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First published online May 21, 2007
Journal of Experimental Biology 210, 1986-1991 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.004291
Allatotropin-like peptide released by Malpighian tubules induces hindgut activity associated with diuresis in the Chagas disease vector Triatoma infestans (Klug)
Centro Regional de Estudios Genomicos (CREG-UNLP) and Catedra Histol. Embriol. Animal (FCNyM-UNLP), La Plata, Argentina
* Author for correspondence (e-mail: jrondero{at}museo.fcnym.unlp.edu.ar)
Accepted 26 February 2007
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: Malpighian tubules, allatotropin, Triatoma infestans, neuropeptide, hindgut, diuresis
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Like other neuropeptides, it is multifunctional, acting as
a myostimulator at the level of the foregut in two lepidopteran species
(Duve et al., 1999;
Duve et al., 2000) and at the
level of the hindgut (HG) in the cockroach Leucophaea maderae
(Rudwall et al., 2000). AT is
also a cardioacceleratory peptide, both in lepidopterans
(Koladich et al., 2002;
Veenstra et al., 1994) and in
cockroaches (Rudwall et al.,
2000). In addition, a member of the AT family was isolated from
male accessory glands of the locust Locusta migratoria, showing
myostimulatory activity at the level of the HG and the oviducts in the species
of origin, as well as in L. maderae
(Paemen et al., 1991).
Haematophagous insects incorporate a large amount of blood with each meal,
inducing them to produce a large quantity of urine in a few hours to eliminate
excess water and mineral ions (Maddrell,
1964; Maddrell,
1978; Maddrell et al.,
1993; O'Donnell et al.,
2003; Ramsay,
1952). This process involves the coordinated activity of the crop,
Malpighian tubules (MTs) and HG, regulated by neurosecretory mechanisms,
including diuretic signals such as serotonin
(Maddrell et al., 1991,
Orchard, 2006). The presence
of diuretic and anti-diuretic peptides acting together with serotonin has been
also documented in the bug Rhodnius prolixus
(Orchard, 2006;
Paluzzi and Orchard, 2006;
Quinlan et al., 1997;
Te Brugge et al., 1999;
Te Brugge et al., 2001;
Te Brugge and Orchard, 2002;
Te Brugge et al., 2005).
Malpighian tubules, the main excretory organ in insects, have traditionally
been regarded as a system involved in water and mineral balance in response to
neuroendocrine stimulus. New roles for this organ have started to emerge, such
as a circadian rhythm regulator
(Giebultowicz and Hege, 1997)
and as an autonomous immune system producing anti-microbial peptides
(Dow and Davies, 2006;
McGettigan et al., 2005). In a
related article, we communicate the ability of the MTs of the kissing bug
Triatoma infestans (Klug) to secrete an AT-like peptide (M.S.S. and
J.R.R., manuscript submitted). This activity was found to respond to changes
in water and mineral composition and it is associated with the critical
process of post-prandial diuresis (M.S.S. and J.R.R., manuscript
submitted).
In the present study, we communicate the activity of AT as a myostimulator at the level of the hindgut and its association with the elimination of urine during post-prandial diuresis in T. infestans, confirming the proposed endocrine activity of MTs and assigning a first function to this new role.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Hindgut peristaltic contractions assay
As a first approach to assess the myostimulatory activity of AT on the HG,
4th-instar insects were dissected under R. prolixus saline
(Maddrell et al., 1993). The
HG was dissected together with MTs and the last portion of the midgut. Basal
contractions were induced by bathing the dissected material in
phosphate-buffered saline (PBS). A large variation in the response of the HG
was observed between insects, apparently associated with nourishment state and
the amount of material accumulated in the HG. After 3 min, samples in which
signs of continued contractile activity were not evident were discarded. Only
those preparations with intact HG (without loss of content during dissection)
were used. Different doses of pure Aedes aegypti allatotropin (kindly
provided by Dr Fernando G. Noriega) were diluted in PBS. Then, 20 µl of
each dilution was applied to each preparation. The same preparation was used
for assays with different doses. Between tests, preparations were washed with
PBS (three washes, 2 min each) to restore basal conditions. Preparations were
finally analysed under a dissection microscope, where the total number of
contractions in a 2-min period was recorded for each dose applied
(Duve et al., 2000). Results
are expressed as number of contractions per minute (frequency of
contractions).
Number of MTs and elapsed time to eliminate HG content after osmotic shock
We have recently shown that isolated MTs release an AT-like peptide into
the incubation media in a constitutive way. The quantity of the peptide
released increases when MTs undergo an osmotic shock (M.S.S. and J.R.R.,
manuscript submitted). In this assay, we analysed the time required by
MTs–HG preparations to start content evacuation after an osmotic shock.
Three groups of insects were used, one group retaining all four MTs (control),
one group with two MTs and the remaining group with only one MT. All groups
were exposed to 25 µl of diluted R. prolixus saline (20% saline,
80% distilled water), undergoing an osmotic shock and inducing AT-like peptide
secretion (M.S.S. and J.R.R., manuscript submitted). The time at which the HG
began eliminating its content was recorded. Results are expressed in
seconds.
In vitro blockade of diuresis
To assess the relationship between AT-like peptide released by MTs and the
ability of the HG to eliminate urine, we assayed the in vitro
blockade of this process with an AT-antiseum (Hernadez-Martinez et al., 2005).
We used two alternative experimental designs. In the first design,
MTs–HG preparations preserving the complete set of renal tubules were
incubated in 25 µl of diluted R. prolixus saline, including A.
aegypti AT-antiserum. Different antiserum dilutions were applied; 1:100
000; 1:10 000; 1:1000 and 1:100. As a control, we used two different antiserum
solutions preadsorbed with pure A. aegypti allatotropin (4°C
overnight) (1:1000/20 nmol AT; 1:100/200 nmol AT). The amount of pure peptide
used to preadsorb the antiserum was calculated based on previous experiments
blocking immunostaining in cytological analysis of MTs (M.S.S. and J.R.R.,
manuscript submitted). The time required to fully evacuate the HG was
recorded.
A second design was applied to corroborate the action of the AT-like peptide. MTs–HG preparations were incubated in a similar solution but, in this case, renal tubules were detached and maintained next to the HG, preventing their secretion into the HG. The presence of MTs in the same in vitro preparation allowed the AT-like peptide to diffuse into the medium.
In vivo blockade of diuresis
After in vitro experiments, we performed an in vivo assay
to test the fate of the post-feeding diuresis alteration induced by AT
blockade. The experimental design involved three groups of 4th-instar T.
infestans larvae. In all treatments (12 insects each), 1 µl of
solution was applied by intra-abdominal (ventral) injections three hours
before offering a meal. We used an estimated haemolymph volume of 10 µl in
non-fed 4th-instar insects to calculate the volume of injected solutions. The
first group received an injection of R. prolixus saline, representing
normal insects (general control). A second group was injected with
AT-antiserum (1:100). The last group was injected with the same antiserum
dilution, preadsorbed overnight (4°C) with pure AT (200 nmol). Of all the
insects injected, only those that fed ad libitum after 30 min and
without evident signs of internal damage caused by injection were used. After
this sorting, none of the groups retained less than four insects. Fed insects
were individually kept in microtubes (1.5 µl). The volume of urine produced
by individual insects was recorded at five different times (45, 60, 90, 120
min and 24 h after blood intake) by the use of micropipettes. Data are
expressed as µl of urine released per insect.
Statistical analysis
Differences between treatments were analysed by one-way or two-way analysis
of variance (ANOVA), depending on the experimental design applied. In those
experiments in which the interest was focused only in comparisons between
treated groups and control, single post-hoc comparisons were tested
by least significant difference (LSD). In experiments involving multiple
comparisons, differences were analysed by the Tukey honest significant
difference (HSD) test. Primary data were transformed to logarithms to improve
normality and homoscedasticity when necessary. Only differences equal or less
than 0.05 were considered significant. None of the samples analysed had less
than three replications. Finally, data are expressed as means ±
standard error. Each experimental design presented in this study was performed
twice or more times showing similar results.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Our results showed a significant increase in the frequency of HG
contractions with AT concentrations as low as 10–18 mol
l–1 (Fig. 1A),
producing a clearly defined peristaltic wave (see Movie in supplementary
material). With a concentration of 10–15 mol
l–1, the peptide produced contractions with a lower frequency
and longer duration of peristaltic waves. Higher concentrations of AT further
increased the intensity of contractions, reaching a full and maintained
contraction with AT concentrations of 
10–12 mol
l–1 (Fig.
1B,C). Again, and as previously described in the tomato moth
Lacanobia oleracea (Duve et al.,
2000), a large variation in the response to AT was observed
between individual insects, apparently associated with nourishment state and
the amount of material accumulated in the HG.
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Time to evacuation was similar in controls and preparations treated with preadsorbed antiserum. By contrast, MTs–HG preparations treated with AT-antiserum (1:100) showed a longterm blockade of the diuresis (>3 h), indicating the need for the AT-like peptide released by MTs to induce voiding. After this long-term blockade, the HG was swollen with liquid drained by MTs, showing that the excretory function continued, unaffected by the experimental blockage (Fig. 2B).
When similar MTs–HG preparations were treated with higher dilutions of the antiserum (1:1000; 1:10 000 and 1:100 000), the delay decreased proportionally, showing a dose–response behaviour. One of the dilution treatments used (1:1000) was compared with the analogous treatment with AT-preadsorbed antiserum. A highly significant difference in the time elapsed was found, suggesting the specificity of the blockade (Fig. 2C).
To validate the action of AT on the HG, a new experimental design involving MTs–HG in vitro preparations was performed. In vitro preparations with unattached MTs placed near the HG in a diluted solution produced results very similar to those previous described, with a similar delayed response blocked by the AT-antiserum (Fig. 2D). The AT-like peptide diffusing through the incubation medium caused a direct response without MTs draining their content directly into the HG.
In vivo AT-like peptide blockade
The volume of urine produced at given times during the first 120 min after
blood intake was lower in insects receiving AT-antiserum than in controls
(Fig. 3A).
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While the cumulative volume of urine eliminated by insects injected with AT-antiserum during the first two hours after a blood meal was lower than that of controls, differences disappear during the next 22 h. The lack of an inhibitory effect after the first 2 h may indicate that the AT-antiserum was either eliminated from the haemocoel or completely saturated by the AT-like peptide released by MTs (Fig. 3C).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Duve et al.,
2000). AT was first isolated in M. sexta
(Kataoka et al., 1989) and then
found in other insect and invertebrate species, showing a highly conserved
sequence among all species. The results presented in the present study show
that A. aegypti AT can modulate muscle contractions in the HG of
T. infestans 4th-instar larvae, suggesting the presence of specific
functional receptors and that the AT-like peptide found in T.
infestans MTs is closely related to that in other insects. In fact, an
alternative splicing of AT was found in the MTs of M. sexta
(Lee et al., 2002).
The response of the HG to AT in all experiments performed shows that after
a maximum increment of contractions with a given dose, the frequency of
contractions returns to that observed in controls. A similar behaviour was
described in cockroaches (Rudwall et al.,
2000). In our experience, this decrease is associated with a
longer duration of contractions. In fact, the highest doses led to a long-term
contraction, possibly due to the saturation of the receptors. With regard to
the direction of the peristaltic waves, as in the lepidopteran Helicoverpa
armigera (Duve et al.,
1999), we found considerable variations among different
individuals. In some insects, the peristaltic waves seemed to be mainly in a
posterior-to-anterior direction, while in other insects a combined pattern of
anterior-to-posterior and posterior-to-anterior directed waves was found. The
variability observed seems to be associated with the amount of material in the
HG. A similar pattern of contractions observed in H. armigera crop
has been proposed as a mechanism to generate a powerful mixing of the contents
(Duve et al., 1999). Likewise,
this pattern of peristaltic contractions in HG of T. infestans could
serve to create a better mix of contents, facilitating the elimination of
urine and faeces.
Our experiments show a correlation between the number of MTs and the time elapsed between an osmotic shock and voiding of HG contents. Our results also provide conclusive evidence that AT-like peptides released into the medium by MTs act directly on the HG, inducing its evacuation. In this way, MTs participate in an endocrine fashion, facilitating the voiding of the HG during post-prandial diuresis. In vitro treatments with different dilutions of AT-antiserum confirm this hypothesis. The use of different antiserum dilutions showed a dose–response pattern, decreasing the elapsed time when antibody dilutions were incremented. The delay must be related to the quantity of neuropeptide blocked by the antiserum applied, being greater when more antiserum is present in the medium. Furthermore, with the highest antiserum concentration tested, a long-term blockade was reached despite the fact that the HG was full with urine, showing that MT excretory function was active and not blocked during the experiment.
Haematophagous insects incorporate a large amount of blood with each meal,
undergoing a critical post-feeding period during which they produce a large
quantity of urine to eliminate the excess water and Na+
incorporated (Maddrell, 1964;
Maddrell et al., 1993). In our
experiments, T. infestans 4th-instar larvae eliminated around 50% of
the total urine produced during the first 24 h of post-prandial diuresis in
the first 2 h.
In vivo blockade of peptide activity by the use of an antiserum
has been previously used to analyse related physiological functions
(Gebhardt, 2004;
Patel et al., 1995;
Tublitz and Evans, 1986). In
our in vivo experiments, insects receiving AT-antiserum showed a
significant decrease in urine eliminated during post-prandial diuresis. The
effect was evident during the first 2 h after blood intake, when the injected
antiserum decreased the accumulated volume of urine. A large intra-group
variation was observed in the volume of urine produced over 24 h in the group
treated with antiserum. This could be due to the fact that blockade depends on
the action of an intra-haemocoelic injection of the antiserum, which could be
biologically degraded at different rates in different insects.
We have shown that T. infestans MTs secrete an AT-like peptide in a constitutive way, increasing reversibly the quantity of the peptide released when the surrounding medium is diluted. Furthermore, the content of AT-like peptide present in MTs decreases during the first hours after a blood meal, when diuresis is occurring at high rates. Taken together, our experiments clearly indicate the physiological nature of the processes involving the effect of AT-like peptides produced by MTs at the HG level and allow us to assign a first function to this new role. The importance of this peptide in the elimination of urine after a blood meal is clearly established.
T. infestans, like other triatominae insects, is implicated in the transmission of Chagas disease in several regions of Latin America, affecting a large number of people in several countries. The infection is naturally transmitted when the insect feeds, releasing, together with urine, faeces containing the infective form of the protozoan Trypanosoma cruzi. The possibility of delaying or even blocking urine elimination after a blood meal provides new ways in which to consider the potential control of this disease.
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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0.05). Each bar represents the mean ± s.e.m. of the
number of contractions m–1 in one of three experiments
performed showing similar results (N=3–5 samples per
treatment). (B) Image obtained from a hindgut preparation maintained in saline
(Control). (C) The same preparation after treatment with pharmacological
concentrations of pure A. aegytpi AT, showing a full and long-term
contraction of the HG.
