|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
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
Distribution, activity and evidence for the release of an anti-diuretic peptide in the kissing bug Rhodnius prolixus
Department of Biology, University of Toronto at Mississauga, Mississauga, ON, L5L 1C6, Canada
* Author for correspondence (e-mail: jeanpaul.paluzzi{at}utoronto.ca)
Accepted 5 January 2006
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Key words: anti-diuresis, CAPA, CAP2b, neuropeptide, neurosecretory cells, immunohistochemistry, insect, Malpighian tubule, Rhodnius prolixus
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
;
Maddrell et al., 1993;
Te Brugge et al., 1999;
Te Brugge et al., 2005) acting
through the intracellular second messenger, cyclic 3',5'-adenosine
monophosphate (cAMP) (see Barrett and
Orchard, 1990; Montoreano et
al., 1990; Te Brugge et al.,
1999). Similar mechanisms controlling diuresis exist in other
insect species (see Coast et al.,
2002) but, in addition, in Drosophila melanogaster and
Manduca sexta, another second messenger, cyclic
3',5'-guanosine monophosphate (cGMP) has also been shown to be
involved in increasing the rate of tubule fluid secretion
(Skaer et al., 2002;
Davies et al., 1995).
Anti-diuresis in R. prolixus, or the cessation of diuresis, has
typically been considered to occur through a decrease in the levels of
diuretic hormones 3–4 h following feeding
(Maddrell, 1964). More recent
studies on R. prolixus have identified MasCAP2b (M. sexta
cardioactive peptide 2b) and cGMP as components of an anti-diuretic mechanism
(Quinlan et al., 1997).
Specifically, cGMP was identified as an intracellular second messenger to
MasCAP2b, and Malpighian tubule cGMP levels were shown to increase in response
to MasCAP2b and also as tubule secretion rates declined in vivo
(Quinlan et al., 1997). In
addition, application of cGMP to tubules elicited effects that were
antagonistic to the secretory effects of cAMP
(Quinlan and O'Donnell, 1998).
It has been proposed that cGMP activates a cAMP phosphodiesterase that
degrades cAMP, thus lowering the level of the second messenger that stimulates
diuresis (O'Donnell and Spring,
2000).
MasCAP2b (pELYAFPRVamide, recently renamed Mas-CAPA-1, see later) is a
cardioactive peptide first isolated in M. sexta
(Huesmann et al., 1995). It is
now known that MasCAP2b is a member of a family of peptides sharing the
C-terminal PRVamide motif (Loi and Tublitz,
2004). In the central nervous system (CNS) these include some
periviscerokinins (see Wegener et al.,
2002) and CAP2b-related peptides in D. melanogaster and
M. sexta (Kean et al.,
2002; Loi and Tublitz,
2004). In the periphery, the PRVamide motif is retained by M.
sexta pre-ecdysis-triggering hormone (MasPETH) from the peripheral
endocrine Inka cells. Some other related peptides have a C-terminal PRXamide
motif (where X=I, L, M or V). For example, in Lepidopteran species, these
include the pheromone biosynthesis activating neuropeptides (PBAN) (see
Teal et al., 1996) within the
central nervous system, and peripherally include ecdysis-triggering hormone
(ETH) (
itna
et al.,
2002).
Recent studies have isolated and sequenced the gene coding for MasCAP2b
(Loi and Tublitz, 2004). Owing
to its high degree of homology with the capability gene in D.
melanogaster (Kean et al.,
2002), it was named the Manduca CAPA gene
(Loi and Tublitz, 2004). This
gene encodes three propeptides, a CAP2b propeptide and two CAP2b-related
propeptides referred to as Mas-CAPA-1, Mas-CAPA-2 and Mas-pyrokinin-1
(Mas-PK-1), respectively (Loi and Tublitz,
2004). The capability gene in D. melanogaster
encodes three neuropeptides termed CAPA-1 and CAPA-2, which are CAP2b related,
while CAPA-3 is PBAN/PK related (Kean et
al., 2002). To avoid confusion, we will follow the more recent
nomenclature and subsequently refer to MasCAP2b as Mas-CAPA-1.
Given the recent finding suggesting a novel anti-diuretic mechanism in
R. prolixus involving a Mas-CAPA-1-like peptide and the intracellular
second messenger, cyclic GMP (Quinlan et
al., 1997), we sought to map the location of putative
Mas-CAPA-1-like immunoreactive cells and to seek evidence for an endogenous
Mas-CAPA-1-like neuropeptide in R. prolixus with anti-diuretic
properties. Here we describe the distribution of PRXamide-like immunoreactive
neurons and neurohaemal sites in R. prolixus using an antiserum
against MasPETH that recognizes Mas-CAPA-1. In addition, we provide evidence
for the presence of an endogenous Mas-CAPA-1-like factor from the central
nervous system (CNS) of R. prolixus that inhibits 5-HT-stimulated
diuresis and elevates cGMP levels in 5-HT-stimulated tubules.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Immunohistochemical staining
The insects were pinned ventral surface down, and the dorsal cuticle,
dorsal diaphragm and digestive tissue removed under physiological saline
(NaCl, 150 mmol l–1; KCl, 8.6 mmol l–1;
CaCl2, 2 mmol l–1; NaHCO3, 4 mmol
l–1; glucose, 34 mmol l–1; MgCl2,
8.5 mmol l–1; Hepes pH 7.0, 5 mmol l–1). The
nervous tissue was fixed in situ with 2% paraformaldehyde (pH 7.0) at
4°C overnight (16–18 h). Following fixation, the tissues were washed
and the CNS and short stretches of peripheral nerves removed under
phosphate-buffered saline (PBS) (Lange et
al., 1988). The nervous tissue was incubated in 4% Triton X-100,
2% bovine serum albumin (BSA) and 10% normal sheep serum (NSS) in PBS for 1 h
at room temperature followed by several washings with PBS. The polyclonal
rabbit antiserum to MasPETH (generously provided by Dr Dusan
itna and Dr Mike Adams) diluted 1:1000 was preincubated in a
0.4% Triton X-100, 2% bovine serum albumin (BSA) and 2% normal sheep serum
(NSS) in PBS at 4°C overnight (16–18 h) prior to use. The nervous
tissue was then incubated in the antiserum for 48 h on a flatbed shaker at
4°C. Following this, tissues were washed several times in PBS, including
an overnight washing at 4°C with shaking. Tissues were subsequently
incubated overnight (16–18 h) with Cy3-labelled sheep anti-rabbit
immunoglobulin G (IgG; Sigma-Aldrich, St Louis, MO, USA) diluted 1:200 with
10% NSS in PBS at 4°C with shaking and then washed numerous times at room
temperature. Tissues were mounted in glycerol on microscope slides and
observed under a Nikon epifluorescence microscope. PRXamide-like
immunoreactivity (PRXa-LI) was mapped with the aid of a drawing tube
attachment and images were obtained using confocal microscopy consisting of a
helium-neon laser (543 nm line) and Zeiss LSM Image Browser software.
Tissues from fed and unfed insects, of identical age, were compared for
intensity of staining, keeping settings on the confocal microscope constant.
All insects, fed or unfed, were kept at room temperature until they were
dissected. To ensure consistency in measurement of intensity of staining,
images of individual immunoreactive cell bodies were taken keeping the nucleus
of the cell in focus. Several post-feeding time points were compared to
insects that had been exposed to the rabbit but not allowed to feed. Staining
intensity was converted into grayscale values using the ImageJ Software
(Rasband, 2005) and then
subjected to statistical analyses including analysis of variance (ANOVA) and
Tukey post-test. Grayscale values of confocal images were analyzed over an
intensity scale from 0 to 255 (minimum to maximum intensity threshold for an
8-bit image, respectively).
Reverse phase high performance liquid chromatography (RP-HPLC)
Central nervous systems were dissected under saline and pooled in a 500
µl volume of methanol:acetic acid:water (90:9:1, by volume) and stored at
–20°C for later use. The CNS tissue from 250 insects was then
sonicated and centrifuged at 10 000 g for 10 min. The
supernatant was collected and dried in a Speed Vac concentrator (Savant,
Farmingdale, NY, USA) and then reconstituted in 0.1% trifluoroacetic acid
(TFA). This sample was then applied to a C18 Sep-Pak cartridge (Waters
Associates, Mississauga, ON, Canada) that had been sequentially equilibrated
with 8 ml of methanol, 8 ml ddH2O, 8 ml 0.1% TFA, and finally 5 ml
0.1% TFA containing 1 µg protease-free bovine serum albumin (BSA; Sigma,
Mississauga, ON, Canada). Once the sample was loaded, the cartridge was first
washed with 0.1% TFA and subsequently extracts were collected by eluting with
5 ml of 60% acetonitrile (ACN; Burdick and Jackson, Muskegon, MI, USA) with
0.1% TFA. The eluant was dried in a Speed Vac concentrator and then
resuspended in high performance liquid chromatography (HPLC) start buffer (9%
acetonitrile, 0.1% TFA) to be fractionated by reverse phase HPLC (RP-HPLC)
using a Brownlee C18 column (Mandel/Alltech, Guelph, ON, Canada) with a linear
gradient of 9–60% ACN over 34 min, beginning 5 min after injection.
Fractions with Mas-CAPA-1-like biological activity were identified by tubule
secretion assays and cGMP RIA.
Malpighian tubule fluid secretion assay
Rhodnius prolixus have four Malpighian tubules (two bilateral
pairs) composed of both upper and lower segments. Whole tubules from fifth
instars were dissected under saline and transferred on glass probes to a
Sylgard-coated Petri dish containing 20 µl drops of saline overlaid with
water-saturated mineral oil. Two tubules were mounted in each 20 µl bathing
droplet. The proximal end of the tubule was pulled out of the saline droplet
and wrapped around a nearby minuten pin. The equilibrating saline was removed
and replaced with saline containing 50 nmol l–1
5-hydroxytryptamine (5-HT; Sigma, Oakville, ON, Canada) alone or combined with
different concentrations of Mas-CAPA-1 (custom synthesized by GenScript Corp.,
Piscataway, NJ, USA) or CNS RP-HPLC fractions. Tubules were allowed to secrete
for 30 min. Droplets of secreted fluid from the nicked end of the tubule were
then collected using an oil-filled micropipette tip. The droplet was then
blown out under oil to be measured on the bottom of the Sylgard-coated Petri
dish. The droplet volume was calculated using the equation V=(
/6)d3, where d is the droplet diameter measured
using an eyepiece micrometer. At the end of the experiment, a maximal rate of
secretion was established by stimulating with 1 µmol l–1
5-HT to check on the viability of the tubules. Values, expressed as mean
± standard errors of the mean (s.e.m.), were then subjected to
statistical analysis using Student's t-test.
Malpighian tubule cyclic GMP radioimmunoassay
Malpighian tubules were dissected under saline and tested as a set,
including all four tubules from fifth instars, and transferred to a
microcentrifuge tube containing saline, 50 nmol l–1 5-HT
alone or 50 nmol l–1 5-HT combined with either Mas-CAPA-1 or
CNS RP-HPLC fractions in a total volume of 50 µl. Tubules were incubated
for 10 min and the experiment terminated by adding 250 µl of boiling 50
mmol l–1 sodium acetate (pH 6.2). The incubation tubes were
then immediately placed in a boiling water bath for 5 min and then stored at
–20°C. To prepare the samples for the assay, tubes were thawed,
sonicated briefly on ice and centrifuged at 4°C for 10 min at 8800
g. The supernatant was then collected and assayed using a
cyclic GMP RIA kit (PerkinElmer/NEN, Boston, MA, USA). Assays were performed
according to the manufacturer's instructions except for some minor changes in
volumes and ratio of reagents.
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
|
Brain and retrocerebral complex
On the dorsal surface of the brain, two main groups of cells showed PRXa-LI
(Fig. 2A). The first group
consists of a bilateral pair of lateral neurosecretory cells (LNCs)
prominently identified in the border region of the optic lobe and brain, which
have processes projecting medially through the brain. A second group of cells
consists of five pairs of medial neurosecretory cells (MNCs) arranged as a
cluster along the boundary between the protocerebral lobes. Immunoreactive
varicosities were also present along the periphery of the protocerebral lobes
originating at the optic lobe/brain boundary and also present anterior to the
MNCs. Some immunoreactivity appeared to be associated with the corpus
cardiacum, however, extensive PRXamide-like immunoreactive processes were
present along the walls of the aorta, decreasing in intensity as they proceed
posteriorly (Fig. 2B).
|
The ventral surface of the brain contains two sets of bilaterally paired cells (Fig. 3A), which are located posterior to the LNCs. These cells project processes posteriorly. Varicosities similar to the pattern visible on the dorsal surface of the brain were also observed on the ventral surface of the brain. Immunoreactive processes were also seen in the recurrent nerve with numerous cell bodies staining for PRXa-LI in the frontal ganglion (Fig. 4A).
|
|
Prothoracic ganglion (PRO)
Processes originating from the SOG are observed in the dorsal prothoracic
ganglion (PRO). The medial processes continue through to the posterior of the
PRO whereas some lateral processes arborise in the central neuropile
(Fig. 2C). On the ventral
surface of the PRO, some faint PRX-amide immunoreactive staining was observed
in the central neuropile (Fig.
3C).
Mesothoracic ganglionic mass (MTGM)
On the dorsal surface of the mesothoracic ganglionic mass (MTGM), a small
number of cell bodies showed faint PRXa-LI in both the mesothoracic and the
abdominal neuromeres (Fig. 2D).
Specifically, in the mesothoracic neuromere there were two cells (bilaterally
paired) with posteriorly projecting processes. In the abdominal neuromeres,
there are four cells (two bilaterally paired) along the lateral margins with
processes projecting medially. The processes originating from the SOG and
passing through the PRO continue into the MTGM where they arborise in the
metathoracic neuromere. Processes from three pairs of strongly staining cell
bodies located on the ventral MTGM project dorsally and then posteriorly
continue into the second, third and fourth abdominal nerves that stem from the
abdominal neuromeres in the posterior MTGM
(Fig. 2D,
Fig. 3D). These immunoreactive
processes form extensive neurohaemal sites over the proximal portion of the
nerves and distally continue as fine processes
(Fig 3D,
Fig. 4B).
Cell bodies with variable PRXa-LI were observed on the ventral surface of the MTGM. Beginning anteriorly, there was a ventral unpaired medial (VUM) neuron within the mesothoracic neuromere, which had strong PRXa-LI. Moving posteriorly, within the metathoracic neuromere, a bilateral pair of cells showed weak PRXa-LI. Finally, within the midline of the abdominal neuromeres, the six (three bilaterally paired) strongly staining cells, referred to earlier, were consistently seen with their processes projecting dorsally and then posteriorly out of corresponding abdominal nerves. Of this strongly staining group, the most anterior pair projects through the second abdominal nerve, whereas the second pair project to the third abdominal nerves, and the last strongly staining pair, and most posterior, project processes through the fourth abdominal nerves. Each cell of the most posterior pair have a diameter of 29 µm, which is considerably larger than the two more anterior pairs of cells (16 µm).
Time-course immunohistochemical analysis
Following a blood meal, extensive changes in the intensity of staining of
PRXamide-like cells and processes are observed. Specifically, over the MTGM,
the staining of the six strongly staining cells within the abdominal
neuromeres become weaker in intensity, beginning as early as 3 h following a
blood meal (Fig. 5). These
cells project into the abdominal nerves, and form neurohaemal sites,
suggesting a location for release into the haemolymph. Changes in PRXa-LI
post-feeding were analyzed by evaluating staining intensity of the six
strongly staining cell bodies over the MTGM using the ImageJ software package.
No significant changes in staining intensity were seen in unfed control
animals across all time points analyzed. In contrast, staining intensity
appears to weaken as early as 3 h following feeding (see
Fig. 5). As time post-feeding
progresses, significant decreases in staining intensity were observed.
Interestingly, the largest and most posterior pair of cells regained staining
before the two smaller and more anterior pairs of cells. This last pair of
cells project processes into the fourth abdominal nerves, which has notably
fewer neurohaemal sites than the second and third abdominal nerves.
Nonetheless, the intensity of staining of the neurohaemal sites and processes
over the abdominal nerves was visibly assessed and was reduced at the same
time points.
|
Malpighian tubule fluid secretion assay
To better characterize the anti-diuretic mechanism in R. prolixus,
we tested various concentrations of Mas-CAPA-1 on 5-HT-stimulated tubules and
observed a dose-dependent inhibition on tubule secretion
(Fig. 6). Threshold was
observed at approximately 0.1 nmol l–1 Mas-CAPA-1 and maximal
inhibition at a dose of 1 µmol l–1 Mas-CAPA-1. In order to
provide further empirical evidence for the presence of a Mas-CAPA-1-like
neuropeptide in R. prolixus, we tested individual fractions from
RP-HPLC against 5-HT-stimulated tubules and identified an anti-diuretic
fraction. This fraction (fraction 25) ran in close proximity to synthetic
Mas-CAPA-1, which eluted from the C18 column at 24.5 min (an acetonitrile
concentration of 38.25%). Tubules incubated in this fraction, in the presence
of 50 nmol l–1 5-HT, showed a dose-dependent decrease in
secretion rate (Fig. 7). Thus,
tubules stimulated with 50 nmol l–1 5-HT and this
anti-diuretic fraction from 1 CNS equivalent inhibited secretion by 11%, from
5 CNS equivalents by 27% and from 10 CNS equivalents by 74%. The ability of a
single CNS equivalent to decrease secretion by only 11% could be due to the
combined influence of losses associated with sonication of tissues and
preparatory steps prior to HPLC as well as impurity of the factor. Since CNS
extracts were run only through a single column, other factors eluting within
this fraction could be contributing to the biological activity observed.
|
|
Malpighian tubule cyclic GMP radioimmunoassay
To further understand the mechanism of action of this endogenous
Mas-CAPA-1-like anti-diuretic neuropeptide in R. prolixus, cyclic GMP
radioimmunoassays were conducted on fifth instars to confirm the previous
observation of an elevation of intracellular cGMP in response to Mas-CAPA-1 in
third-instar tubules stimulated with 5-HT. 5-HT (50 nmol l–1)
lowered cGMP levels of fifth-instar tubules and these levels were restored to
control values by Mas-CAPA-1 at 500 nmol l–1
(Fig. 8). Similarly, fraction
25 at 10 CNS equivalents also increased the cGMP levels of 5-HT-stimulated
tubules (Fig. 8). The levels of
cGMP in tubules stimulated with fraction 25, in the presence of 50 nmol
l–1 5-HT, were found to be significantly higher than
unstimulated tubules, suggesting that the actions of this endogenous
Mas-CAPA-1-like factor involve augmenting levels of intracellular cGMP.
|
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
itna et al.,
2003)], we have demonstrated the presence of cell bodies and
processes having PRXa-LI throughout the central and peripheral nervous system
of fifth-instar R. prolixus. Several insect neuropeptides sharing
this common C-terminal motif have been identified and the CAPA gene codes
three extended PRXamides, including CAPA-1 (a PRVamide). Of these closely
related neuropeptides, the PRXa-LI observed in fifth-instar R.
prolixus closely resembles the immunocytochemical localization of the
CAPA gene peptides in D. melanogaster
(Kean et al., 2002). More
specifically, the strongly immunoreactive ventral medial cells over the
abdominal neuromeres in the posterior MTGM closely resemble three pairs of
abdominal neurosecretory cells in D. melanogaster, which stain for
the CAPA precursor protein, and thus provides evidence that these cell types
produce CAP2b-related peptides (Kean et
al., 2002). In addition, the distribution in R. prolixus
of PRXamide-like immunoreactive cells over the MTGM resembles CAPA
gene-expressing cells in the abdominal ganglia of larval and adult M.
sexta (Loi and Tublitz,
2004). Medial neurosecretory cells showing PRXa-LI over the dorsal
brain of fifth-instar R. prolixus resemble cells over the dorsal
brain of M. sexta larvae that express CAPA transcripts
(Loi and Tublitz, 2004). Taken
together, these similarities in immunoreactivity along with the abolition of
immunoreactivity following preincubation of the antiserum with Mas-CAPA-1,
suggests that the immunoreactivity observed indicates the presence of
Mas-CAPA-1-like neuropeptides in the CNS of R. prolixus. More
importantly, the medial ventral cell bodies in the MTGM project processes into
the abdominal nerves, well-known neurohaemal release sites
(Miksys and Orchard, 1994),
and thus provides a location for release of these peptides into the
haemolymph. Furthermore, the intensity of staining of immunoreactivity in
these cell bodies and their neurohaemal release sites is greatly reduced
3–4 h post feeding – a time when the cessation of diuresis is
observed (Maddrell, 1964),
suggesting the release of an anti-diuretic factor. We suggest that the strong PRXa-LI observed over the MTGM and abdominal nerves provides evidence for a CAPA-like neuropeptide in the CNS of R. prolixus, which includes a Mas-CAPA-1-like peptide. The presence of PRXamide-like immunoreactive cell bodies in addition to processes and neuropiles over the length of the CNS suggest additional roles as neurotransmitters and/or neuromodulators. Future studies will help elucidate whether the PRXamide-like peptide functions as a neurotransmitter and/or neuromodulator in R. prolixus. Certainly, the results indicate that the PRXamide-like neuropeptide in R. prolixus acts as a neurohormone since there is evidence of release from the MTGM and abdominal nerves as well as activity on a non-innervated visceral tissue (Malpighian tubules).
Previous analyses on third-instar R. prolixus tubules showed
dose-dependent effects of Mas-CAPA-1in the nanomolar range
(Quinlan et al., 1997). To
better understand the response of tubules to Mas-CAPA-1-like peptides in
fifth-instar R. prolixus, we tested a broad range of physiological
doses of Mas-CAPA-1 to determine the dose-dependency on isolated tubules. At
0.1 nmol l–1, the lowest dose tested, Mas-CAPA-1 caused a 5%
decrease in secretion. This neuropeptide had a maximal effect on tubules at 1
µmol l–1, inhibiting secretion by over 75%. A higher dose
(10 µmol l–1) of this neuropeptide was slightly less
effective at inhibiting fluid secretion, possibly indicating the beginning of
receptor desensitization.
Further evidence for the presence of a Mas-CAPA-1-like neuropeptide in R. prolixus sharing similar characteristics to Mas-CAPA-1 was revealed by bioassay of native material. Analysis of RP-HPLC fractions from 250 CNSs revealed a factor with anti-diuretic effects on Malpighian tubules stimulated with 5-HT. This factor eluted from the C18 column at a similar time and acetonitrile concentration to Mas-CAPA-1, suggesting that this factor shares similar chromatographic properties to Mas-CAPA-1. Doses as low as a single CNS equivalent were adequate in eliciting an anti-diuretic effect on tubules. Furthermore, tubules stimulated with higher doses of this factor demonstrated a greater inhibition of secretion, indicating the effects of this factor are dose dependent. To our knowledge, this is the first study to show direct evidence for the presence of an endogenous anti-diuretic factor in R. prolixus, which significantly inhibits 5-HT-stimulated secretion in a dose-dependent manner.
This same fraction elevated intracellular cyclic GMP levels in tubules
stimulated with 5-HT, indicating that this second messenger may be exploited
by the native Mas-CAPA-1-like anti-diuretic peptide in R. prolixus.
Moreover, this fraction not only reversed the effects of 5-HT on cGMP, but at
this dose also increased cGMP above its original saline control values. This
result implies that this factor is actively involved in the synthesis of
intracellular cGMP, which, as suggested previously, may involve the actions of
a guanylate cyclase belonging to the class of membrane-bound enzymes
(Quinlan et al., 1997).
Interestingly, Mas-CAPA-1 has been shown to increase the synthesis of nitric
oxide and cGMP leading to an increase in fluid production in D.
melanogaster tubules (Davies et al.,
1995; Davies et al.,
1997). Expression of the receptor for Mas-CAPA-1 in tubules has
been shown in a number of dipterans (see
Pollock et al., 2004). It is
interesting that there has been a divergence in signalling between these
organisms.
In conclusion, this study investigated the distribution of PRXa-LI
throughout the CNS of R. prolixus. It is probable that many of these
cells, especially those in the abdominal neuromeres, are Mas-CAPA-1-like
since: (1) preincubation of the antiserum with Mas-CAPA-1 peptide eliminated
all immunoreactivity within the CNS; (2) immunoreactivity was significantly
reduced beginning 3–4 h post-feeding in accordance with the time of
anti-diuretic behaviour (Maddrell,
1964), which suggests the release of an anti-diuretic peptide from
the putative neurohaemal release sites on the abdominal nerves; (3) this
study, as well as previous studies on third-instar R. prolixus, have
shown that Mas-CAPA-1 elicits an anti-diuretic effect on R. prolixus
tubules (Quinlan et al.,
1997); (4) tubule secretion assay utilizing CNS fractions from a
C18 HPLC run identified a factor with Mas-CAPA-1-like biological activity,
which inhibits 5-HT-induced tubule secretion; lastly, (5) this same RP-HPLC
fraction containing an anti-diuretic factor was also effective at increasing
levels of intracellular cGMP in Malpighian tubules.
|
Acknowledgments |
|---|
itna for
their generous gift of the anti-PETH antiserum. This research was supported
through an NSERC grant to I.O. |
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Barrett, F. M. and Orchard, I. (1990). Serotonin-induced elevation of cyclic AMP levels in the epidermis of the blood-sucking bug, Rhodnius prolixus. J. Insect Physiol. 36,625 -633.[CrossRef]
Coast, G. M., Orchard, I., Phillips, J. E. and Schooley, D. A. (2002). Insect diuretic and antidiuretic hormones. Adv. Insect Physiol. 29,279 -409.[CrossRef]
Davies, S. A., Huesmann, G. R., Maddrell, S. H. P., O'Donnell, M. J., Skaer, N. J. V., Dow, J. A. T. and Tublitz, N. J. (1995). CAP2b, a cardioaccelatory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP. Am. J. Physiol. 269,R1321 -R1326.
Davies, S. A., Stewart, E. J., Huesmann, G. R., Skaer, N. J., Maddrell, S. H., Tublitz, N. J. and Dow, J. A. (1997). Neuropeptide stimulation of the nitric oxide signaling pathway in Drosophila melanogaster Malpighian tubules. Am. J. Physiol. 273,R823 -R827.
Huesmann, G. R., Cheung, C. C., Loi, P. K., Lee, T. D., Swiderek, K. M. and Tublitz, N. J. (1995). Amino acid sequence of CAP2b, an insect cardioaccelatory peptide from the tobacco hawkmoth Manduca sexta. FEBS Lett. 371,311 -314.
Kean, L., Cazenave, W., Costes, L., Broderick, K. E., Graham, S., Pollock, V. P., Davies, S. A., Veenstra, J. A. and Dow, J. A. (2002). Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster. Am. J. Physiol. 269,R1297 -R1307.
Lange, A. B., Orchard, I. and Llyod, R. J. (1988). Immunohistochemical and electrochemical detection of serotonin in the nervous system of the blood-feeding bug, Rhodnius prolixus. Arch. Insect Biochem. Physiol. 8, 187-201.[CrossRef]
Loi, P. K. and Tublitz, N. J. (2004). Sequence and expression of the CAPA/CAP2b gene in the tobacco hawkmoth, Manduca sexta. J. Exp. Biol. 207,3681 -3691.
Maddrell, S. H. P. (1964). Excretion in the
blood-sucking bug, Rhodnius prolixus Stål. II. The normal
course of diuresis and the effect of temperature. J. Exp.
Biol. 41,163
-176.
Maddrell, S. H. P., Pilcher, D. E. M. and Gardiner, B. O. C.
(1971). Pharmacology of the Malpighian tubules of
Rhodnius and Carausius: the structure-activity relationship
of tryptamine analogues and the role of cyclic AMP. J. Exp.
Biol. 54,779
-804.
Maddrell, S. H. P., Herman, W. S., Farndale, R. W. and Riegel, J. A. (1993). Synergism of hormones controlling epithelial fluid transport in an insect. J. Exp. Biol. 174, 65-80.[Abstract]
Miksys, S. and Orchard, I. (1994). Immunogold labelling of serotonin-like and FMRFamide-like immunoreactive material in neurohaemal areas on abdominal nerves of Rhodnius prolixus. Cell Tissue Res. 278,145 -151.
Montoreano, R., Triana, F., Abate, T. and Rangel-Aldao, R. (1990). Cyclic AMP in the Malpighian tubule fluid and in the urine of Rhodnius prolixus. Gen. Comp. Endocrinol. 77,136 -142.
O'Donnell, M. J. and Spring, J. H. (2000). Modes of control of insect Malpighian tubules: synergism, antagonism, cooperation and autonomous regulation. J. Insect Physiol. 46,107 -117.[CrossRef][Medline]
Pollock, V. P., McGettigan, J., Cabrero, P., Maudlin, I. M.,
Dow, J. A. T. and Davies, S. A. (2004). Conservation of capa
peptide-induced nitric oxide signaling in Diptera. J. Exp.
Biol. 207,4135
-4145.
Quinlan, M. C. and O'Donnell, M. J. (1998). Anti-diuresis in the blood-sucking insect Rhodnius prolixus Stål: antagonistic actions of cAMP and cGMP and the role of organic acid transport. J. Insect Physiol. 44,561 -568.[CrossRef][Medline]
Quinlan, M. C., Tublitz, N. J. and O'Donnell, M. J. (1997). Anti-diuresis in the blood-sucking insect Rhodnius prolixus Stål: the peptide CAP2b and cyclic GMP inhibit Malpighian tubule fluid secretion. J. Exp. Biol. 200,2363 -2367.[Abstract]
Rasband, W. S. (2005). ImageJ, US National Institutes of Health, Bethesda, Maryland, USA.
Skaer, N. J. V., Nässel, D. R., Maddrell, S. H. P. and Tublitz, N. J. (2002). Neurochemical fine tuning of a peripheral tissue: peptidergic and aminergic regulation of fluid secretion by Malpighian tubules in the tobacco hawkmoth M. sexta. J. Exp. Biol. 205,1869 -1880.
Teal., P. E., Abernathy, R. L., Nachman, R. J., Fang, N., Meredith, J. A. and Tumlinson, J. H. (1996). Pheromone biosynthesis activating neuropeptides: Functions and chemistry. Peptides 17,337 -344.[CrossRef][Medline]
Te Brugge, V. A., Miksys, S. M., Coast, G. M., Schooley, D. A. and Orchard, I. (1999). The distribution of a CRF-like diuretic peptide in the blood-feeding bug Rhodnius prolixus. J. Exp. Biol. 202,2017 -2027.
Te Brugge, V. A., Lombardi, V. C., Schooley, D. A. and Orchard, I. (2005). Presence and activity of a Dippu-DH31-like peptide in the blood-feeding bug, Rhodnius prolixus. Peptides 26, 29-42.
Wegener, C., Herbert, Z., Eckert, M. and Predel, R. (2002). The periviscerokinin (PVK) peptide family in insects: evidence for the inclusion of CAP(2b) as a PVK family member. Peptides 23,605 -611.[CrossRef][Medline]
itna, D., Hollar, L., Spalovská, I.,
Takác, P., Zitnanová, I., Gill, S. S. and Adams, M. E.
(2002). Molecular cloning and function of ecdysis-triggering
hormones in the silkworm Bombyx mori. J. Exp. Biol.
205,3459
-3473.
itna, D., Zitnanová, I.,
Spalovská, I., Takác, P., Park, Y. and Adams, M. E.
(2003). Conservation of ecdysis-triggering hormone signaling in
insects. J. Exp. Biol.
206,1275
-1289.
This article has been cited by other articles:
|
|
 |
|
  J.-P. Paluzzi, W. K. Russell, R. J. Nachman, and I. Orchard Isolation, Cloning, and Expression Mapping of a Gene Encoding an Antidiuretic Hormone and Other CAPA-Related Peptides in the Disease Vector, Rhodnius prolixus Endocrinology, September 1, 2008; 149(9): 4638 - 4646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. T. Brugge, D. A. Schooley, and I. Orchard Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus J. Exp. Biol., February 1, 2008; 211(3): 382 - 390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Santini and J. R. Ronderos Allatotropin-like peptide released by Malpighian tubules induces hindgut activity associated with diuresis in the Chagas disease vector Triatoma infestans (Klug) J. Exp. Biol., June 1, 2007; 210(11): 1986 - 1991. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
) than tubules stimulated with Mas-CAPA-1 or saline alone
(P<0.05).