RESEARCH ARTICLE Allatotropin Modulates Myostimulatory and Cardioacceleratory Activities in Rhodnius prolixus (Stal). María José Villalobos-Sambucaro1,2, Alicia Nieves Lorenzo-Figueiras4, Fernando Luis Riccillo1,2, Luis Anibal Diambra2, Fernando Gabriel Noriega3, Jorge Rafael Ronderos1,2* 1 Cátedra Histología y Embriología Animal (FCNyM-UNLP), La Plata, Argentina, 2 Centro Regional de Estudios Genómicos (CREG-UNLP), La Plata, Argentina, 3 Department of Biological Sciences, Florida International University, Miami, Florida, United States of America, 4 Departamento de Biodiversidad y Biología Experimental (IBBEA, FCEyN-UBA), Buenos Aires, Argentina * jrondero@museo.fcnym.unlp.edu.ar Abstract Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the ex- cretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circula- tion as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circu- lation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle con- tractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post- prandial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus. Introduction Juvenile individuals of the kissing bug Rhodnius prolixus (Stal) (Hemiptera: Reduviidae) can ingest a volume of blood up to 12.5 times its unfed weight in a single meal [1]. Consequently large quantities of salts and water must be quickly eliminated in the urine in order to decrease the weight and restore water and mineral balance. Large volumes of urine are produced during PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 1 / 14 OPEN ACCESS Citation: Villalobos-Sambucaro MJ, Lorenzo- Figueiras AN, Riccillo FL, Diambra LA, Noriega FG, Ronderos JR (2015) Allatotropin Modulates Myostimulatory and Cardioacceleratory Activities in Rhodnius prolixus (Stal). PLoS ONE 10(4): e0124131. doi:10.1371/journal.pone.0124131 Academic Editor: Pedro Lagerblad Oliveira, Universidade Federal do Rio de Janeiro, BRAZIL Received: September 10, 2014 Accepted: March 10, 2015 Published: April 21, 2015 Copyright: © 2015 Villalobos-Sambucaro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are available within the paper and its Supporting Information files. Funding: This study was supported by Universidad Nacional de La Plata N559 and N673 to JRR. Competing Interests: The authors have declared that no competing interests exist. http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0124131&domain=pdf http://creativecommons.org/licenses/by/4.0/ the first few hours after feeding [2–6]. Malpighian tubules (MTs) respond by increasing their rate of secretion to produce hypo-osmotic urine and re-establish the osmotic balance [2, 7]. This physiological process is controlled by diuretic and antidiuretic hormones; being serotonin an important regulator of MTs activity [7–9]. Water and ion homeostasis also depends on the ability of the dorsal vessel (DV), composed by the heart and the aorta, to pump haemolymph in a posterior-anterior direction from the abdominal segment 6th along the abdomen to the thorax and head [10]. Since the pioneering studies of Gerould [11] it is known that in several insect species the heart beat alternates between anterograde and retrograde directions. In the mosquito Anopheles gambiae, this reversal pattern of contractions is restricted to the adults [12]. It was later confirmed that this alternating pattern of contractions is also present in other taxa including Orthoptera and Hemiptera [13]. On the contrary, in R. prolixus the DV normal- ly pumps haemolymph only in a postero-anterior direction. Diuresis also depends on the abili- ty of the anterior midgut (crop) to move haemolymph in an antero-posterior direction [2]. In fact immediately after the ingestion of blood, the number of peristaltic waves of the crop in- creases, facilitating haemolymph recirculation and diuresis [2]. In several insect species serotonin is also involved in the regulation of visceral and cardiac muscle contractions. Serotonin increases the heart beat frequency of Drosophila melanogaster larvae [14]. In Agrius convolvuli larvae (Lepidoptera: Sphingidae), serotonin increases the rate of anterograde contractions, and reverses to anterograde direction when applied during the posterograde phase [15]. Serotonin regulates several physiological processes in R. prolixus. In addition to the role as a diuretic factor, serotonin controls other processes during feeding, in- cluding salivation and plasticization of the cuticle [9], serotonin increases the heart rate of con- tractions at a concentration of 10-8 M [16]. Allatotropin (AT), a neuropeptide isolated on the basis of its activity stimulating juvenile hormone synthesis in the lepidoteranManduca sexta [17], has also proved to be multifunction- al, acting in different insect species as myoregulator and cardioaccelerator [18–22]. In R. pro- lixus, AT has no effect modulating heart beat frequency or contractions of the digestive tract under basal conditions [23]. In the related species Triatoma infestans AT increases the frequen- cy of contractions of the DV, crop and hindgut [24, 25]. In unfed adult males of T. infestans, AT has no myoregulatory effect by itself, but synergizes the stimulatory effect of serotonin on the frequency of the DV contractions [25]. Previous studies described that AT released by the MTs controls rectum muscle activity, stimulating peristaltic waves that mix and helps voiding urine and feces [24, 26]. Treatment of 4th-instar larvae with AT-antiserum decreased urine elimination [24]. In T. infestans, AT is present in the nervous system, midgut epithelium and MTs [25–27]; and the production of the peptide in these tissues undergoes daily rhythmic ac- tivities, with highest levels on the afternoon as insects prepare to blood-fed [28]. In the present study, we provide evidence that in R. prolixus AT increases the frequency of contractions of the aorta only when insects have been previously treated with serotonin; sug- gesting a synergistic relationship between these two hormones. The expression of the AT re- ceptor in the target organs (i.e. hindgut/rectum, DV and midgut); and the blockade of urine voiding with AT antiserum, suggest that as in T. infestans, this peptide is involved in physiolog- ical processes associated with post-prandial diuresis, facilitating haemolymph recirculation and deposition of urine and feces. Material and Methods 2.1 Insects Adult males and 4th-instar larvae of R. prolixus were obtained from a colony maintained at 28 ± 2°C, 45% relative humidity, and 12:12 hour light-dark period. All the experiments were Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 2 / 14 performed in vivo. Adult males were immediately isolated after molting and starved during 14 to 21 days before experiments were performed. 4th-instar larvae were immediately isolated after molt (3rd to 4th instar) and also starved during 14 to 21 days. All the experiments were performed with experimental groups conformed by 6 to 14 insects. Insects were fed on phos- phate buffer saline in an artificial feeder and immediately sacrificed after the experiments. 2.2 Myoregulatory bioassays The effect of AT on the contractions of the aorta and the anterior midgut was analyzed in vivo. To perform these experiments, the wings of the insects were removed to expose the dorsal cuti- cle of the abdomen. Due to the transparent nature of the cuticle, the contractions of the aorta and the peristaltic waves of the anterior midgut were clearly recorded [25] (S1 Movie). We test- ed the effect of Aedes aegypti AT (APFRNSEMMTARGF) (Biopeptide, San Diego, CA) [29] which shares a 58.3% identity and 83.3% similarity with the corresponding R. prolixus peptide. The concentrations tested were 10-9 and 10-6 M. Peptides were diluted in 3 μl of R. prolixus sa- line [4]. Controls received only saline. AT was administered through an incision of the conex- ive tissue in the first abdominal segment. To minimize the effect of the stress caused by handling, insects were rested for 30 minutes before the administration of the first treatment. The contractions of the aorta and peristaltic waves of the anterior midgut were observed through the dorsal cuticle (segments IV and V of the abdomen) under a dissection microscope. The number of contractions in a 3-min period was recorded at 5, 15 and 30 minutes after treat- ments [24, 25]. To evaluate the effect on the peristaltic waves of the crop, only those contrac- tions that produce a complete anterior-posterior wave through the abdomen were recorded. Local contractions (usually observed at the level of the segments II and III of the abdomen) were not recorded. All data were collected by the same operator. Results are expressed as num- ber of contractions or peristaltic waves per minute. For experiments involving fed insects, bugs were allowed to feed for 15 minutes, and only those insects fed ad libitum were selected. 2.3 Effect of feeding AT antiserum on diuresis Insects were fed using an artificial feeder developed by Nuñez and Lazzari [30]. Groups of 4th-instar larvae of R. prolixus were fed: 1) buffer phosphate saline (PBS) (controls), 2) AT-anti- serum whose specificity was previously confirmed [24, 26–27, 29] diluted in a PBS solution (1/100) [24], and 3) A solution containing AT-antiserum that was preadsorbed overnight at 4°C with synthetic AT (200 nmol) [24]. All insects were fed during 15 min, and only those in- sects fed ad libitum were selected. Each fed insect was individually placed in microtubes. The volume of urine was measured at 15, 30, 45, 60, 90, 120, 180, 240 min, and 24 h (1440 min). To calculate the quantity of urine produced, the weight of each microtube was recorded before the insect was caged in it. After each period (i.e. 0–15 mins; 15–30; etc.) the insect was removed from the tube and the weight of the tube was recorded. To evaluate the quantity of urine re- leased by the insect, the final weight of the microtube was compared to the original weight. After that, each insect was placed in a new microcentrifuge tube previously weighed to evaluate changes in the next period. In addition, the frequencies of aorta contractions and crop peristal- tic waves were recorded at 15, 30, 45, 60, 90, 120 and 240 min on male insects that were fed ei- ther PBS or a PBS-AT-antiserum solution. 2.4 Identification and characterization of the AT receptor The sequence of theManduca sexta AT receptor (ADX66344.1) was used to search the R. pro- lixus genome (https://www.vectorbase.org) using the TBLASTN algorithm and the Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 3 / 14 https://www.vectorbase.org/ BLOSUM62 matrix. The structure of the RpATr gene was predicted using the Augustus soft- ware (http://augustus.gobics.de). Allatotropin receptor sequences were obtained from databases and used for the align- ments. We aligned the allatotropin receptor sequences using Clustal W (http://www.ebi.ac. uk/Tools/msa/clustalw2/) and JalView 2.7 [31]. The accession numbers of the AT receptors utilized are:Manduca sexta (ADX66344.1), Danaus plexippus (EHJ74388.1), Bombyx mori (NP_001127714.1), Aedes aegypti (AEN03789.1), Tribolium castaneum (XP_973738.2), Bombus terrestris (XP_003402490.1), Bombus impatiens (XP_003486747.1), Apis florea (XP_003690070.1),Megachile rotundata (XP_003708421.1), Harpegnathos saltator (EFN76143.1), Nasonia vitripennis (XP_001604582.2), Schistocerca gregaria (AEX08666.1). The seven transmembrane domains of the AT receptors were determined using the online software InterProScan [32]. AT receptor mRNA tissue expression was studied using RNA extracted from whole 4th in- star larvae and adult R. prolixusMalpighian tubules, DV, hindgut/rectum, ovaries, and midgut. RNA was isolated using RNAeasy kit for RNA isolation according to the specifications of the manufacturers (Qiagen). The RNA was first treated with RNAse-free DNAse Set (Qiagen) to prevent amplification of genomic DNA. First strand cDNA was synthesized using Revert Aid First Strand cDNA Synthesis Kit (Fermentas, USA) and used as template in a PCR reaction with the following two set of primers: 1) Primer Forward 5´—ATGTCCGATGAAGACTATCTG—3´ and primer Reverse 5´—TGTA GATAAGAGGATTAGTGGC—3´. 2) Primer Forward 5´—ATGTCCGATGAAGACTATCTG—3´ and primer Reverse 5´—GTAG AGCACTAATTTGCAGAG—3´ The sequence of the PCR products was confirmed using cDNA sequencing (Unidad de Genó- mica—Instituto de Biotecnología—CICVyA—CNIA—INTA, Argentina). 2.5 Statistical analysis Significant differences were evaluated by multifactorial Analysis of Variance (ANOVA) con- sidering two factors acting on the evaluated variables (i.e. treatment and time of frequency re- cording). Single post-hoc comparisons were tested by the Tukey test. Only differences equal or less than 0.05 were considered significant. Data are expressed as means ± standard error. Results 3.1 Allatotropin does not increase the frequency of aorta contractions under basal conditions Treatment with two different concentrations of AT (10-9 and 10-6 M) did not significantly in- crease the basal frequency (Fig 1A). 3.2 Daily rhythmic contraction of the aorta The basal frequency of aorta contractions displayed daily rhythmic activity; with maximum frequencies in the afternoon (Fig 1B). To analyze variations in the activity of AT associated to daily rhythms, we tested the effect of 10-6 M AT in the morning (low rate) and in the afternoon (high rate). AT did not show effect neither in the morning nor in the afternoon (Fig 1C). Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 4 / 14 http://augustus.gobics.de/ http://www.ebi.ac.uk/Tools/msa/clustalw2/ http://www.ebi.ac.uk/Tools/msa/clustalw2/ Fig 1. Daily rhythmic contraction of the aorta and AT effect on the basal contractions. A) Allatotropin did not showmyoregulatory effect on the basal frequency of contractions of the aorta at doses of 10-9 and 10-6 M. B) Basal frequency of contractions of the aorta of unfed adult males measured during the morning and afternoon. C) AT (10-6 M) does not showmyoregulatory activity on the basal frequency of contractions of the aorta when assayed during the morning or the afternoon. Each bar represents Mean ± standard error (n = 6 for each treatment). Asterisks represent statistically significant differences between frequencies. doi:10.1371/journal.pone.0124131.g001 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 5 / 14 3.3 Allatotropin and serotonin synergistically increased aorta activity To test the synergistic effect of AT and serotonin on aorta contractions, insects were treated first with serotonin (10-9 M), and subsequently injected with AT (10-9 and 10-6 M). Serotonin increased the frequency of aorta contractions, and injection of AT 10-9 M resulted in an addi- tional increment on the frequency of contractions (Fig 2A). The treatment with the highest dose previously tested (10-6 M) caused a maximum and sustained contraction of the DV (data not shown). In the same group of insects the rate of peristaltic waves of the crop was evaluated, but no significant differences were observed (Fig 2B). 3.4 Ingestion of AT antiserum decreased urine elimination, aorta frequency of contractions and rate of peristaltic waves of the crop The amount of urine voided after feeding was significantly lower in 4th-instar larvae fed with anti-AT when compared with those insects fed with saline or with the preadsorbed AT antise- rum (Fig 3A and 3B). The frequencies of contraction of the aorta and the crop peristaltic waves were significantly modified by ingestion of AT-antiserum (Figs 4 and 5). After a period of 90 mins during which the rate of waves was significantly decreased, crop contraction frequencies returned to control levels (Fig 4A and 4B). The behavior of the DV was similar, but covering a shorter period of time (Fig 5A and 5B). 3.5 Genomic characterization and expression of the R. prolixus AT receptor The R. prolixus AT receptor is composed of five exons and four introns (Fig 6A). The predicted mature RNA encodes a 334 amino acid protein that includes the classical seven transmem- brane domains characteristic of this family of receptors (Fig 6A; S1 File). The expression of the receptor was confirmed in several organs, including midgut, hindgut/rectum and DV (Fig 6B). Comparison of the fragment sequenced of the R. prolixus AT receptor with Allatotropin/orexin orthologous from other insect species revealed a high degree of conservation such as 60% of identity and 74% of similarity withM. sexta (S1 Fig). Discussion R. prolixus and T. infestans are major vectors of Chagas disease in Latin America. Transmission occurs when insects feed, releasing together with urine and feces the infective form of Trypano- soma cruzi. Understanding the regulation of diuresis is therefore critical. The use of specific an- tiserums provided with the meal to alter the biology of blood-sucking vectors has been already demonstrated in several species [33–35], but not in triatominae insects. We have previously re- ported in the related species T. infestans that the injection of the AT-antiserum in the haemo- coel, compared with insects that receive saline or a preadsorbed AT-antiserum solution, prevents the release of urine inhibiting the voiding of the rectum, showing that the presence of the antiserum in the haemocoel is able to specifically capture the peptide and block of its activi- ty [24]. The experiments performed in this study show similar results strongly suggesting that, the antiserum administrated with the meal reaches the haemolymph, capturing the circulating peptide and inhibiting the ability of the rectum to release the urine, and also inhibiting the fre- quency of contractions of the DV. The possibility of delaying, or even blocking the elimination of urine and feces after a blood meal, provides new potential avenues for the control of diseases transmitted by Triatominae insects [24]. It has been previously reported the presence of allatotropic axons innervating the aorta, midgut and hindgut in R. prolixus. In these studies AT stimulated muscles in the ducts and the Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 6 / 14 Fig 2. Synergistic activity of AT on the increase of the frequency of contractions of the aorta induced by serotonin. A) AT treatment (10-9 M) induces a further increase on the frequency of contractions of the aorta after the increment produced by serotonin (10-9 M). B) The rate of the peristaltic waves of the anterior midgut are not significantly modified by serotonin or AT treatments. Each bar represents Mean ± standard error. Different letters represent statistically significant differences between treatments (n = 6 for each treatment). doi:10.1371/journal.pone.0124131.g002 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 7 / 14 Fig 3. Allatotropin antiserum supplied with the meal decreases urine elimination, aorta frequency of contractions and rate of peristaltic waves of the crop. A) Effect of AT-antiserum on the urine released during the first 24 h (1440 min) after a blood meal.B) Effect of anti-AT supplied with the meal on the accumulated urine released by 4th-instar larvae. Each data represents Mean ± standard error. Asterisks represent statistically significant differences between treatments. doi:10.1371/journal.pone.0124131.g003 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 8 / 14 Fig 4. Inhibition of the peristaltic wave rate of the crop induced by the administration of the AT-antiserumwith the meal. A) Peristaltic wave rate in each time recorded along 240 mins after meal.B) Comparison of the peristaltic wave rate of the crop along the first 120 mins after a meal. Each point represents Mean ± standard error. Asterisks in each graph represent statistically significant differences between treatments. doi:10.1371/journal.pone.0124131.g004 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 9 / 14 Fig 5. Decrease of the frequency of contractions of the aorta after the administration of the AT-antiserumwith the meal. A) Frequency of contractions in each time recorded along 240 mins after a meal.B) Comparison of the peristaltic wave rate of the crop along the first 90 mins after a meal. Each point represents Mean ± standard error. Asterisks represent statistically significant differences between treatments. doi:10.1371/journal.pone.0124131.g005 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 10 / 14 secretory portions of the salivary glands, increasing the expulsion of saliva, but failed to exert any myostimulatory effects on the digestive system or the DV under basal conditions [23]. In T. infestans, AT immunoreactivity is present in the aorta and the anterior midgut of 4th-instar lar- vae [27] and adults [25]. Allatotropic cells resembling open type secretory cells were found in the epithelial sheet of the crop in 4th-instar larvae and adults [25, 27]. In T. infestans, AT in- creased the contractions of the digestive tract (midgut and hindgut/rectum) and the DV [24, 25] only when the tissues were previously stimulated. AT regulatory activities on the peristaltic waves of the rectum were also confirmed by injecting 4th-instar larvae with anti-AT antiserum; a treatment that resulted in a decrease in the total quantity of urine eliminated [24]. In the pres- ent study we observed a similar phenomenon when 4th-instar larvae of R. prolixus were fed with a saline solution containing the same anti-AT antiserum; while controls fed with saline or AT- antiserum preadsorbed with the peptide displayed no differences in the quantity of urine voided. When the effect of feeding AT-antiserum was evaluated in adults, we observed a significant de- crease in the peristaltic activity of the crop and the frequency of contractions of the aorta, show- ing that the antiserum was able to pass across the wall of the crop reaching the haemolymph. These results matched those of previous studies in T. infestans, that revealed myostimulatory ac- tivities of AT on the crop, associated with the presence of open type epithelial cells, which sug- gested that the peptide was acting in a paracrine mode [25]. The existence of daily rhythms in triatominae insects is widely documented [36, 37]. The basal frequency of aorta contractions displayed daily rhythmic contractions, with maximum frequencies in the afternoon when insects were preparing for blood-feeding. In T. infestans, the levels of AT in the midgut and MTs vary along a 24 h period, reaching their highest amounts a few hours previous to the beginning of the dark period when the insect is preparing for blood feeding [28]. AT did not display cardioacceleratory effects when it was assayed on non-stimulated tissues (basal frequency), neither when assayed on high nor low frequencies (i.e. morning and after- noon respectively), confirming results previously reported by Masood and Orchard [23]. How- ever, we found that AT was very effective when applied after the DV has been previously Fig 6. Characterization and expression of the R. prolixusAT receptor. A) Structure of the RpATr showing the existence of five exons and a predicted protein containing the typical seven transmembrane domains.B) RpATr expression in whole 4th-instar larvae and aorta, mid-gut and hindgut/rectum of adult. doi:10.1371/journal.pone.0124131.g006 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 11 / 14 stimulated with serotonin. These results confirm those previously described for T. infestans, re- porting that AT synergizes the effect of serotonin [25]. This synergistic activity between seroto- nin and neuropeptides was also observed in D.melanogaster with the FMRFamide-related peptides. In fact, it was shown that the effect of FMRFamide varies when it is administered alone (having no effect), but causing an increment of the frequency of the aorta when it is ap- plied together con serotonin [38]. The R. prolixus AT receptor is a GPCR that shares a high degree of sequence similarity with AT receptors from other insects [39–42]. A highly conserved sequence in the TM7 (YAN- SCAN[V/I/T]PI), which is considered a mammalian orexin receptor signature [36], is still recognisable in the insect receptors (S1 Fig). Although allatotropin and orexin are not structur- ally related, it is interesting that both peptides have been implicated with the regulation of food intake, starvation and energy metabolism, and their receptors seem to share a common ances- tor [43]. The receptor, expressed in aorta, midgut and hindgut/rectum tissues, is the first AT re- ceptor characterized for a hemimetabolous insect species, and supports the hypothesis that AT is acting as a myomodulator in R. prolixus, being involved in post-feeding diuresis. In summary all together in the present work we provide evidence that AT acting synergistically with seroto- nin plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus. Supporting Information S1 Fig. RpATr sequences from several holometabolous and hemimetabolous species. (PDF) S1 File. Comparison of RpATr genomic and cloned nucleotide sequences; and predicted amino acid sequence.Highlighted letters correspond to differences between predicted and cloned sequences. (PDF) S1 Movie. Movie showing the anterograde contractions of the aorta (running from top to bottom in the movie) and the peristaltic waves of the crop, running in the opposite direc- tion (from bottom to top in the movie). (MP4) Acknowledgments This research was supported by funds provided by the National University of La Plata. Author Contributions Conceived and designed the experiments: JRR. Performed the experiments: MJVS FLR. Ana- lyzed the data: JRR MJVS FGN LAD. Contributed reagents/materials/analysis tools: JRR FGN LAD. Wrote the paper: JRR FGN. Wrote the paper: JRR. Critically revised the manuscript: FGN. References 1. Buxton PA (1930). The biology of a blood-sucking bug, Rhodnius prolixus. Trans R Entomol Soc Lon- don 78: 227–236 2. Maddrell SHP (1964). Excretion in the blood-sucking bug Rhodnius prolixus Stal. II. The normal course of diuresis and the effect of temperature. J Exp Biol 41: 163–176 PMID: 14161607 3. Maddrell SHP (1978). Physiological discontinuity in an epithelium with an apparently uniform structure. J Exp Biol 75: 133–145 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 12 / 14 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0124131.s001 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0124131.s002 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0124131.s003 http://www.ncbi.nlm.nih.gov/pubmed/14161607 4. Maddrell SHP, O'Donnell MJ, Caffrey R. (1993). The regulation of haemolymph potassium activity dur- ing initiation and maintenance of diuresis in fed Rhodnius prolixus. J Exp Biol 177: 273–285. PMID: 8486999 5. O'Donnell M J, Ianowski JP, Linton SM, Rheault MR (2003). Inorganic and organic anion transport by insect renal epithelia. Biochim Biophys Acta 1618: 194–206 PMID: 14729156 6. Ramsay JA (1952). The excretion of sodium and potassium by the Malpighian tubules of Rhodnius. J Exp Biol 29: 110–126 7. Maddrell SHP, Phillips JE (1975). Secretion of hypo-osmotic fluid by the lower Malpighian tubules of Rhodnius prolixus. J Exp Biol 62: 671–683 8. Maddrell SHP, HermanWS, Mooney RL, Overton JA (1991). 5-hydroxytryptamine: a second diuretic hormone in Rhodnius prolixus. J Exp Biol 156: 557–566 PMID: 2051136 9. Orchard I (2006). Serotonin: A coordinator of feeding-related physiological events in the blood-gorging bug, Rhodnius prolixus. Comp Biochem Physiol A 144: 316–324 PMID: 16377224 10. Chiang RG, Chiang JA, Davey KG (1990). Morphology of the dorsal vessel in the abdomen of the blood-feeding insect Rhodnius prolixus. J Morphol 204: 9–23 PMID: 2187100 11. Gerould JH (1933). Orders of insects with heart-beat reversal. Biol Bull. 64: 424–431 12. League GP, Onuh OC, Hillyer JF (2015). Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae. J Exp Biol. 218: 370–380 doi: 10.1242/jeb.114942 PMID: 25524976 13. Glenn JD; King JG; Hillyer JF (2010) Structural mechanics of the mosquito heart and its function in bidi- rectional hemolymph transport. J Exp Biol. 213: 541–550 doi: 10.1242/jeb.035014 PMID: 20118304 14. Dasari S; Cooper RL (2006). Direct influence of serotonin on the larval heart of Drosophila melanoga- ster. J Comp Physiol B. 176: 349–357 PMID: 16362307 15. Matsushita T; Kuwasawa K; Uchimura K; Ai H; Kurokawa M (2002). Biogenic amines evoke heartbeat reversal in larvae of the sweet potato hornworm, Agrius convolvuli. Comp Biochem Physiol A. 133: 625–636 16. Chiang RG; Chiang JA; Davey K.G. (1992). A sensory input inhibiting heart rate in an insect, Rhodnius prolixus. Experientia 48: 1122–1125 17. Kataoka H, Toschi A, Li JP, Carney RL, Schooley DA, Kramer SJ (1989). Identification of an allatotropin from adultManduca sexta. Science 243: 1481–1483. PMID: 17839751 18. Duve H, East PD, Thorpe A (1999). Regulation of lepidopteran foregut movement by allatostatins and allatotropin from the frontal ganglion. J Comp Neurol 413: 405–416 PMID: 10502248 19. Duve H, Audsley N, Weaver RJ, Thorpe A (2000). Triple co-localization of two types of allatostatin and an allatotropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut. Cell Tissue Res 300: 153–163 PMID: 10805084 20. Koladich PM, Cusson M, BendenaWG, Tobe SS, McNeil JN (2002). Cardioacceleratory effects of Manduca sexta allatotropin in the true armywormmoth Pseudaletia unipuncta. Peptides 23: 645–651 PMID: 11897383 21. Rudwall AJ, Sliwowska J, Nässel DR (2000). Allatotropin-like neuropeptide in the cockroach abdominal nervous system: Myotropic actions, sexually dimorphic distribution and co-localization with serotonin. J Comp Neurol 428: 159–173 PMID: 11058230 22. Veenstra JA, Lehman HK, Davis NT (1994). Allatotropin is a cardioacceleratory peptide inManduca sexta. J Exp Biol 188: 347–354. PMID: 7964382 23. Masood M, Orchard I (2013). Molecular characterization and possible biological roles of allatotropin in Rhodnius prolixus. Peptides. 53: 159–171. doi: 10.1016/j.peptides.2013.10.017 PMID: 24177575 24. Santini MS, Ronderos JR (2007). Allatotropin-like peptide released by Malpighian tubules induces hind- gut activity associated to diuresis in the Chagas disease vector Triatoma infestans (Klüg). J Exp Biol 210: 1986–1991. PMID: 17515423 25. Sterkel M, Riccillo FL, Ronderos JR (2010). Cardioacceleratory and myostimulatory activity of allatotro- pin in Triatoma infestans (Klüg). Comp Biochem Physiol A 155: 371–377. doi: 10.1016/j.cbpa.2009.12. 002 PMID: 19995615 26. Santini MS, Ronderos JR (2009). Allatotropin-like peptide in Malpighian tubules: Insect renal tubules as an autonomous endocrine organ. Gen Comp Endocrinol 160: 243–249 doi: 10.1016/j.ygcen.2008.12. 002 PMID: 19118556 27. Riccillo FL, Ronderos JR (2010). Allatotropin expression during the development of the fourth instar lar- vae of the kissing-bug Triatoma infestans (Klüg). Tissue Cell 42: 355–359 doi: 10.1016/j.tice.2010.07. 011 PMID: 20817237 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 13 / 14 http://www.ncbi.nlm.nih.gov/pubmed/8486999 http://www.ncbi.nlm.nih.gov/pubmed/14729156 http://www.ncbi.nlm.nih.gov/pubmed/2051136 http://www.ncbi.nlm.nih.gov/pubmed/16377224 http://www.ncbi.nlm.nih.gov/pubmed/2187100 http://dx.doi.org/10.1242/jeb.114942 http://www.ncbi.nlm.nih.gov/pubmed/25524976 http://dx.doi.org/10.1242/jeb.035014 http://www.ncbi.nlm.nih.gov/pubmed/20118304 http://www.ncbi.nlm.nih.gov/pubmed/16362307 http://www.ncbi.nlm.nih.gov/pubmed/17839751 http://www.ncbi.nlm.nih.gov/pubmed/10502248 http://www.ncbi.nlm.nih.gov/pubmed/10805084 http://www.ncbi.nlm.nih.gov/pubmed/11897383 http://www.ncbi.nlm.nih.gov/pubmed/11058230 http://www.ncbi.nlm.nih.gov/pubmed/7964382 http://dx.doi.org/10.1016/j.peptides.2013.10.017 http://www.ncbi.nlm.nih.gov/pubmed/24177575 http://www.ncbi.nlm.nih.gov/pubmed/17515423 http://dx.doi.org/10.1016/j.cbpa.2009.12.002 http://dx.doi.org/10.1016/j.cbpa.2009.12.002 http://www.ncbi.nlm.nih.gov/pubmed/19995615 http://dx.doi.org/10.1016/j.ygcen.2008.12.002 http://dx.doi.org/10.1016/j.ygcen.2008.12.002 http://www.ncbi.nlm.nih.gov/pubmed/19118556 http://dx.doi.org/10.1016/j.tice.2010.07.011 http://dx.doi.org/10.1016/j.tice.2010.07.011 http://www.ncbi.nlm.nih.gov/pubmed/20817237 28. Santini MS, Ronderos JR (2009). Daily variation of an allatotropin-like peptide in the Chagas disease vector Triatoma infestans (Klug). Biol Rhythm Res 40: 299–306. 29. Hernández-Martínez S, Li Y, Lanz-Mendoza H, Rodriguez MH, Noriega FG (2005). Immunostaining for allatotropin and allatostatin-A and -C in the mosquitoes Aedes aegypti and Anopheles albimanus. Cell Tissue Res 321: 105–113. PMID: 15909164 30. Nuñez JA, Lazzari CR (1990). Rearing of Triatoma infestans Klug (Heteroptera, Reduviidae) in the ab- sence of a live host I. Some factors affecting the artificial feeding. J Appl Ent 109: 87–92. 31. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009). JalView version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191. doi: 10.1093/ bioinformatics/btp033 PMID: 19151095 32. Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam H, et al. (2014). InterProScan 5: genome-scale protein function classification. Bioinformatics 30: 1236–1240 doi: 10.1093/ bioinformatics/btu031 PMID: 24451626 33. Allingham PG, Kerling RL, Tellam RL, Briscoe SJ, Standfast HA (1992). Passage of host immunoglobu- lin across the mid-gut epithelium into the haemolymph of blood-fed buffalo flies Haematobia irritans exi- gua. J Insect Physiol 38: 9–17 34. Ben-Yakir D (1989). Quantitative studies on host immunoglobulin G in the hemolymph of ticks (Acari). J Med Entomol 26: 243–246 PMID: 2769701 35. Kumari A, Gakhar SK, Hooda V (2009). Antibodies raised against hemolymph of Anopheles culicifacies reduce the fecundity and malaria parasite development. J. Vector Borne Dis. 46: 255–260 PMID: 19959850 36. Steel CGH, Vafopoulou X (2006). Circadian orchestration of developmental hormones in the insect, Rhodnius prolixus. Comp Biochem Physiol A. 144: 351–364 37. Vafopoulou X, Terry KL, Steel CGH (2010). The Circadian Timing System in the Brain of the Fifth Larval Instar of Rhodnius prolixus (Hemiptera). J Comp Neurol 518: 1264–1282 doi: 10.1002/cne.22274 PMID: 20151359 38. Nichols R (2006). FMRFamide-related peptides and serotonin regulate Drosophila melanogaster heart rate: Mechanisms and structure requirements. Peptides 27: 1130–1137. PMID: 16516344 39. Vuerinckx K, Verlinder H, Lindermans M, Vanden Broeck J, Huybrechts R (2011) Characterization of an allatotropin-like peptide receptor in the red flour beetle, Tribolium castaneum. Insect BiochemMolec Biol. 41: 815–822. 40. Horodyski FM, Verlinder H, Filkin N, Vandermissen HP, Fleury C, Reynolds SE, et al. (2011). Isolation and functional characterization of an allatotropin receptor fromManduca sexta. Insect BiochemMolec Biol. 41: 804–814 41. Nouzova M, Mayoral JM, Brockhoff A, Goodwin M, Meyerhof W, Noriega FG (2012). Functional charac- terization of an allatotropin receptor expressed in the corpora allata of mosquitoes. Peptides. 34:201–208. doi: 10.1016/j.peptides.2011.07.025 PMID: 21839791 42. Voisin T, Rouet-Benzineb P, Reuter N, Laburthe M (2003). Orexins and their receptors: structural as- pects and role in peripheral tissues. Cell Mol Life Sci 60: 72–87. PMID: 12613659 43. Alzugaray ME, Adami ML, Diambra AL, Hernández-Martínez S, Damborenea C, Noriega FG, et al. (2013). Allatotropin: An Ancestral Myotropic Neuropeptide Involved in Feeding. PLoS One. 8: e77520 doi: 10.1371/journal.pone.0077520 PMID: 24143240 Myoregulatory Peptides in Rhodnius PLOSONE | DOI:10.1371/journal.pone.0124131 April 21, 2015 14 / 14 http://www.ncbi.nlm.nih.gov/pubmed/15909164 http://dx.doi.org/10.1093/bioinformatics/btp033 http://dx.doi.org/10.1093/bioinformatics/btp033 http://www.ncbi.nlm.nih.gov/pubmed/19151095 http://dx.doi.org/10.1093/bioinformatics/btu031 http://dx.doi.org/10.1093/bioinformatics/btu031 http://www.ncbi.nlm.nih.gov/pubmed/24451626 http://www.ncbi.nlm.nih.gov/pubmed/2769701 http://www.ncbi.nlm.nih.gov/pubmed/19959850 http://dx.doi.org/10.1002/cne.22274 http://www.ncbi.nlm.nih.gov/pubmed/20151359 http://www.ncbi.nlm.nih.gov/pubmed/16516344 http://dx.doi.org/10.1016/j.peptides.2011.07.025 http://www.ncbi.nlm.nih.gov/pubmed/21839791 http://www.ncbi.nlm.nih.gov/pubmed/12613659 http://dx.doi.org/10.1371/journal.pone.0077520 http://www.ncbi.nlm.nih.gov/pubmed/24143240