on slides in glycerol and viewed witha confocal microscope (Zeiss, LSM 510).

Heart assayFifth instar R. prolixus were placed dorsal surface down intoperiphery wax and dissected under physiological saline from theventral surface, as described above. Segments 5–10 of the dorsalcuticle were removed and transferred to a Sylgard (PaisleyProducts, Scarborough, ON, Canada)-coated dish, covered with100·�l of physiological saline and secured by minutin pins.Electrodes from an impedance converter (UFI model 2991, MorroBay, CA, USA) were placed on either side of the anterior-most pairof the heart’s alary muscles. Contractions of the heart, detected bythe impedance monitor (set on alternating current, AC, short), wererecorded on a chart recorder (Linear 1200; VWR, Mississauga, ON,

Canada). The tissue was equilibrated in 100·�l of saline for 20·minat room temperature, during which the surrounding fluid wasreplaced with fresh saline every 5·min. After this period, the salinewas removed and replaced with an equal volume of either saline ortest solutions. The Rhopr/Dippu-DH31 was tested at concentrationsranging from 10–12 to 10–6·mol·l–1 (N=5–9).

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Fig.·2. Purification of DH31-like material from 150 R. prolixus CNS throughRPLC system A. The active fraction 28 was then run through system B.Aliquots were tested by a DH31 ELISA to determine DH31-likeimmunoreactive fractions. The elution times of Drome-DH31 and Dippu-DH31 are indicated by the asterisks.

2974.0 2986.6 2999.2 3011.8 3024.4 3037.0

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determined for Dippu-DH31. M,mass number; Z, atomic number.


386

Hindgut assayHindgut assays were conducted on isolated R. prolixus 5th instarhindguts maintained under physiological saline. The hindgut wasdissected along with a portion of the ventral cuticle from abdominalsegments 5–10 including the cuticle surrounding the anus. Theventral cuticle was used to secure the hindgut to a dish, coated withSylgard. The anterior end of the hindgut, at the junction with theposterior midgut, was tied by a fine silk thread to a miniature forcetransducer (Aksjeselskapet Mikro-elektronikk, Horten, Norway).The output from the transducer was connected to a data acquisitionsystem (Biopac MP 100 workstation, Biopac Systems Inc., SantaBarbara, CA, USA) for measuring changes in basal tonus as wellas phasic contractions. Tissues were equilibrated in 50·�l of salinefor 20·min, then the saline was removed and replaced with eithersaline or peptide solutions. Rhopr/Dippu-DH31 was tested atconcentrations ranging from 10–12 to 10–5·mol·l–1 (N=5–9).

RESULTSQuantification of DH31-like material in 5th instar R. prolixus

CNSThe DH31 ELISA is a competitive ELISA that is sensitive (able todetect) over a range of 25 to 1000·fmol Dippu-DH31. The sameantibody has been tested and shown to be specific for DH31-likepeptides with some cross-reactivity with vertebrate calcitonin-likepeptides (Te Brugge et al., 2005). Dilutions of the R. prolixus CNSextracts paralleled the Dippu-DH31 standard curve (Fig.·1A). Fifthinstar R. prolixus CNS contained 1.2±0.17·pmol of DH31-likematerial. There was no difference found in the amount of DH31-like material in the CNS of male and female 5th instar R. prolixus(data not shown). The brain and MTGM contained the majority ofthe Dippu-DH31-like activity (595±45 and 325±54·fmol,respectively; Fig.·1B). Smaller amounts of DH31-like material weremeasured in the suboesophageal ganglion (SOG) and prothoracicganglion (PRO) and in the neurohaemal areas of the CC andabdominal nerves (ABN).

RPLC purification of R. prolixus DH31-like materialDH31-like material extracted from 150 R. prolixus CNS andseparated by using RPLC over a C18 column (system A) eluted intwo major peaks of activity; one in fractions 27, 28 and 29(40.5–45% acetonitrile) and a second much smaller peak infractions 36, 37 and 38 (54–58.5% acetonitrile; Fig.·2A). TheDH31-like immunoreactive material in the largest peak, fraction 28,


was dried in a SpeedVac and run again on RPLC using a phenylcolumn (system B). These fractions were collected, and aliquotstested in the DH31 ELISA. DH31-like material eluted from thephenyl column in fraction 40 (approximately 42% acetonitrile;Fig.·2B). The estimated amount of DH31-like activity in fraction 40,using the DH31 ELISA, was approximately 34.6·pmol. The DH31

immunoreactive material purified from the batch of 250 CNS elutedat a similar time and percentage acetonitrile to the batch of 150CNS in both the C18 and phenyl RPLC runs. An estimated85.6·pmol of DH31-like material was found in fraction 40 from thisrun. Known DH31-like peptides were run on system B after the R.prolixus samples. The D. melanogaster (Drome-DH31) standardeluted from the column at 37.9·min (fraction 38) while the Dippu-DH31 standard eluted at 39.3·min (fraction 40).

DH31 analysis: MALDI-TOF MS, MALDI-TOF MS/MS andEdman degradation

An aliquot of fraction 40 from the phenyl RPLC run of 150 CNSwas analysed by MALDI-TOF MS and shown to contain a masscorresponding to the mass of Dippu-DH31. This peak was furtheranalysed by MALDI-TOF MS/MS and trypsin digestion; the resultswere consistent with a peptide identical to Dippu-DH31 except forthe inability to distinguish between leucine and isoleucine.

Fraction 40 from the second batch of 250 CNS tissues wasfurther separated by RPLC using a Zorbax C8 column and peakswere collected based on their absorbance at 280·nm and aliquotsanalysed by MALDI-TOF MS. A peak eluting at 29.4·min hadMH+ 2986.61 (Fig.·3). A portion of the sample was digested withtrypsin and fragments were analysed by tandem MS; a number offragment peptides were observed with MH+ of 830.48 (residues1–8), 852.43 (residues 9–17), 1341.69 (residues 18–31), 1663.90(residues 1–17) and 2176.10 (residues 9–31). The intact peptidewas submitted for Edman degradation sequencing and was foundto have the sequence GLDLGLSRGFSGSQAAKHLMGLAAAN -YAGGP, a result identical to the sequence for Dippu-DH31. The C-terminal residue was identified as Pro-amide based on the observedMr.

Thus, this R. prolixus peptide, Rhopr-DH31, is identical to Dippu-DH31.

Cyclic AMP assaysThe cyclic AMP content of the Malpighian tubules incubated withsaline, Rhopr/Dippu-DH31 or serotonin was measured in the

presence or absence of IBMX. Therewas no significant increase in thecyclic AMP content of the Malpighiantubules incubated with 10–6 or10–8·mol·l–1 Rhopr/Dippu-DH31 for1·min, 10·min or 10·min in IBMX,whereas 10–6·mol·l–1 serotoninsignificantly increased cyclic AMPunder each of these conditions

Table 3. Cyclic AMP content of dorsal vessel and hindgut

10·min incubation with IBMX 10·min incubation with IBMX(pmol per dorsal vessel) (pmol per hindgut)

Saline 2.5±0.65 3.1±1.26Serotonin (10–6·mol·l–1) 12.4±0.78* 5.6±1.31*Rhopr/Dippu DH31 (10–6·mol·l–1) 7.5±0.93* 7.7±1.09*

IBMX, isobutyl methylxanthine. *Significantly different from saline control (Studentʼs unpaired t test, P<0.05).

Table 2. Cyclic AMP content of Malpighian tubules

1 min incubation 10 min incubation 10 min incubation with IBMX(pmol per tubule set) (pmol per tubule set) (pmol per tubule set)

Saline 5.7±0.79 7.8±1.09 9.6±1.07Serotonin (10–6·mol·l–1) 9.3±0.84* 9.7±1.04* 18.0±1.07*Rhopr/Dippu-DH31 (10–8·mol·l–1) 7.6±0.51 6.1±1.16 9.7±0.65Rhopr/Dippu-DH31 (10–6·mol·l–1) 5.1±0.81 7.5±0.48 8.3±0.97

IBMX, isobutyl methylxanthine. *Significantly different from saline control values (Studentʼs unpaired t test, P<0.05).



(Table·2). Both Rhopr/Dippu-DH31 and serotonin did, however,increase the cyclic AMP content of dorsal vessel and of hindgut,when incubated for 10·min in the presence of IBMX (Table·3).

DH31-like immunoreactivity of the heart and alary musclesThe R. prolixus dorsal vessel and heart are located within the dorsaldiaphragm. The heart and alary muscles are confined to theposterior portion of the abdomen in segments 5–10 (Fig.·4A).Phalloidin staining of the dorsal diaphragm outlines the structureof the heart and alary muscles (Fig.·4B). The alary muscles areattached to both the ventral and dorsal cuticle in these segments.

The dorsal vessel (aorta) at the CC continues anteriorly towards thehead forming a hood-like structure over the CC. DH31-likeimmunoreactivity was seen over the anterior portion of the dorsalvessel, at the CC, and continuing along the dorsal vessel to theposterior end of the thorax (not shown). No DH31-likeimmunoreactivity was seen directly innervating the heart, alarymuscles or posterior dorsal vessel; however, there was DH31-likeimmunoreactivity in processes, derived from the abdominal nerves,over the dorsal diaphragm (Fig.·4C) that come in close proximityto the alary muscles and heart.

Heart bioassayContractions recorded with the impedance monitor were seen tostart with a contraction of the alary muscles, followed by therelaxation of the alary muscles combined with the contraction ofthe heart, and finally the contraction of the dorsal vessel (Fig.·5).The majority of the contractions under our experimental conditionswere anterograde, with no consistent pattern of retrogradecontractions observed in either saline or saline containingRhopr/Dippu-DH31. The mean frequency of contractions in salinewas 5.6±1.2·contractions·min–1 (N=7). Addition of Rhopr/Dippu-DH31 increased the frequency of contractions over the salinecontrols in a dose-dependent manner (Figs·6 and 7), with athreshold (lowest concentration to produce a significant increase infrequency) of approximately 10–11·mol·l–1.

Hindgut bioassayContractions of the R. prolixus hindgut are composed ofcontractions of circular and longitudinal muscles, resulting in acomplex and variable pattern of contraction (Fig.·8). The frequencyof contractions in R. prolixus saline was 2.3±0.3·contractions·min–1

Heart

Dorsal vessel

Alarymuscles

200 µmB

100 µmC

A

Fig.·4. Rhodnius prolixus dorsal cuticle with dorsal diaphragm, dorsalvessel and alary muscles. (A) Line diagram of dorsal abdominal segments.Box indicates the approximate location of images B and C. (B) Phalloidinstaining of the heart/dorsal vessel (filled arrow) and alary muscles (openarrow). (C) Dippu-DH31-like staining of the dorsal diaphragm and alarymuscles (open arrow).

a

b

c

Fig.·5. Trace of a single contraction of the heart alary muscles and thedorsal vessel. Deflections in the trace from the impedance converterindicate the contraction of the alary muscles (a), heart (b) and dorsalvessel (c).


388

(N=9). The variability appeared, in part, to be dependent on thefeeding state of the bug and the amount of residual material in thehindgut. Rhopr/Dippu-DH31 increased the frequency ofcontractions in a dose-dependent manner (Figs·8 and 9) with athreshold of 10–11·mol·l–1.

DISCUSSIONWe have isolated and sequenced a calcitonin-like DH31 peptidefrom the CNS of 5th instar R. prolixus. This is the first neuropeptideto be sequenced from the CNS of R. prolixus. The peptide isidentical to Dippu-DH31 sequenced from the cockroach D. punctata(Furuya et al., 2000), and is therefore called Rhopr/Dippu-DH31.Identical sequences have now been isolated and sequenced (D.punctata and R. prolixus) or predicted from genomes (bee), whileother insect DH31-like peptides have a high degree of identity intheir amino acid sequences (Table·1). These results suggest that theDH31 peptides are highly conserved across orders of insects as wellas between insects utilising various feeding strategies, suggestingthat this family of peptides plays an important role in diuresis andpossibly feeding. In species of insects other than R. prolixus, onlyone form of DH31 peptide has been detected. However, it isinteresting that in R. prolixus a second, smaller peak of DH31-likeimmunoreactive material elutes later in the RPLC purification.Whether this represents a second DH31-like peptide or representscross-reactivity with another peptide must await further study.

Quantification of the DH31-like material in the R. prolixus CNSdemonstrated approximately 1.2·pmol per CNS of DH31-likeimmunoreactive material. The distribution of DH31-likeimmunoreactive material is consistent with theimmunohistochemical staining of CNS neurons and processes, withthe number of neurons highest in the brain and MTGM, and smalleramounts of DH31-like material in the putative neurohaemal sites ofthe CC and ABN. No other studies have quantified DH31 in insectCNS and so no comparisons can be made.

DH31-like immunoreactivity is co-localised with serotonin in theDUM cells of the MTGM and in the associated neurohaemalterminals of the abdominal nerves (Te Brugge et al., 2005).


Serotonin is a true diuretic hormone in R. prolixus (Maddrell et al.,1991), released from these neurohaemal terminals in response tofeeding, with its titre increasing in the haemolymph during the first5·min of feeding (Lange et al., 1989). Thus, it is intriguing thatDH31-like immunoreactivity is co-localised with serotonin in theseDUM cells and terminals as it suggests that Rhopr-DH31 is probablyco-released with serotonin at the start of feeding (Te Brugge et al.,2005), and that it is a neurohormone associated with feeding.However, there are other neurons in the CNS that express DH31-like immunoreactivity but do not co-express serotonin. It istherefore possible that Rhopr/Dippu-DH31 can be released on itsown, or in combination with serotonin, suggesting that fine levelsof control are possible.

Previously we have tested Dippu-DH31 on R. prolixus Malpighiantubules (Te Brugge et al., 2005). Since the sequence of the R.prolixus peptide is identical to that of the cockroach peptide we can

10–6 mol l–1 Dippu-DH31



1 min

A

B

C

Fig.·6. Examples of heart bioassay for R.prolixus. The first 5·min of a recording insaline applied at the open arrow and thesecond 5·min in peptide applied at the filledarrow. (A) Rhopr/Dippu-DH31 tested at10–10·mol·l–1; (B) Rhopr/Dippu-DH31 testedat 10–8·mol·l–1; (C) Rhopr/Dippu-DH31 testedat 10–6·mol·l–1. Note the increase infrequency and amplitude of heartbeat.

10–12 10–11 10–10 10–9 10–8 10–7 10–6

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cent

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of s

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e co

ntro

l

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300

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Fig.·7. Heart bioassay dose–response curve as percentage increase in thefrequency of contractions over saline controls. Concentrations were testedbetween 10–12 and 10–6·mol·l–1 (N=5–9; note the log scale).



now confirm that the native R. prolixus DH31 peptide does notstimulate the high rates of secretion (1000-fold) seen for serotoninon R. prolixus Malpighian tubules. However, Rhopr/Dippu-DH31

does stimulate a 14-fold increase in secretion. It may, of course, alsoplay other roles in Malpighian tubules, such as modifying the ioniccomposition of the primary urine, but this is yet to be tested.

The second messenger pathway for DH31-like peptides has beenstudied in the Malpighian tubules of several species. In both D.melanogaster (Coast et al., 2001) and A. gambiae (Coast et al.,2005) the native DH31-like peptide increases the production ofcyclic AMP, while cyclic GMP production is unchanged in thepresence of IBMX. Dippu-DH31 increases the cyclic AMP contentof Schistocerca americana tubules, but not tubules of Manducasexta (Furuya et al., 2000) or of D. punctata (Tobe et al., 2005).Interestingly, Rhopr/Dippu-DH31 does not elevate the cyclic AMPcontent of R. prolixus tubules, despite being capable of increasingsecretion 14-fold. In this manner, R. prolixus tubules act more likeM. sexta and D. punctata tubules.

In previous studies DH31-like immunoreactivity was observed onthe anterior dorsal vessel and the hindgut suggesting thatRhopr/Dippu-DH31 could be acting directly on these tissues, and/orreleased from these terminals into the haemolymph (Te Brugge etal., 2005). Interestingly, Rhopr/Dippu-DH31 stimulates an increase

in the cyclic AMP content of the dorsal vessel and the hindgut butdoes not increase the cyclic AMP content of the Malpighian tubulesof R. prolixus. This difference, in R. prolixus, between theMalpighian tubules, dorsal vessel and hindgut in the production ofcyclic AMP suggests the possibility of a difference in theRhopr/Dippu-DH31 receptors on the tubules versus the dorsal vesseland hindgut, or possibly a difference in the G-proteins that coupleto the receptor for DH31.

DH31-like immunoreactivity was observed on the anteriorportion of the dorsal vessel, in processes that are putativeneurohaemal release sites and in processes that extend over thedorsal diaphragm close to the alary muscles, heart and dorsal vessel.No direct innervation of the alary muscles or heart was observed.The heart and dorsal vessel play an important role in movinghaemolymph from the posterior to the anterior (or occasionally thereverse) of the bug. This is potentially all the more important afteringestion of the large blood-meal, since the expanded crop mayconfine haemolymph to anterior or posterior parts of the engorgedbug. The dorsal vessel is capable of producing rates of flowapproaching 4·�l·min–1 (Amaka Enh, personal communication)and so may play a significant role in the circulation of haemolymphand hormones. Previous studies have examined the contraction ofthe heart and dorsal vessel of R. prolixus (Chiang et al., 1992;Sarkar et al., 2003). Interestingly, both serotonin (Chiang et al.,1992) and Rhopr/Dippu-DH31 (this study) increase the frequencyof contractions in a dose-dependent manner, with cyclic AMPpossibly acting as the second messenger.

DH31-like immunoreactivity is also present over the dorsalhindgut and this immunoreactivity is diminished in intensity 1·hafter feeding, suggesting that the DH31-like material in theseterminals had been released (Te Brugge et al., 2005). The hindgutof R. prolixus plays an integral role during rapid post-feedingdiuresis. The Malpighian tubules empty their contents into thehindgut through the ampullae. Contractions of the hindgut are thennecessary to expel the hindgut contents and are also potentiallyimportant for mixing of both the hindgut contents and thesurrounding haemolymph. During rapid post-feeding diuresis thehindgut expels its contents every few minutes. Since the hindgut ofR. prolixus is where the mature Trypanosome parasites reside priorto being expelled following diuresis/excretion, it is of someconsiderable interest to understand the factors that might affect this




60 s

A

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0.01 g

60 s

0.01 g

60 s

0.01 g

Fig.·8. Examples of hindgut muscle bioassay for R. prolixus. The first 5·minof a recording in saline applied at the open arrow, and the second 5·min inthe peptide applied at the filled arrow. (A) Rhopr/Dippu-DH31 tested at10–10·mol·l–1; (B) Rhopr/Dippu-DH31 tested at 10–8·mol·l–1; (C) Rhopr/Dippu-DH31 tested at 10–6·mol·l–1. Note the increase in frequency and the variablechange in amplitude.

10–12 10–11 10–10 10–9 10–8 10–7 10–6 10–5

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Fig.·9. Hindgut dose–response curve as percentage increase in thefrequency of contractions over saline controls. Concentrations were testedbetween 10–12 and 10–5·mol·l–1 (N=5–9; note the log scale).


tissue. In unfed R. prolixus 5th instars, the spontaneous contractionsof the hindgut are highly variable in frequency and nature, andrespond to a variety of neuropeptides including leucokinin (TeBrugge et al., 2002; Sarkar et al., 2003) and tachykinin (Kwok etal., 2005) which both increase basal tonus, and increase theamplitude and frequency of spontaneous contractions. In thepresent study, Rhopr/Dippu-DH31 stimulated an increase in thefrequency of hindgut contractions, but did not cause a change inbasal tonus. Again, in contrast to Malpighian tubules, but in amanner similar to dorsal vessel, Rhopr/Dippu-DH31 might act viacyclic AMP as a second messenger in hindgut muscle. Theseresults, taken together, suggest that Rhopr/Dippu-DH31 may beplaying an active role in modulating the hindgut during the rapidpost-feeding diuresis/excretion in R. prolixus.

The DH31 family of peptides in insects is highly conserved andis involved in ion and water balance as well as muscle contraction.The native R. prolixus DH31, Rhopr/Dippu-DH31, would appear toplay an integral role in this diuresis, stimulating low levels ofsecretion by the Malpighian tubules, and stimulating contractionsof both the heart and hindgut (tissues that are associated with thediuretic behaviour). The interaction of Rhopr/Dippu-DH31 withother factors, in particular serotonin, and its role in Malpighiantubules and the digestive system will be of importance for futurestudy; Dippu-DH31 is a potent synergist of the action of the CRF-like DH Dippu-DH46 in Diploptera punctata, and converselyDippu-DH46 is a potent synergist of Dippu-DH31 (Furuya et al.,2000). Diuresis in R. prolixus appears to involve complex andtightly integrated events that incorporate a variety of tissues thatare under neurohormonal/neuromodulatory control of both aminesand neuropeptides.

We thank Dr Yi-Min She (Advanced Protein Technology Centre, Hospital for SickChildren, Toronto Canada), Ms Rebekah Woolsey (Nevada Proteomics Center)and Jack Presley (UC Davis Molecular Structure Facility) for peptide analysis andJen Juno and Dewan Arefeen for assistance with the dorsal vessel and hindgutbioassays. This work was supported by the Natural Sciences and EngineeringResearch Council of Canada.

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V. A. Te Brugge, D. A. Schooley and I. Orchard390


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