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Cardioacceleratory and myostimulatory activity of allatotropin in Triatoma infestans

Marcos Sterkel a, Fernando Luis Riccillo b, Jorge Rafael Ronderos a,b,⁎a Centro Regional de Estudios Genomicos (CREG-UNLP), Argentinab Catedra Histol. Embriol. Animal (FCNyM-UNLP) La Plata, Argentina

a b s t r a c ta r t i c l e i n f o

Article history:Received 20 August 2009Received in revised form 10 November 2009Accepted 3 December 2009Available online 6 December 2009

Keywords:MyostimulatoryAllatotropinTriatoma infestansCardioacceleratorySerotonin

Haematophagous insects incorporate a large quantity of blood with each meal, producing a big quantity ofurine in a few hours. The activity of the Malpighian tubules (MTs) is facilitated by the increase of thecirculation of the haemolymph produced by the increase of the aorta contractions as well as, of the peristalticwaves of the anterior midgut. MTs of Triatoma infestans secrete an allatotropin-like peptide, which has amyostimulatory effect on the hindgut, inducing the mixing and voiding of the content during post-prandialdiuresis. We are reporting now the activity of allatotropin (AT) as a cardioacceleratory and a myostimulatorypeptide at the level of the anterior midgut. The peptide induced the increase of the rate of contractions of theanterior midgut and the aorta in a wide range of concentrations. The cardioacceleratory effect of AT wasdependent on the feeding state of the insects and on the presence of serotonin. The response showed theexistence of a differential behavior between sexes, inducing a higher increase on the frequency ofcontractions, as well as, the width of the aorta in males than in females. Finally, our results suggest that ATinteracts with serotonin to facilitate the circulation of haemolymph during post-prandial diuresis.

© 2009 Elsevier Inc. All rights reserved.

1. Introduction

During feeding, haematophagous insects incorporate a largequantity of blood. In the following few hours, high volumes of urineare produced to eliminate the excess of water and mineral ions(Ramsay, 1952; Maddrell, 1964; Maddrell, 1978; Maddrell, et al.,1993; O'Donnell et al., 2003). In the kissing-bug Rhodnius prolixus(Hemiptera:Reduviidae) a decrease of the osmotic concentration ofthe haemolymph occurs during feeding (Maddrell, 1964). Malpighiantubules (MTs) respond to this physiological stress increasing their rateof secretion producing hypo-osmotic urine to re-establish water andmineral balance. (Maddrell, 1964, Maddrell and Phillips, 1975). Allthese processes are regulated by neurohormonal mechanisms,involving the integrated activity of the anterior midgut, MTs andhindgut. They include diuretic signals, like serotonin (Maddrell andPhillips, 1975, Maddrell et al., 1991, Orchard, 2006), which alsoinduces the re-absorption of K+ at the lower MTs (Maddrell, et al.,1993). Furthermore, the presence of other diuretic and even anti-diuretic peptides acting together with serotonin has been proposed(Te Brugge et al., 1999; 2002; 2005; Orchard, 2006). Indeed, theamino acid sequence of a calcitonin-like diuretic hormone (DH31)

was recently characterized in R. prolixus (Te Brugge et al., 2008). Tofacilitate the high rate of activity of the MTs, circulation of thehaemolymph increases, ensuring that, around the renal tubules, thehaemolymph is constantly stirred, facilitating also the access of thehormones regulating its function. In fact, immediately after the insectbegins to feed the peristaltic activity of the anterior midgut increases,causing an acceleration of the flow of hemolymph in an antero–posterior direction (Maddrell, 1964).

Serotonin, the most studied and characterized diuretic hormone intriatominae insects has multiple functions, reaching high titers in thehaemolymph few minutes after the beginning of the meal (Langeet al., 1989). This neurohormone not only acts as a diuretic hormone.Itisalsoinvolvedinanumberofprocessesoccurringduringbloodintake,such as the plasticisation of the cuticle (Barrett andOrchard, 1990), theincrease of ion transport from the lumen of the anterior midgut to thehaemolymph(Farmeretal.,1981),theincreaseoftheperistalticwavesofthemidgutandalsooftheheartbeatrate(Orchard,2006;TeBruggeetal.,2009). Serotonin is also involved in the regulation of the expression ofaquaporinsintheMTs(Martinietal.,2004).

We have recently shown that Triatoma infestans MTs secrete anallatotropin-like peptide (AT-like) which stimulates peristaltic con-tractions of the hindgut in an endocrine way, facilitating the mixing ofurine and feces, and inducing the voiding during post-prandialdiuresis (Santini and Ronderos, 2007; 2009a). Furthermore, thecontent of the peptide in the renal tubules and midgut shows dailyvariations, reaching a maximum previously to the beginning of thedark phase of the day, when the insect begin to prepare itself for thecritical process of blood intake (Santini and Ronderos, 2009b).

Comparative Biochemistry and Physiology, Part A 155 (2010) 371–377

⁎ Corresponding author. Centro Regional de Estudios Genomicos (CREG) UniversidadNacional de La Plata, Parque Tecnologico Florencio Varela, Av Calchaqui km 23,500(1888) Florencio Varela, Buenos Aires, Argentina. Tel./fax: +54 11 42758100.

E-mail address: jrondero@museo.fcnym.unlp.edu.ar (J.R. Ronderos).

1095-6433/$ – see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpa.2009.12.002

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Allatotropin (AT) is a neuropeptide isolated because of its ability toinduce juvenile hormone synthesis in Manduca sexta (Kataoka et al.,1989). It has also been extensively characterized in other insectspecies (Veenstra and Costes, 1999; Truesdell et al., 2000; Lee et al.,2002; Park et al., 2002; Abdel-latief et al., 2003) while members of thefamily are also present in several invertebrate phyla (Elekonich andHorodyski, 2003). As other neuropeptides, AT is multifunctional,acting as a myostimulator at the level of the foregut in lepidopteranspecies (Duve et al., 1999; Duve et al., 2000). Also, it has been shownthat AT acts as a myostimulator at the level of the hindgut in thecockroach Leucophaea maderae (Rudwall et al., 2000) and in T.infestans (Santini and Ronderos, 2007). It has also been associatedwith the control of ion transport in epithelial cells of the digestivesystem (Lee et al., 1998). AT has also demonstrated to be car-dioacceleratory in lepidopteran species as M. sexta (Veenstra et al.,1994) and Pseudaletia unipuncta (Koladich et al., 2002) (Lepidoptera),as well as in the cockroaches L. maderae and Periplaneta americana(Rudwall et al., 2000).

In the present study we analyze the activity of AT as both acardioacceleratory, as well as, a myostimulatory peptide in adults(males and females) of the kissing-bug T. infestans, the main Chagasdisease vector in Latin-America.

2. Materials and methods

2.1. Insects

T. infestans (Klug) adults (males and females) were obtained froman artificial colony maintained at 28±2 °C, 45% relative humidity anda 12:12 hour light–dark period. For those experiments performedwith non-fed insects, those individuals which reached the adult stagewere starved during 14 days. For experiments with fed insects, thesame period of starvation was applied. After this period, meal wasoffered and only those insects fed ad libitum were selected forexperiments. Insects were fed on live chickens during the light period.All the experiments were performed with experimental groupsconformed by 5 to 8 insects. All the treatments were performedduring the light period.

2.2. Cardioacceleratory and midgut peristaltic contraction assays

To assay the effect of AT on the contractions of the aorta andanterior midgut, male and female adults (fed and non-fed) wereanalyzed in vivo. Those experiments involving fed insects wereperformed 24–48 h after blood intake. The wings of the insects werecut to expose the dorsal cuticle of the abdomen. In this way, due to thetransparent nature of the cuticle, the contractions of the aorta and theperistaltic waves of the anterior midgut are clearly seen and recorded.As the hormone has previously proved to produce myostimulatoryeffects at low doses (Duve et al., 1999; Santini and Ronderos, 2007),we decided to assay it at different concentrations, starting with lowones. The following doses of Aedes aegypti pure allatotropin(APFRNSEMMTARGF) synthesized by Biopeptide Company (SanDiego, CA, USA) (Hernandez-Martinez et al., 2007) (kindly providedby Dr. Fernando G. Noriega) were assayed (10−18; 10−15; 10−12; 10−9

and 10−6 M). The peptide was administered through the femoralsegment of the leg,whichwas cut at thebeginningof theexperiment. ATwas diluted in 5 µl of R. prolixus saline (Maddrell et al., 1993). Controlsreceived only saline. As both pairs of wings and one leg of the insects arecut at the beginning of the experiment, the pressure of the injection ineach treatment, displaces a similar volume of haemolymph which iseliminated, causing the final volume to remain constant throughout theexperiment. To minimize the effect of the stress caused by handling,insects were left for 30 min prior to the administration of the first dose.The insects were maintained under a dissection microscope. Thecontractions of the aorta and peristaltic waves of the anterior midgut

were observed by transparency through the dorsal cuticle of theinsects (segments IV and V of the abdomen). The number of con-tractions in a 2-min period was recorded for each dose applied (Duveet al., 2000; Santini and Ronderos, 2007) at 5, 15 and 30 min after theapplication of the treatment. To assay the effect on the peristalticwaves,only those contractions which produced an anterior–posterior wavethrough the abdomen were recorded. Local contractions (usuallyobserved at the level of segments II and III of the abdomen) were notrecorded. All data were recorded by the same operator.

Alternatively, two different kinds of controls were performed.First, following the same experimental design, some insects wereinjected only with saline solution, mimicking the same number oftreatments as the groups treated with allatotropin. In this case, thefrequency of contractions was not modified, showing that thehandling does not produce any effect. Furthermore, other groups ofinsects were only treated with doses of 10–9 M, reaching the samefrequency as the individuals which received the complete dose–response treatment.

For serotonin–allatotropin assay, the same doses of AT were used,but now diluted in a solution with serotonin (10−9 M), which isknown to produce an increase of contractions at the level of theanterior midgut and the aorta (Orchard, 2006; Te Brugge et al., 2009).After 40 min, the frequency of contractions resembled the frequencyof the control, showing that the insects tend to return to basalconditions (see supplementary files). The same insect was used toassay different doses. Finally, results are expressed as number ofcontractions per minute (frequency of contractions).

To analyze the effect of the peptide on the amplitude of the aorta,two groups of insects (3 individuals each) injected with saline (control)or 10−9 M of AT were randomly selected, and the contractions of theaorta were recorded during 1 min with an Olympus digital cameraannexed to an Olympus stereo microscope. All the movies wererecorded with the same magnification, which implies that the pixelsin every movie have the same size, allowing comparisons betweentreatments.When all themovieswere recorded, each individualfilewasanalyzed frame by frame, and the maximum and minimum widths ofthe aorta was recorded as the number of pixels contained in a linecrossing from one side to the opposite of the aorta. The width of theaortawas finally evaluated by the use of the Image J 1.23 image analysissoftware (National Institute of Health, USA) and defined as the numberof pixels of the maximum and minimum cross sections of the aorta foreach treatment analyzed.

2.3. AT-like immunoreactivity in the anterior midgut and aorta

The cephalic portion of the aorta and the anterior midgut ofinsects were dissected under a binocular microscope and fixed informaldehyde-phosphate buffered saline (PBS) (4%) at 4 °C for 12 h.Tissuewas thenwashed in PBS-Tween (0.05%) (PBS-T), permeabilisedwith Triton X-100 (1%) (24 h at 4 °C) and then blockedwith 3% bovineserum albumin (BSA) for 2 h. Then the tissue was incubated overnightat 4 °C with a polyclonal antiserum against A. aegypti AT (Hernandez-Martinez et al., 2005) (1/100 in 3% BSA diluted in PBS-T) whichpreviously proved to be specific in T. infestans (Santini and Ronderos,2007, 2009a). Finally, tissues were incubated with FITC-labelled goatanti-rabbit secondary antibody (1/200 in blocking-buffer) for 3 h atroom temperature. After every step, tissue was washed (3 time-s×20 min) with PBS-T (0.05%). As mentioned above, primaryantibody specificity was previously assayed both for immunohisto-chemistry, as well as in the whole animal. For secondary antibodycontrol, primary antibody incubation was replaced with PBS. Theresulting material was analyzed with a Laser Scan Microscope ZeissLSM 510 Meta. When primary antibody was omitted, no staining wasobserved showing that the secondary antibody does not cross-reactwith insect tissues.

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2.4. Statistical analysis

Differences between treatments were analyzed by multifactorialanalysis of variance, taking into account two factors acting on thefrequency of contractions of the aorta and peristaltic waves of theanterior midgut: dose and time after injection (effect of AT in each sexindividually); and sex and doses (differential response betweensexes). Single post-hoc comparisons were tested by the Tukey honestsignificant difference (HSD) test. Primary data were transformed tologarithms to improve normality and homoscedasticity when neces-sary. Only differences equal to or less than 0.05 was consideredsignificant. Finally, data are expressed as means±standard error.

3. Results

3.1. Anterior midgut contractions assay in fed animals

The analysis of the number of peristaltic waves of the anteriormidgut under the effect of AT showed that the frequency was

increased in both sexes (Fig. 1A). The number of contractions wasdifferent in males and females, with a higher frequency of contrac-tions reached in males than in females (Table 1).

3.2. Cardioacceleratory effect in fed animals

AT also caused a significant increase of the frequency ofcontractions of the aorta both in males and in females. The responsewas dependent on the dose applied (Fig. 1B), showing also adifferential response between sexes (Table 1). The basal frequencies(i.e. number of contractions in insects injected with saline) weresimilar in males and females, but the maximum rate of contractions ofthe aorta was higher in males.

3.3. Cardioacceleratory effect of AT in non-fed animals

Interestingly when the same approach was performed with non-fed insects, there were no responses in males or in females. We

Fig. 1. Effect of different concentrations of AT on peristaltic waves of the anterior midgut(A)and the frequencyof contractionsof theaorta (B) inmalesand females of fedT. infestans,showing the response of the variables to the action of the peptide. Each bar representsmean±standard error (n=5–8). (*): significant difference with saline treated insects.

Table 1Analysis of variance showing the differential response to allatotropin between sexes on the anterior midgut and aorta in fed insects, and the effect of allatotropin applied with orwithout serotonin in non-fed insects. df: degrees of freedom; MS: median squares; F: Fisher value; treatments: allatotropin with or without serotonin.

Aorta df effect MS effect df error MS error F p-level

Sex 1 7408.63965 252 236.541595 31.3206635 5.6967E-08Doses 5 3652.67505 252 236.541595 15.4419985 3.021E-13Sex by doses 5 510.816833 252 236.541595 2.15952206 0.0591111Aorta males

Treatments 1 4560.25391 250 216.515305 21.0620403 7.0428E-06Aorta females

Treatments 1 0.65966976 250 0.11163302 5.90927124 0.01576527

Fig. 2. Effect of AT on the rate of contractions of the aorta in non-fed insects (males andfemales). The effect of AT in males (A) and females (B) is only observed when thepeptide is applied simultaneously with serotonin (10−9 M), suggesting an interactionbetween the activity of these two neurohormones. Each bar represents mean±standard error (n=5–6). (*): significant difference with saline treated insects.

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decided to test the existence of a relationship between the activities ofAT and serotonin. While the application of AT did not show anyresponse in non-fed individuals when the peptide was administeredalone, treatments of the insects with the peptide, applied togetherwith serotonin (10−9 M), caused an effect similar to that observed bythe administration of AT in fed insects, suggesting the existence of aninteractive relationship between the activity of these two hormones(Fig. 2A and B; Table 1).

3.4. Effect of AT on the width of the aorta

Regarding the frequency of contractions of the aorta and theperistaltic waves of the anterior midgut a differential responsebetween sexes was found. Taking into account that the accelerationof the flow of haemolymph is critical during the post-prandial diuresisin both sexes, as an approach to analyze the underlying mechanisms,the minimum and maximum widths of the aorta were measured ininsects undergoing control conditions (i.e. injected with R. prolixussaline) and those receiving a dose that causes a significant effect onheart rate (10−9 M). The results show that there is a significantincrease in the maximum width of the aorta in males, while thisparameter does not undergo changes in females (Fig. 3A). Moreover,while the rate between themaximum andminimumwidths increased1.7 in males, the value in females was only 0.6 (Fig. 3B). Finally, whenthe minimum and maximum widths for males and females werecompared, the results clearly showed that females showed always thehighest amplitudes (Fig. 4A to F).

3.5. Presence of AT on the aorta and anterior midgut

Histological analysis of the anterior midgut and aorta showed thepresence of the AT-like peptide in both organs. The presence ofepithelial cells was evident at the level of the anterior midgut. Thesecells were found exclusively at the anterior portion, and themorphology corresponds to the open-type cells (Fig. 5A and B). Atthe level of the aorta the presence of immunoreactive material inneural processes both, in the surface as well as, going across the wallswere found (Fig. 5C and D).

Fig. 3. Changes in the width of the aorta in males and females after the treatment withAT. (A) Comparison between the effect of the peptide in males and females. Whilemales respond to AT with the increase of the width, females do not show any response.(B) Percentage of increase between maximum andminimum amplitudes of the aorta inmales and females. Each bar represents mean±standard error (n=3). (*): significantdifference with saline treated insects.

Fig. 4. Differences in the minimum and maximum amplitude of the aorta betweenmales and females. Arrows in (B), (C), (E) and (F) show the aorta at the level ofsegments IV of the abdomen in males (B and E) and females (C and F) respectively.Pictures are horizontally oriented with the cephalic region of the abdomen located atthe right side of the picture (segment III). Each bar represents mean±standard error(n=3). (*): significant difference. Scale bar in segment V: 1 mm.

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4. Discussion

Post-prandial diuresis is a critical process in haematophagousinsects. A few minutes after the animal begins to feed, the levels ofserotonin in haemolymph increase (Lange et al., 1989; Orchard, 1989;Barrett et al., 1993) and produce an unusual increase in the activity ofthe MTs, generating a big quantity of urine during the next few hours,to re-establish water and mineral balance. Triatominae insects havefour MTs: two of these run backwards, closely associated to thehindgut. The other two run in opposite directions, maintaining aclosely relationship with the midgut (Wigglesworth, 1931). MTs arenot innervated, being stimulated by serotonin (Maddrell, 1969;Maddrell et al., 1993; Lange et al., 1989; Barrett and Orchard, 1990)and other diuretic hormones, like a calcitonin-like diuretic hormone(DH31) (Te Brugge et al., 2008) released as neurohormones. Tofacilitate the access of diuretic hormones, the circulation of thehaemolymph must be accelerated during and after blood meal,ensuring the presence of high titers of diuretic hormones, andmaintaining constantly stirred the fluid around the MTs. Indeed,immediately after beginning to feed; the anterior midgut intensifiesboth the frequency and also the intensity of contractions, generating afaster movement of the insect fluids in an antero–posterior direction(Maddrell, 1964).

Previous studies have shown that AT has amyostimulatory activityat different levels of the digestive system in several insect species(Duve et al., 1999; Duve et al., 2000, Rudwall et al., 2000) including T.infestans (Santini and Ronderos, 2007). Furthermore, AT has alsoproved to act as a cardioacceleratory peptide in two lepidopteranspecies (Veenstra et al., 1994; Koladich et al., 2002) and in

cockroaches (Rudwall et al., 2000). Our results show for the firsttime that AT has a cardioacceleratory effect in Hemiptera. Further-more, a myostimulatory effect at the level of the anterior midgut isshown. Indeed, in fed insects, the peptide increases the basalfrequency of contractions, mimicking the frequency observed duringpost-prandial diuresis.

When the hormone was applied in insects that had never been fedduring the adult stage the peptide did not induce any response,neither in the aorta nor in the anterior midgut. Interestingly, thesimultaneous application of AT and serotonin, which inducescontractions at the level of the heart and the anterior midgut inR. prolixus (Orchard, 2006, Te Brugge et al., 2009) produced anincrease in the frequency of contractions of the aorta, suggesting theexistence of an interactive effect of both hormones. At the level of theanterior midgut, the simultaneous administration of both hormonesdid not cause any effect suggesting that other factors could beinvolved. In fact, new studies by Te Brugge et al. (2009) have shownthat some diuretic hormones also act as myostimulatory factors at thelevel of the anterior midgut in the related species R. prolixus.

Regarding the range of concentrations in which AT produces itseffect, it was not surprising that the peptide has caused a slight effectwith low doses at the level of the aorta. In fact, this kind of phe-nomenonwas previously described by Duve et al. (1999)who showedthat this peptide causes an increase greater than 100% in the frequencyand amplitude of contractions of the foregut of Helicoverpa armigera(Lepidoptera:Noctuidae) with concentrations as low as 10−16 M.Furthermore, in our experience, under in vitro conditions AT inducesa significant increase in the frequency of contractions of the hindgutat a concentration of 10−18 M (Santini and Ronderos, 2007). In this

Fig. 5. Immunohistochemical analysis of the AT-like expression in the anterior midgut and aorta in males (similar images were found in females). (A and B) Confocal images showingthe presence of the AT-like peptide in open-type cells located at the first portion of the anterior midgut. (C and D) Immunoreactive processes running across the wall of the aorta(arrow heads) (C) and 3D reconstruction of the aorta showing the presence of the neural processes with AT-like peptide (arrow heads) and nephrocytes (arrow) (D).

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particular case, we have shown that AT is present either in epithelialopen-type cells at the level of the anterior midgut, and also in neuralprocesses which reach the aorta. Indeed, the effect observed could bethe result of the addition of the peptide produced by the insect itselfplus the pure peptide applied in each treatment.

A differential response between sexes was found in some of thevariables analyzed, with the males reaching the highest frequency ofcontractions of the aorta. A similar phenomenon was observedpreviously regarding the cardioacceleratory effect of the AT incockroaches, showing also the highest rate of contractions in males(Rudwall et al., 2000). When the changes in the width of the aortawere analyzed, again, a differential response was found. In this case,only males showed a significant increase after treatments. Indeed,when the increase between maximum and minimum widths of theaorta was compared it was higher in males than in females. When theminimum and maximum widths in males and females werecompared, we found that both variables were higher in females.This difference between sexes could represent a biologically adaptivecharacter. In fact, the results show an inverse relationship betweenrate of contractions and width in males and females. While thehighest frequencies were observed in males, which present the lowestwidths, the females showed the opposite. This inverse relationshipcould compensate the rate of flux of haemolymph in the insect(i.e. lower width associated with higher frequencies and vice versa),needing a lower frequency in the females, due to the greater volumeof haemolymph pumped by the aorta in each contraction. Further-more, females reached the highest frequency of contractions withlower doses. In this respect, taking into account the myostimulatoryactivity of AT, it could be probable that this multifunctional peptidemay also act as a myoregulator at the level of other organs. In fact, theLom-AG-myotropin, an AT-related peptide isolated from the acces-sory glands of the reproductive system of the male of Locustamigratoria, induces contractions at the level of the oviducts in locustsand cockroaches (Paemen et al., 1991; Rudwall et al., 2000; Elekonichand Horodyski, 2003). In this way, females could solve the problem ofthe increase of the circulation of the haemolymph without compro-mise of the activity of the oviducts during post-prandial diuresis.Indeed, during the course of experiments with females, some eggshave been occasionally deposited during post-prandial diuresis.

In previous studies, we have demonstrated that MTs produce anAT-like peptide, which is secreted in response to changes of the waterand mineral balance of the haemolymph (Santini and Ronderos,2009a). We have also shown that this peptide, secreted by the renaltubules, positively modulates the rate of the peristaltic contractions atthe level of the hindgut, inducing in this way the voiding in anendocrine way (Santini and Ronderos, 2007). To try to find out if theAT-like peptide is acting on the aorta and the midgut by an endocrineway, we injected an antiserum against A. aegypti AT (Hernandez-Martinez et al., 2005) which previously proved to be specific in T.infestans (Santini and Ronderos, 2007, 2009a) in insects undergoingpost-prandial diuresis. The use of a similar concentration of theantiserum used to block the AT-like activity of the hindgut in nymphs(1/100) (Santini and Ronderos, 2007) and even a greater one (1/50)did not induce significant changes in the aorta or in the anteriormidgut, suggesting that AT is not acting at this level by the endocrineway (data not shown). The presence of open-type cells producing anallatostatin-like peptide in both, the anterior and posterior midgut ofthe related species R. prolixus was previously demonstrated (Sarkaret al, 2003). In this regard, we have recently shown in IV instar larvaeof T. infestans, that an AT-like peptide is present in the anterior midgutshowing daily variations in its content (Santini and Ronderos, 2009b).We are now showing the presence of epithelial open-type cellsproducing an AT-like peptide, in the anterior midgut of the adult ofthe kissing-bug T. infestans. This finding corroborates the previous onein the larval stage. The presence of open-type cells in the anterior wallof the anterior midgut is an interesting finding, suggesting that these

cells could be involved in the regulation of peristaltic movements ofthe anterior midgut. The production of AT by the open-type cellslocated in the first portion of the anterior midgut should be the sourceof the peptide. These AT producing cells would act in a paracrine wayby sensing changes in the lumen of the organ, regulating in a fasterand accurate way the peristaltic waves of the anterior midgut duringblood intake and post-prandial diuresis. Regarding the source of thepeptide acting on the aorta our results, showing the presence ofimmunoreactive neural processes at the level of the wall, stronglysuggests that the myostimulatory activity of AT is regulated by thenervous system.

Finally, our results show for the first time in Hemiptera and in fact,in species other than Lepidoptera and Dictioptera, the activity of AT asa cardioacceleratory peptide. Moreover, our data suggest that thispeptide is involved in the process of accelerating the circulation ofhaemolymph that occurs during post-prandial diuresis.

Acknowledgements

The authors wish to thank Dr. Fernando G. Noriega (FloridaInternational University Florida—USA) for generously supplying uswith allatotropin and allatotropin-antibody.

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