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First published online January 18, 2008
Journal of Experimental Biology 211, 382-390 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.013771
Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus
1 Department of Biology, University of Toronto at Mississauga, 3359 Mississauga
Road, Mississauga, Ontario, Canada, L5L 1C6
2 Department of Biochemistry, University of Nevada, Reno, NV 89557, USA
* Author for correspondence (e-mail: victoria.tebrugge{at}utoronto.ca)
Accepted 30 October 2007
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: amino acid, Rhodnius prolixus, calcitonin, diuretic hormone
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
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 (CAP2b)-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
DH31-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).
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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 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-DH31-like immunoreactive
processes were also observed over the anterior dorsal vessel, dorsal hindgut
and the salivary glands. Immunohistochemical analysis 1 h after feeding
demonstrated a reduction in the staining intensity of processes on both the
abdominal nerves and dorsal hindgut, suggesting that the DH31-like
material may be released during the rapid 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
other insects, 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 this increase in secretion by
DH31-like peptides is through a cyclic AMP-dependent pathway
(Coast et al., 2001;
Coast et al., 2005). It is
possible that the native R. prolixus DH31 is sufficiently
different from the other peptides such that the non-native peptides could be
relatively inactive in R. prolixus. It is also possible that
stimulation of Malpighian tubule secretion is not the main role for a
DH31-like peptide in R. prolixus, and that it plays other
roles during diuresis, such as modifying the ionic composition of the urine,
gut contraction or heart contraction. The presence of
Dippu-DH31-like immunoreactivity on the hindgut and the anterior
dorsal vessel and its potential release as a neurohormone suggest a role for
DH31-like peptides 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 appreciate the cocktail of hormones (neuropeptides and amines) involved, we have quantified, analysed the distribution of, isolated and sequenced a native DH31, Rhopr/Dippu-DH31. We have tested this peptide for its possible activation of cyclic AMP pathways in Malpighian tubules, and also shown that Rhopr/Dippu-DH31 has potential roles in other tissues involved in rapid post-feeding diuresis.
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MATERIALS AND METHODS |
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Tissue collection
CNS, whole or parts, from 5th instar male and female R. prolixus
were 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 acidified methanol were frozen at
–20°C, and later thawed, sonicated and centrifuged at 8800
g for 10 min. The supernatant was decanted and dried in a
SpeedVac (Savant, Farmingdale, NY, USA) and frozen at –20°C until
use.
DH31 enzyme-linked immunosorbant assay
The competitive DH31 enzyme-linked immunosorbant assay (ELISA)
employed the same affinity-purified antibody as was used for
immunohistochemistry and the direct ELISA described previously
(Te Brugge et al., 2005). In
brief, the procedure used 100 µl 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 to each 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 times with 200 µl per well of washing
buffer (0.05% Tween 20 in HBS, pH 7.4). The contents of the plate were then
discarded and the plate blocked by adding 100 µl of 5% normal goat serum in
HBS to each well 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 µl–1
Dippu-DH31), samples and alkaline phosphatase
[Cys-32]Dippu-DH31 conjugate (1:2000) were made up in HBS with 0.1%
bovine serum albumin (BSA). Standards and samples, run in duplicate, were
added in a volume of 50 µl followed by the addition of an equal volume of
the DH31 alkaline phosphatase conjugate to each well. The plate was
covered and incubated at room temperature for 3 h on the flatbed shaker.
Subsequently, the contents of the plate were discarded and the plate was
washed three times with 200 µl of washing buffer and then blotted. To each
well, 100 µl of p-nitrophenyl phosphate (pNPP) liquid substrate system
(Sigma-Aldrich, St Louis, MO, USA) was added, and the plate covered and then
placed on a flatbed shaker for 1.5 h at room temperature. The plate was read
in a Molecular Devices Spectramax microplate reader at 405 nm (Molecular
Devices, Sunnyvale, CA, USA).
Tissue collection for DH31 purification
Four-hundred 5th instar R. prolixus CNS were dissected under
physiological saline. The tissues were extracted in batches of 50 in 1 ml of
ice-cold acidified methanol. The tissues in acidified methanol were frozen at
–20°C, then later thawed, sonicated and centrifuged at 8800
g (10 000 r.p.m.) for 10 min. The supernatant was decanted and
dried in a SpeedVac. These tissue extracts were brought up in 0.1%
trifluoroacetic acid (TFA; BDH, Toronto, ON, Canada) in water and applied to a
C18 Sep-Pak cartridge (Waters Associates, Mississauga, ON, Canada)
that had previously been equilibrated with sequential applications of 8 ml
methanol, then 8 ml water, then 8 ml water containing 0.1% TFA, and finally 2
ml of 0.1% TFA in water with 10 µg of protease-free BSA (Sigma-Aldrich).
The cartridge was then washed sequentially with 8.0 ml each of water with 0.1%
TFA then 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 neuropeptide families, since our interests extend
beyond DH31 alone. The collected extracts 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 two batches
containing 150 and 250 CNS equivalents. Two RPLC purification steps were used
and are described below.
System A
The dried eluents were brought into solution in 9% acetonitrile (Burdick
and Jackson) with 0.1% TFA and filtered using a 0.22 µm Spin-X® filter
(Corning). The filtrate was then applied to an RPLC system 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 B
Active fractions, as determined by DH31 ELISA, were further
purified through an RPLC system that utilised a Brownlee Spheri 5 phenyl
column (Mandel/Alltech). The fractions were brought into solution in 1 ml of
18% acetonitrile with 0.1% TFA. The RPLC gradient was from 18% to 60%
acetonitrile over 60 min (0.7% acetonitrile per min) collected in 1 ml
fractions. The fractions were aliquoted, dried and stored at –20°C
until use.
Mass spectrometry and Edman degradation sequencing
The isolated sample with DH31-like immunoreactivity from 150 CNS
was dried by SpeedVac and analysed by the Centre of Advanced 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-TOF analysis of specific molecular
weights were subjected to further analysis using tandem mass spectrometry
(MALDI-TOF MS/MS) and trypsin digestion.
The second batch of 250 CNS were dried and processed on RPLC in a similar manner, with the DH31-like immunoreactive fraction further subjected to molecular mass determination and sequencing. This sample was dissolved in 1% aqueous TFA with sonication and then injected onto a 3.5 µm Zorbax 300SB C8 column, 2.1 mm x 100 mm, using a 5 ml loop (Rheodyne Model 7125, Rohnert Park, CA, USA) fitted to a Hewlett Packard Model 1050 chromatograph. The sample was eluted with a gradient starting 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 at 280 nm, and aliquots analysed by MALDI-TOF MS using an Applied Biosystems Model 4700 spectrometer (Framingham, MA, USA). Aliquots of peaks of interest were then digested with trypsin and fragments were analysed by tandem MS (Applied Biosystems Model 4700 spectrometer). Approximately half of the intact peptide was submitted for sequence analysis by Edman degradation using an Applied Biosystems Procise Model 494 sequencer (Foster City, CA, USA).
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The cyclic AMP content of the supernatant was measured using an RIA kit
(Perkin Elmer, Woodbridge, ON, Canada) with modifications as previously
described (Lange and Orchard,
1986). The means ± s.e.m. were calculated and compared
using Student's unpaired t-test (N=5–10).
DH31-like immunoreactivity of the R. prolixus 5th instar dorsal diaphragm and heart
Fifth instar R. prolixus were placed dorsal surface down into
periphery wax and dissected under physiological saline from the ventral
surface. Cuticle from ventral abdominal segments 1–7 was removed, as was
the ventral diaphragm, gut and Malpighian tubules. This exposed the dorsal
vessel and heart without damaging the alary muscles or the dorsal diaphragm.
Dorsal abdominal segments posterior to segment 5 were transferred to a
solution of 2% paraformaldehyde in Millonig's buffer. Immunohistochemistry was
performed as described previously (Tsang
and Orchard, 1991; Te Brugge
et al., 2005), with only minor modifications. The primary
antiserum solution utilised the affinity-purified rabbit anti-DH31
at 1:500 in 0.4% Trition X-100 with 10% normal sheep serum and incubation was
carried out at 4°C for 48 h. Secondary antibody was Cy3-labelled sheep
anti-rabbit immunoglobulin at 1:200 dilution (Sigma-Aldrich). In order to show
more clearly the alary muscles, heart and dorsal vessel, some fixed
preparations were exposed to a solution containing phalloidin conjugated to
TRITC (Sigma-Aldrich) at a concentration of 1:1000 in PBS for 0.5 h at room
temperature, followed by a wash in PBS for 18 h at 4°C. All preparations
were mounted on slides in glycerol and viewed with a confocal microscope
(Zeiss, LSM 510).
Heart assay
Fifth instar R. prolixus were placed dorsal surface down into
periphery wax and dissected under physiological saline from the ventral
surface, as described above. Segments 5–10 of the dorsal cuticle were
removed and transferred to a Sylgard (Paisley Products, Scarborough, ON,
Canada)-coated dish, covered with 100 µl of physiological saline and
secured by minutin pins. Electrodes from an impedance converter (UFI model
2991, Morro Bay, CA, USA) were placed on either side of the anterior-most pair
of the heart's alary muscles. Contractions of the heart, detected by the
impedance monitor (set on alternating current, AC, short), were recorded on a
chart recorder (Linear 1200; VWR, Mississauga, ON, Canada). The tissue was
equilibrated in 100 µl of saline for 20 min at room temperature, during
which the surrounding fluid was replaced with fresh saline every 5 min. After
this period, the saline was removed and replaced with an equal volume of
either saline or test solutions. The Rhopr/Dippu-DH31 was tested at
concentrations ranging from 10–12 to 10–6
mol l–1 (N=5–9).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Dilutions of the R. prolixus CNS extracts paralleled the
Dippu-DH31 standard curve (Fig.
1A). Fifth instar R. prolixus CNS contained
1.2±0.17 pmol of DH31-like material. 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 of the Dippu-DH31-like activity
(595±45 and 325±54 fmol, respectively;
Fig. 1B). Smaller amounts of
DH31-like material were measured in the suboesophageal ganglion
(SOG) and prothoracic ganglion (PRO) and in the neurohaemal areas of the CC
and abdominal nerves (ABN).
RPLC purification of R. prolixus DH31-like material
DH31-like material extracted from 150 R. prolixus CNS
and separated by using RPLC over a C18 column (system A) eluted in
two major peaks of activity; one in fractions 27, 28 and 29 (40.5–45%
acetonitrile) and a second much smaller peak in fractions 36, 37 and 38
(54–58.5% acetonitrile; Fig.
2A). The DH31-like immunoreactive material in the
largest peak, fraction 28, was dried in a SpeedVac and run again on RPLC using
a phenyl column (system B). These fractions were collected, and aliquots
tested in the DH31 ELISA. DH31-like material eluted from
the phenyl 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 eluted at a similar time and
percentage acetonitrile to the batch of 150 CNS in both the C18 and
phenyl RPLC runs. An estimated 85.6 pmol of DH31-like material was
found in fraction 40 from this run. Known DH31-like peptides were
run on system B after the R. prolixus samples. The D.
melanogaster (Drome-DH31) standard eluted 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 and Edman degradation
An aliquot of fraction 40 from the phenyl RPLC run of 150 CNS was analysed
by MALDI-TOF MS and shown to contain a mass corresponding to the mass of
Dippu-DH31. This peak was further analysed by MALDI-TOF MS/MS and
trypsin digestion; the results were consistent with a peptide identical to
Dippu-DH31 except for the inability to distinguish between leucine
and isoleucine.
Fraction 40 from the second batch of 250 CNS tissues was further separated by RPLC using a Zorbax C8 column and peaks were collected based on their absorbance at 280 nm and aliquots analysed by MALDI-TOF MS. A peak eluting at 29.4 min had MH+ 2986.61 (Fig. 3). A portion of the sample was digested with trypsin and fragments were analysed by tandem MS; a number of fragment peptides were observed with MH+ of 830.48 (residues 1–8), 852.43 (residues 9–17), 1341.69 (residues 18–31), 1663.90 (residues 1–17) and 2176.10 (residues 9–31). The intact peptide was submitted for Edman degradation sequencing and was found to have the sequence GLDLGLSRGFSGSQAAKHLMGLAAANYAGGP, a result identical to the sequence for Dippu-DH31. The C-terminal residue was identified as Pro-amide based on the observed Mr.
Thus, this R. prolixus peptide, Rhopr-DH31, is identical to Dippu-DH31.
Cyclic AMP assays
The cyclic AMP content of the Malpighian tubules incubated with saline,
Rhopr/Dippu-DH31 or serotonin was measured in the presence or
absence of IBMX. There was no significant increase in the cyclic AMP content
of the Malpighian tubules incubated with 10–6 or
10–8 mol l–1 Rhopr/Dippu-DH31 for
1 min, 10 min or 10 min in IBMX, whereas 10–6 mol
l–1 serotonin significantly increased cyclic AMP under each
of these conditions (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).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), 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), while other insect
DH31-like peptides have a high degree of identity in their amino
acid sequences (Table 1). These
results suggest that the DH31 peptides are highly conserved across
orders of insects as well as between insects utilising various feeding
strategies, suggesting that this family of peptides plays an important role in
diuresis and possibly feeding. In species of insects other than R.
prolixus, only one form of DH31 peptide has been detected.
However, it is interesting that in R. prolixus a second, smaller peak
of DH31-like immunoreactive material elutes later in the RPLC
purification. Whether this represents a second DH31-like peptide or
represents cross-reactivity with another peptide must await further study. Quantification of the DH31-like material in the R. prolixus CNS demonstrated approximately 1.2 pmol per CNS of DH31-like immunoreactive material. The distribution of DH31-like immunoreactive material is consistent with the immunohistochemical staining of CNS neurons and processes, with the number of neurons highest in the brain and MTGM, and smaller amounts of DH31-like material in the putative neurohaemal sites of the CC and ABN. No other studies have quantified DH31 in insect CNS and so no comparisons can be made.
DH31-like immunoreactivity is co-localised with serotonin in the
DUM cells of the MTGM and in the associated neurohaemal terminals 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 to
feeding, with its titre increasing in the haemolymph during the first 5 min of
feeding (Lange et al., 1989).
Thus, it is intriguing that DH31-like immunoreactivity is
co-localised with serotonin in these DUM cells and terminals as it suggests
that Rhopr-DH31 is probably co-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 is therefore possible
that Rhopr/Dippu-DH31 can be released on its own, or in combination
with serotonin, suggesting that fine levels of control are possible.
Previously we have tested Dippu-DH31 on R. prolixus
Malpighian tubules (Te Brugge et al.,
2005). Since the sequence of the R. prolixus peptide is
identical to that of the cockroach peptide we can now confirm that the native
R. prolixus DH31 peptide does not stimulate the high rates
of secretion (1000-fold) seen for serotonin on R. prolixus Malpighian
tubules. However, Rhopr/Dippu-DH31 does stimulate a 14-fold
increase in secretion. It may, of course, also play other roles in Malpighian
tubules, such as modifying the ionic composition of the primary urine, but
this is yet to be tested.
The second messenger pathway for DH31-like peptides has been
studied 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 of cyclic AMP, while
cyclic GMP production is unchanged in the presence of IBMX.
Dippu-DH31 increases the cyclic AMP content of Schistocerca
americana tubules, but not tubules of Manduca sexta
(Furuya et al., 2000) or of
D. punctata (Tobe et al.,
2005). Interestingly, Rhopr/Dippu-DH31 does not elevate
the cyclic AMP content of R. prolixus tubules, despite being capable
of increasing secretion 14-fold. In this manner, R. prolixus tubules
act more like M. sexta and D. punctata tubules.
In previous studies DH31-like immunoreactivity was observed on
the anterior dorsal vessel and the hindgut suggesting that
Rhopr/Dippu-DH31 could be acting directly on these tissues, and/or
released from these terminals into the haemolymph
(Te Brugge et al., 2005).
Interestingly, Rhopr/Dippu-DH31 stimulates an increase in the
cyclic AMP content of the dorsal vessel and the hindgut but does not increase
the cyclic AMP content of the Malpighian tubules of R. prolixus. This
difference, in R. prolixus, between the Malpighian tubules, dorsal
vessel and hindgut in the production of cyclic AMP suggests the possibility of
a difference in the Rhopr/Dippu-DH31 receptors on the tubules
versus the dorsal vessel and hindgut, or possibly a difference in the
G-proteins that couple to the receptor for DH31.
DH31-like immunoreactivity was observed on the anterior portion
of the dorsal vessel, in processes that are putative neurohaemal release sites
and in processes that extend over the dorsal 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
moving haemolymph from the posterior to the anterior (or occasionally the
reverse) of the bug. This is potentially all the more important after
ingestion of the large blood-meal, since the expanded crop may confine
haemolymph to anterior or posterior parts of the engorged bug. The dorsal
vessel is capable of producing rates of flow approaching 4 µl
min–1 (Amaka Enh, personal communication) and so may play a
significant role in the circulation of haemolymph and hormones. Previous
studies have examined the contraction of the 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 frequency of
contractions in a dose-dependent manner, with cyclic AMP possibly acting as
the second messenger.
DH31-like immunoreactivity is also present over the dorsal
hindgut and this immunoreactivity is diminished in intensity 1 h after
feeding, suggesting that the DH31-like material in these terminals
had been released (Te Brugge et al.,
2005). The hindgut of R. prolixus plays an integral role
during rapid post-feeding diuresis. The Malpighian tubules empty their
contents into the hindgut through the ampullae. Contractions of the hindgut
are then necessary to expel the hindgut contents and are also potentially
important for mixing of both the hindgut contents and the surrounding
haemolymph. During rapid post-feeding diuresis the hindgut expels its contents
every few minutes. Since the hindgut of R. prolixus is where the
mature Trypanosome parasites reside prior to being expelled following
diuresis/excretion, it is of some considerable interest to understand the
factors that might affect this tissue. In unfed R. prolixus 5th
instars, the spontaneous contractions of the hindgut are highly variable in
frequency and nature, and respond to a variety of neuropeptides including
leucokinin (Te Brugge et al.,
2002; Sarkar et al.,
2003) and tachykinin (Kwok et
al., 2005) which both increase basal tonus, and increase the
amplitude and frequency of spontaneous contractions. In the present study,
Rhopr/Dippu-DH31 stimulated an increase in the frequency of hindgut
contractions, but did not cause a change in basal tonus. Again, in contrast to
Malpighian tubules, but in a manner similar to dorsal vessel,
Rhopr/Dippu-DH31 might act via cyclic AMP as a second messenger in
hindgut muscle. These results, taken together, suggest that
Rhopr/Dippu-DH31 may be playing an active role in modulating the
hindgut during the rapid post-feeding diuresis/excretion in R.
prolixus.
The DH31 family of peptides in insects is highly conserved and
is involved in ion and water balance as well as muscle contraction. The native
R. prolixus DH31, Rhopr/Dippu-DH31, would
appear to play an integral role in this diuresis, stimulating low levels of
secretion by the Malpighian tubules, and stimulating contractions of both the
heart and hindgut (tissues that are associated with the diuretic behaviour).
The interaction of Rhopr/Dippu-DH31 with other factors, in
particular serotonin, and its role in Malpighian tubules and the digestive
system will be of importance for future study; Dippu-DH31 is a
potent synergist of the action of the CRF-like DH Dippu-DH46 in
Diploptera punctata, and conversely Dippu-DH46 is a potent
synergist of Dippu-DH31 (Furuya
et al., 2000). Diuresis in R. prolixus appears to involve
complex and tightly integrated events that incorporate a variety of tissues
that are under neurohormonal/neuromodulatory control of both amines and
neuropeptides.
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Coast, G. M., Webster, S. G., Schegg, K. M., Tobe, S. S. and Schooley, D. A. (2001). The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J. Exp. Biol. 204,1795 -1804.[Abstract]
Coast, G. M., Garside, C. S., Webster, S. G., Schegg, K. M. and
Schooley, D. A. (2005). Mosquito natriuretic peptide
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Furuya, K., Milchak, R. J., Schegg, K. M., Zhang, J., Tobe, S.
S., Coast, G. M. and Schooley, D. A. (2000). Cockroach
diuretic hormones: characterization of a calcitonin-like peptide in insects.
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  G. M. Coast Neuroendocrine control of ionic homeostasis in blood-sucking insects J. Exp. Biol., February 1, 2009; 212(3): 378 - 386. [Abstract] [Full Text] [PDF]
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) 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 5th instar R. prolixus (male
and female). Abbreviations: CC, corpus cardiacum; SOG, suboesophageal
ganglion; PRO, prothoracic ganglion; MTGM, mesothoracic ganglionic mass; and
ABN, abdominal nerves.
