Mechanisms controlling haemolymph circulation under resting conditions in the Chagas disease vector Rhodnius prolixus Free

It was shown that individuals of Rhodnius prolixus (Hemiptera: Reduviidae) can ingest up to 12 times their weight in a few minutes (Buxton, 1930). Consequently, during and after feeding, the insect is challenged with sudden physiological changes, such as an increase in weight, an imbalance of ionic equilibrium, an excess of water, changes in the osmotic concentration of the haemolymph, etc. (Orchard, 2006). Once the insect starts feeding, a physiologically critical period begins (post-prandial diuresis). In fact, to re-stablish homeostasis, the insect eliminates urine at high rates for more than 180 min (Maddrell, 1963, 1964a). This process is driven by diuretic hormones, as serotonin (5-HT) (Maddrell, 1964b), which activates Malpighian tubules (MTs) (Maddrell et al., 1991, 1993a).

MTs constitute the main excretory system in insects. They consist of a variable number of epithelial tubules located in the haemocoel bathed by the haemolymph. In triatomine insects, the MT system is made up of four independent tubules, each one presenting a blind distal end and a proximal one, opening at the junction between the posterior midgut and the hindgut (Wigglesworth, 1931; Wigglesworth and Salpeter, 1962). The urine produced by MTs drains into the rectum together with faeces coming from the midgut, where they are mixed by peristaltic movements and periodically eliminated (Santini and Ronderos, 2007).

MTs are not innervated, as they are modulated in a neuroendocrine way. 5-HT is released from the mesothorax ganglion to the haemolymph immediately after the insect begins to feed (Maddrell, 1964b), stimulating MT activity, which express a serotonin type-2b receptor (Paluzzi et al., 2015). To ensure the proper activity of the MTs, the secretion of diuretic hormones is accompanied by the increase of the speed of haemolymph recirculation (Maddrell, 1964a). To do that, powerful peristaltic movements of the anterior midgut (crop) constitute the main factor causing a fast movement of haemolymph in an antero-posterior direction (Maddrell, 1964a). The recirculation is completed by an increase of the frequency of contractions of the dorsal vessel (DV) (Sterkel et al., 2010; Villalobos Sambucaro et al., 2015).

As stated above, the haemolymph moves in a postero-anterior direction by mean of the DV contractions. The DV is a muscular system constituted by the heart, which is located at the final segments of the abdomen, associated with the alary muscles (Fig. 1A–C), and the aorta (Fig. 1D), which runs throughout the abdomen up to the thorax, opening at the level of the head bathing all the organs (Chiang et al., 1990; Villalobos Sambucaro et al., 2015). The heart and the aorta present little holes (ostia) by which haemolymph enters the circuit, from the haemocoel, to be pumped (Fig. 1C,D). Briefly, the alary muscles and the heart contract alternately. When the heart is relaxed, alary muscle contraction results in an expansion of the cardiac chamber, producing a difference of pressure and inducing haemolymph entry into the heart across the ostia. Finally, muscle heart contraction pumps the haemolymph to the aorta. Unlike some species that alternate flow direction through the DV (Gerould, 1933; Glenn et al., 2010; League et al., 2015), in the Triatominae this organ normally pumps only in the anterograde direction (Villalobos Sambucaro et al., 2015).

Previous research has shown that during post-prandial diuresis, MTs secrete the neuropeptide allatotropin (AT), which stimulates the muscle walls of the rectum, inducing peristaltic contractions and voiding (Santini and Ronderos, 2007, 2009a,b). It was also shown that, during this period, at least three different cellular messengers are involved in a highly synchronized cross-talk. Indeed, 5-HT triggers an increase of the aorta contractions and the peristaltic activity of the crop, and this effect is increased by AT (Sterkel et al., 2010; Villalobos Sambucaro et al., 2015). Remarkably, under in vivo conditions AT does not produce any effect by itself (Masood and Orchard, 2014; Sterkel et al., 2010; Villalobos Sambucaro et al., 2015), as its myostimulatory effect depends on the presence of 5-HT (Sterkel et al., 2010; Villalobos Sambucaro et al., 2015, 2016). This effect is inhibited by allatostatin-C (AST-C), which counteracts the effect of AT, decreasing both the rate of peristaltic waves of the crop and the frequency of contractions of the aorta (Villalobos Sambucaro et al., 2016). Both the aorta and crop express receptors for these three messengers (i.e. 5-HT, AT and AST-C) (Paluzzi et al., 2015; Villalobos Sambucaro et al., 2015, 2016). Moreover, the crop and the cephalic portion of the aorta are innervated with allatotropic fibres (Masood and Orchard, 2014; Riccillo and Ronderos, 2010; Sterkel et al., 2010). Allatotropic cells are also present in the epithelial sheet of the crop (Riccillo and Ronderos, 2010; Sterkel et al., 2010).

Triatomine insects can spend long times without food, thus post-prandial diuresis only occurs a few times in life. Between meals, the insects are exposed to different stimuli, such as changes in temperature, which may be transduced by the insect as the presence of a potential host (endothermic animal) (Lazzari and Nuñez, 1989), presence of predators, etc., modifying insect activity. Although most of the previous studies were focused on the post-prandial period, the resting period between meals has been largely ignored. Understanding its physiology could be of interest to identify new and alternatively control methodologies for triatomine populations.

The main goal of this study was to understand how cardiac and crop muscle activity is controlled during the resting period between meals, which represents the longest period during insect life. We analysed different canonical ways that might be acting to maintain a beating rhythm to sustain haemolymph circulation during periods in between meals. We show that, besides the existence of myogenic activity, the rate of contractions is modulated by peptidic messengers, but also by proteins sensing mechanical forces associated with plasma membranes that modulate electric activity of the cell (i.e. Piezo proteins). Piezo proteins are a recently identified family of mechanosensitive, cation channel proteins originally characterized in mice (Piezo1 and Piezo2). Similar proteins have been found in all organisms, including plants (Coste et al., 2010). Indeed, it has been proposed in Drosophila melanogaster that Piezo proteins regulate heart contractions by modulating intracellular Ca²⁺ concentration of the cardiomyocytes (Kim et al., 2012; Zechini et al., 2022). Based on experimental designs involving Piezo protein agonists, we found that DV contractions, as well as peristaltic movements of the crop, depend on Piezo-like proteins. Moreover, we found that the R. prolixus genome predicts the expression of a protein that presents homology with other previously characterized Piezo proteins.

Briefly, the main hypotheses of this study are: (1) myogenic activity of the heart maintains a basal rhythm of contractions acting by L-type voltage-gated calcium channels (VGCCs) that facilitate an influx of calcium inducing the opening of ryanodine receptors to increase cytosolic Ca²⁺ concentration; (2) the myogenic activity may be modulated by peptidic signals such allatotropin and allatostatin-C; and (3) the peristaltic activity of the crop, which is necessary to move haemolymph in an antero-posterior direction depends on the activity of mechanosensitive proteins (i.e. Piezo proteins). Mechanisms controlling haemolymph circulation under resting conditions involves a complex system of signals. The understanding of these regulatory mechanisms may open new windows to develop alternative methodologies of vector control.

Because reproductive processes in female Rhodnius prolixus Stål 1859 involve more physiological variables, we decide to perform the experiments using males, which are easier to standardize. Adult males were obtained from a colony maintained at 28±2°C and 30% relative humidity under a 12 h:12 h light:dark period. Recently emerged, non-fed adult males were starved for at least 21 days to ensure that individuals were in a resting state and ready to look for food.

As in previous studies (Sterkel et al., 2010; Villalobos Sambucaro et al., 2015, 2016), assays were designed to study the frequency of contractions of the DV and the rate of peristaltic contractions of the crop in vivo. After emergence, insects were starved for at least 21 days ensuring that the crop was empty, considering this situation as the resting condition (Riccillo and Ronderos, 2010; Santini and Ronderos, 2007).

Briefly, each individual was placed under a stereoscopic microscope with a digital camera annexed. The legs were held with plasticine on a Petri dish and the wings removed, to allow aorta and crop visualization through the translucent dorsal cuticula. All the treatments were prepared in R. prolixus saline (modified from Maddrell et al., 1993b) and applied directly into the abdomen through a small incision in the conexive using a Hamilton micro-syringe (5 µl). To minimize the effect of the stress caused by handling, insects were allowed to rest for 30 min before treatment administration. The contractions of the aorta and crop were observed through the dorsal cuticle (segments III to V of the abdomen). The number of contractions in a 3-min period was registered at 5, 15 and 30 min after treatments, and all data were recorded by the same operator. Results are expressed as number of contractions per minute.

The calcium ion is a very important factor in cellular physiology. Indeed, it acts as a second messenger, and induces exocytosis and muscle contraction, among others. With the goal of determining the relevance and mechanism of action of Ca²⁺, a set of drugs that modulate calcium availability were tested.

Different compounds previously assayed in our laboratory have been used to study the relevance of Ca²⁺ in the activity of the aorta and crop (Alzugaray and Ronderos, 2018; Alzugaray et al., 2019, 2021). The drugs employed and the concentrations used are as follows: EDTA (Sigma-Aldrich, 40 mmol l⁻¹), a calcium chelator that does not penetrate the cell; BAPTA/AM [1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid; Tocris Bioscience; 300 and 400 µmol l⁻¹], a high-affinity and membrane-permeable (intracellular) Ca²⁺ chelator (Tsien, 1981); nifedipine (Sigma-Aldrich; 30 and 60 µmol l⁻¹), a blocker of L-type voltage-sensitive calcium channels (Guazzi et al., 1977; Zsotér and Church, 1983); xestospongin-C (Xe-C) (Tocris Bioscience; 6 μmol l⁻¹), a membrane-permeable compound isolated from the sponge Xestospongia sp., that inhibits inositol 1,4,5 triphosphate calcium receptor/channel (IP₃R) in the endoplasmic reticulum (ER) (Gafni et al., 1997; Kiselyov et al., 1998); and ryanodine (Ry) (Tocris Bioscience; 10 μmol l⁻¹ and 10 nmol l⁻¹), a diterpenoid alkaloid of plant origin that specifically modifies the activity of the ryanodine receptor (RyR) calcium channels in the ER, maintaining them in an open state at nanomolar concentrations, and closed at micromolar concentrations (Meissner, 1986). According to Bull et al. (1989), this phenomenon might be due to the existence of high and low affinity binding sites for Ry in the ER.

To test whether the G-protein-coupled receptor (GPCR) signalling cascade modulates the frequency of contractions of the organs under study, we assayed the effect of the following drugs, which are associated with GPCR signalling and transduction. Sulfhydrylreactive hydrobromide (SCH-202676) (Tocris Bioscience; 1, 5 and 10 μmol l⁻¹) reversibly inhibits agonist and antagonist binding to a variety of GPCRs in mammalian and non-mammalian cells (Alzugaray et al., 2021; Fawzi et al., 2001; Gao et al., 2004; Hartz et al., 2008; Lewandowicz et al., 2006). U73122 is an inhibitor of phospholipase-C (PLC) (Tocris Bioscience; 10 μmol l⁻¹), an enzyme that produces IP₃ when activated through a GPCR coupled to a G_αq, resulting in an increase of cytosolic Ca²⁺ level by activation of IP₃R in the ER (Bleasdale et al., 1990; Smith et al., 1990).

To determine for the probable interaction between AT (Kataoka et al., 1989), which induces an increase of aorta contractions depending on the presence of 5-HT, and AST-C (Kramer et al., 1991), which antagonises its activity, we assayed the effect of AT (10⁻⁹ mol l⁻¹) (Center for Biotechnology Research, Kansas State University) (Li et al., 2003) on the activity of the DV and the crop, in the presence of an AST-C-antiserum (1/500) (Genemed Synthesis, San Francisco, CA, USA) (Li et al., 2004).

To analyse the involvement of mechanical factors and Piezo proteins in the myoregulatory control of aorta and crop, we assayed the effect of two Piezo agonists, Jedi1 and Jedi2 (Sigma-Aldrich; 250 and 2.5 μmol l⁻¹, respectively), which act as Piezo1 chemical activators (Wang et al., 2018).

To determine the existence of Piezo-like proteins in R. prolixus, we searched for homologues of Piezo proteins in the genome and transcriptome public databases (www.vectorbase.org). The sequences corresponding to Mus musculus (Chordata: Mammalia) Piezo1 (ADN28064) and Piezo2 (ADN28065), were used to look for putative piezo proteins in R. prolixus, and several phyla of Opisthokonta (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences corresponding to Piezo1 and Piezo2, the species included and their accession numbers are: R. prolixus (Arthropoda) RPRC014024; Lingula anatina (Brachiopoda) XP_013404042; Owenia fusiformis (Annelida) CAH1780572; Octopus sinensis (Mollusca) XP_029647893; Trichoplax sp. (Placozoa) RDD46920; Hydra vulgaris (Cnidaria) XP_047142278; Rozella allomycis (Fungi) EPZ31590; Amphimedon queenslandica (Porifera) XP_019849310; Beroe abyssicola (Ctenophora) AQX17753; and Salpingoeca rosetta (Choanoflagellata) XP_004997271.

To determine Piezo protein characteristic domains, the sequence was further analysed with InterproScan (www.ebi.ac.uk/interpro/result/InterProScan/#table). The transmembrane domain analysis was performed using TMHMM 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/).

The selected sequences were aligned using the Clustal Omega multiple sequence alignment program (www.ebi.ac.uk/Tools/msa/clustalo/) and post analysed with JalView 2.7 (Waterhouse et al., 2009).

Finally, the probable phylogenetic relationship and the evolutionary analyses were performed by means of the maximum likelihood methodology (500 bootstrap replicates) using MEGAX64 software (Tamura et al., 2013).

Significant differences were evaluated by one-way ANOVA. Single post hoc comparisons were tested by the least significant difference (LSD) test. Only differences equal to or less than 0.05 were considered significant. All experimental groups constituted 4 to 10 individuals. Data are expressed as means±s.e.m.

An increase of cytosolic calcium concentration is necessary to induce muscle contraction. As a first approach to test the relevance of cytosolic calcium on the frequency of contractions of the aorta, we analysed the effect of BAPTA/AM, a cell membrane permeable chelator, at two different concentrations. As expected, the results show that at both concentrations (300 and 400 µmol l⁻¹), the frequency of contractions diminished (the effect being longer at the highest concentration tested), showing that an increase of cytosolic calcium is necessary to cause the contraction of the aorta (Fig. 2A,B; Table S1). The use of an extracellular chelator as EDTA, completely avoid aorta contraction suggesting that a Ca²⁺ influx from the extracellular matrix is necessary to cause a contraction (Fig. 2C; Table S1).

In view of the results obtained using EDTA, showing the relevance of the extracellular source of Ca²⁺, we tested one of the most common pathways of calcium entry, the L-type VGCCs located at the plasma membrane. To do that, we used the compound nifedipine, a molecule that selectively blocks VGCCs. The results showed a statistically significant transient decrease of the frequency of contractions, confirming the importance of the extracellular Ca²⁺ influx. Indeed, these results suggest that L-type VGCCs are a key mechanism for the muscle contraction of the aorta (Fig. 3A; Table S2).

Extracellular calcium entry induces an increase of the cytosolic concentration of this ion, which triggers the opening of RyR channels at the level of the ER. To test the existence of this pathway, the effect of micromolar concentrations of Ry was tested. Although at nanomolar concentrations Ry stimulates the opening of RyRs (facilitating the outcome of Ca²⁺ from the ER), at micromolar concentrations it has an inhibitory effect (Meissner, 1986). The results showed that inhibitory concentration of Ry (10 μmol l⁻¹) decreased contractile activity, suggesting that Ca²⁺ influx facilitates the increase of cytosolic calcium through the opening of RyR to induce muscle contraction (Fig. 3B; Table S2). To confirm the presence of RyR, we performed a similar assay challenging the aorta to a stimulatory concentration of Ry (10 nmol l⁻¹) (Fig. 3C; Table S2), which stimulated aorta contractions as expected.

As both treatments (i.e. nifedipine and Ry) produce a decrease but do not completely inhibit the contractile activity of the aorta, the existence of a response mediated by extracellular messengers was investigated. Considering that the activity of numerous cellular messengers is mediated by GPCRs, which involve the IP₃/IP₃R pathway to increase the cytosolic Ca²⁺ concentration, we tested the effect of Xe-C (a compound originally isolated from the sponge Xestospongia exigua, which selectively inhibits the opening of IP₃R at the level of ER). A statistically significant decrease of the frequency of contractions was observed (Fig. 4A; Table S3). Interestingly, the use of U73122, a compound that inhibits the synthesis of IP₃, did not have any effect (Fig. 4B; Table S3). To check the proper activity of the compound, a similar experiment using fed individuals was performed. In this case, the activity of the aorta was diminished, showing that U73122 effectively alters PLC activity in this species (Table S3).

As it was shown, neither the compounds altering pathways of RYRs and L-type VGCCs nor those acting on GPCR/IP₃R pathways completely blockaded the aorta frequency of contractions. This suggests that both could act complementarily to regulate the activity of the DV. To attempt an explanation, a new experiment evaluating the blockade of both pathways simultaneously was performed, testing the effect of Xe-C, nifedipine and Ry. Again, the frequency of contractions of the DV was not completely blockaded (Fig. 4C; Table S3), suggesting the presence of another pathway acting to ensure a minimal but constant activity of the DV.

Taking into account that the IP₃R pathway seems to participate in the regulation of the activity of the aorta during resting conditions, suggesting the involvement of cellular messengers acting via GPCRs, we performed a new experiment using an inhibitor of agonists binding to GPCR (SCH-202676) (Fawzi, et al., 2001). If a stimulatory signal is acting through the IP₃R pathway, the expected result using this compound would be the inhibition of the signal, diminishing the rate of aorta contractions. The use of 10 µmol l⁻¹ SCH-202676 caused a statistically significant increase of the frequency, suggesting the participation of an inhibitory cellular messenger also acting through a GPCR (Fig. 5A; Table S4).

Based on the results described above, the interaction of two independent signals, acting complementarily, was investigated. Although several cellular messengers were proposed as modulators of the contractile activity of the aorta, our laboratory demonstrated under in vivo conditions that in R. prolixus at least three messengers synchronously interact to regulate the frequency of aorta contractions during post-prandial diuresis (serotonin, allatotropin and allatostatin-C). Indeed, in the presence of 5-HT, AT increases the frequency of contractions of the aorta (Sterkel et al., 2010; Villalobos Sambucaro et al., 2015), this mechanism being antagonized by AST-C, which diminishes the availability of Ca²⁺ necessary for muscle contraction stimulated by AT (Villalobos Sambucaro et al., 2016). Indeed, it might be possible that AT and AST-C interact to modulate the rhythm of contraction also during the resting condition. To test this hypothesis, a group of unfed individuals was treated with AT in the presence of an anti-AST-C polyclonal antiserum. As a control, a second group was treated only with the antiserum. The results showed that in the presence of the antiserum, AT caused a statistically significant increase like the one observed in the presence of serotonin (Villalobos Sambucaro et al., 2016), suggesting that AST-C constitutes a canonical mechanism that counteracts the activity of AT (Fig. 5B; Table S4). The group treated only with the antiserum (control) did not show any difference, showing that the antiserum does not have any effect by itself and that, under resting conditions, AST-C would be also acting as an AT modulator (Fig. 5C; Table S4).

As stated above, the retrograde flow of haemolymph is caused by the peristaltic contractions of the crop (Maddrell, 1964a). As in previous studies, we also analysed the peristaltic rate of this organ, which receives and processes the blood during feeding. The results showed that the rate of contractions was only altered by chelators (BAPTA/AM and EDTA) and Ry (Fig. 6; Table S5). These results suggest that there is another mechanism controlling peristaltic activity of the crop during resting conditions when compared with the DV.

A complex and complementary system involving both myogenic and peptidergic mechanisms seems to act to maintain the activity of the aorta during the resting conditions. However, neither GPCRs nor L-type VGCC- and RyRs-related pathways seem to represent the complete machinery of the system modulating DV and crop activity. In fact, treatments applied together to alter different pathways did not completely inhibit the contractile activity of the DV. Furthermore, the activity of the crop does not show rhythmic contractions. On the contrary, the variability of the peristaltic activity appears to be caused by a reflex response.

To further understand the complete machinery acting to modulate aorta and crop contractions during resting conditions, we analysed the probable involvement of Piezo proteins.

An in silico search for Piezo-like proteins in R. prolixus showed that the genome predicts the expression of a transcript showing homology with this family of proteins (accession number RPRC014024). Indeed, it was found that this transcript is expressed in the midgut (including crop) (Ribeiro et al., 2014) and sensory system (Latorre-Estivalis et al., 2017). The protein predicted is highly conserved, presenting two domains considered characteristic of Piezo proteins: one is located between residues 1163 and 1294, and the second one corresponds to the mechanosensitive ion channel (residues 1779 to 2368) (Fig. 7). The transcript predicts a protein containing 36 transmembrane domains.

The predicted protein shows a high degree of conservation with members of the family corresponding to several Eukaryota groups, including non-metazoan organisms such as Fungi and Choanoflagellata (Fig. 8). Indeed, the protein includes the PFEW motif proposed as a signature of the Piezo family (Prole and Taylor, 2013) (Fig. 7A). To delve into the evolutionary analysis, we looked for the degree of conservation of other motifs (Figs 7A and 8A), and found that the motif FLYRSPET at the C-terminal domain is conserved in Bilateria, Cnidaria and Placozoa.

The use of two Piezo protein agonists (Jedi1 and Jedi2) physiologically confirms the presence of this family of proteins as is suggested by the increase of contractile activity of both the aorta and crop (Fig. 9; Table S6). When the activity of the aorta was evaluated, results showed that both agonists caused a similar response. On the contrary, regarding the peristalsis of the crop, Jedi1 caused a major increase while Jedi2 induced a slight but still statistically significant response (Fig. 9B; Table S6).

Chagas disease affects more than 6.5 million people around the world and is considered a neglected infectious disease by the World Health Organization (2015). Despite the existence of different mechanisms of transmission, the more relevant is through the elimination of urine and faeces by hematophagous insects such as T. infestans and R. prolixus. Although several approaches have been investigated and proposed, insecticides are still the most widely used methodology for control. Even though more and better formulae have been developed, the emergence of resistant populations is still a problem to solve (Mougabure-Cueto and Picollo, 2021). Taking this into account, a better understanding of the mechanisms that regulate triatomine physiology is necessary to develop alternative methods of control.

During and after feeding, the insect undergoes an intense diuretic process. To ensure that, the rate of contractions of the DV and the crop is higher, causing the increase of haemolymph circulation velocity. Triatominae insects can spend several weeks without feed, being the process of diuresis limited to short periods along its lifetime. In this regard, the resting situation (i.e. period between two consecutive meals) is the most frequent physiological condition. The physiological changes associated with diuresis (Maddrell et al., 1993a,b) and those related to haemolymph circulation during feeding have been extensively studied (Santini and Ronderos, 2007, 2009a; Sterkel et al., 2010; Villalobos Sambucaro et al., 2015, 2016). On the contrary, the processes related to the mechanisms that modulate haemolymph flow during the resting condition have been largely ignored. Therefore, the knowledge of the mechanisms ruling that phenomenon is of interest to develop new approaches tending to the control of Triatominae populations.

In the present study, we delved into the mechanisms ruling haemolymph circulation during resting conditions in R. prolixus, analysing several signalling pathways that regulate visceral muscle contraction.

As a first approach to analyse the myogenic activity, we decided to check the role and sources of Ca²⁺. As expected, the use of an intracellular chelator decreased the frequency of contractions. Interestingly, the use of an extracellular chelator demonstrated that Ca²⁺ influx is necessary to maintain the activity of the heart, as occurs in other insect species (Markou and Theophilidis, 2000; Feliciano et al., 2011) as well as in vertebrates (Gilbert et al., 2020).

The relevance of the extracellular calcium influx was confirmed using a specific blocker of L-type VGCCs. This observation is compatible with the existence of action potentials driving the basal activity of the aorta. One of the most common responses to the extracellular Ca²⁺ influx is the further increase of the cytosolic level owing to its release from the ER mediated by specific channels as RyRs. When these channels were blockaded, the contractile activity of the aorta also decreased.

Regarding the myogenic contractions of the DV, there are not specific studies in R. prolixus, but the existence of heart automatism in insects has been proposed by several studies over the years (Maloeuf, 1935; Markou and Theophilidis, 2000; Sláma, 2006, 2012; Sláma and Lukás, 2011).

Interestingly, when the DV of R. prolixus is maintained ex vivo, a rhythm of contractions is still evident (Movie 1), supporting the existence of a myogenic mechanism driving the basal rhythm of the DV.

Besides the myogenic nature of DV beating, it is known that the rhythmicity is also modulated by neuropeptides and neurotransmitters (for a review, see Hillyer, 2018). To analyse the probable incidence of cellular messengers during the resting conditions, we assayed the effect of two compounds that interfere transduction pathways associated with some GPCRs. Our results showed that the inhibition of IP₃R channels at the level of the ER causes a transient decrease of the frequency of contractions of the aorta, suggesting the involvement of a GPCR associated with a G_q subunit.

Surprisingly, the inhibition of PLC (the enzyme responsible for the synthesis of IP₃) by the use of U73122 did not cause the expected effect on the contractile activity under resting conditions. Although we were unable to fully explain this result, a similar result was previously reported on the spontaneous rhythmic contractions of the oviduct of Grillus bimaculatus (Tamashiro and Yoshino, 2014). In fact, in that study, the authors showed that this compound does not modify the rhythm of contractions, but their amplitude. Furthermore, it was also reported that under certain conditions, U73122 does not inhibit PLC activity, but rather causes an increase of the activity of some isoforms (Klein et al., 2011).

To go further in the understanding of the GPCR pathway, we analysed the effect of an allosteric modulator of this family of receptors. This compound inhibits the binding to GPCRs, altering the activity of both stimulator and inhibitor ligands in vertebrates and invertebrates (Alzugaray et al., 2021; Fawzi et al., 2001; Gao et al., 2004; Hartz et al., 2008; Lewandowicz et al., 2006). Our results showed that insects treated with SCH-202676 underwent an increase of the frequency of contractions, suggesting the predominance of an inhibitory signal. As it is known, physiological processes are usually modulated by complementary signals (i.e. stimulatory and inhibitory). Thus, our results could suggest an interaction between both kinds of signals. As stated above, the existence of several cardioactive peptides in triatomine insects have already been proposed. Among others, our team reported that AT and AST-C peptides interact to modulate the frequency of contractions of the aorta during post-prandial diuresis, as the stimulatory effect of AT is antagonized by AST-C (Villalobos Sambucaro et al., 2016). It was also reported that, in vivo, the myostimulatory activity of AT is mediated by the presence of 5-HT, but this peptide has no effect when applied by itself (Masood and Orchard, 2014; Sterkel et al., 2010; Villalobos Sambucaro et al., 2015, 2016). Regarding this, we decided to check the probable existence of this functional relationship between AT and AST-C under resting conditions. The results showed that in the presence of an anti-AST-C antiserum, AT was able to stimulate the frequency of contractions of the aorta, bringing a probable explanation for the effect caused by the allosteric modulator of GPCRs. This result also shows that the AT–AST-C interaction might be a canonical pathway acting to regulate and maintain the homeostasis of the visceral muscle activity at the level of the aorta and other organs. In fact, a similar regulatory mechanism was recently proposed acting on the activity of the muscle tissues of the female reproductive system of R. prolixus (Villalobos Sambucaro et al., 2023).

As mentioned above, under in vivo conditions, AT increases the contractile activity of the aorta only when 5-HT is present (Masood and Orchard, 2014; Sterkel et al., 2010; Villalobos Sambucaro et al., 2015), as occurs during diuresis (Maddrell et al., 1991). Regarding the results obtained in this series of experiments, and considering that the aorta expresses both AT and AST-C receptors (Villalobos Sambucaro et al., 2015, 2016), we assayed the response of the aorta in the presence of AT (10⁻⁹ mol l⁻¹) ex vivo (i.e. with no influence of the nervous and neuroendocrine systems) (Movie 1). Interestingly, under ex vivo conditions, the aorta reacts to AT without 5-HT. In this regard, the phenomena observed in vivo could be the result of some kind of cross-talk between 5-HT and AST-C. AST-C activity is mediated by a GPCR homologue to somatostatin receptors in vertebrates (Alzugaray et al., 2016; Birgül et al., 1999). Similarly, the same kind of functional relationship between 5-HT and somatostatin was observed in mammals; in fact, it was shown that 5-HT inhibits somatostatin secretion at the level of the stomach, facilitating gastrin secretion (Koop and Arnold, 1984), and on delta cells in human pancreatic islets (Almaça et al., 2016).

The frequency of contractions of the aorta depends on both myogenic processes as well as cellular messenger. Neither the compounds that alter action potentials response nor those associated with GPCR signalling completely block the contractile activity by themselves. Trying to understand this, we attempted to induce a complete blockage of the activity by treating the insects simultaneously with those compounds that proved to alter both pathways (i.e. action potentials and GPCR signalling). Again, a complete blockage of the contractile activity was not evident. These results suggest that besides action potentials and cellular messengers, another route must be involved to ensure the function of the circulatory circuit.

As in previous studies, we also evaluated the rate of peristaltic contractions of the anterior midgut. As expected, owing to the relevance of calcium on muscle contraction, the system was sensitive to the lack of this ion. This was evident after treatment with both extracellular and intracellular chelators. In contrast to what was observed in the DV, only Ry was effective altering peristaltic activity. Interestingly, the opening of RyRs did not show to be associated with L-type VGCC, as nifedipine did have any effect. Taken together, these results suggest that other membrane-associated proteins might be involved in facilitating calcium influx to open RyRs and to induce muscle contraction. Moreover, spontaneous muscle activity does not seem to occur in the crop. Furthermore, in contrast to the aorta, crop behaviour seems to be variable and random. As the DV pumps regularly in an anterograde direction, causing an increase in the volume of haemolymph at the level of the thorax and the head, this increment could cause a slight but significant increase of the haemolymph pressure to trigger a reflex contraction of the muscle wall. To delve into the mechanisms associated with this phenomenon, we looked for the existence of Piezo proteins in the genome of R. prolixus. Piezo proteins are a recently characterized family of proteins associated with cellular membranes. They are sensitive to mechanical forces, causing the influx of cations (Coste et al., 2010, 2012) including Ca²⁺ (Zechini et al., 2022), and they are associated with multiple functions (see Wu et al., 2017). Originally characterized in vertebrates, they were also found in invertebrates. In D. melanogaster, these proteins proved to be involved in nociception (Kim et al., 2012), control of meal size (Min et al., 2021) and cardiomyocyte activity (Zechini et al., 2022).

Looking for a probable reflex response, we searched for Piezo-like proteins in R. prolixus. We found that the R. prolixus transcriptome predicts the expression in the midgut and other organs (Latorre-Estivalis et al., 2017; Ribeiro et al., 2014) of a protein sharing a high level of homology with the Piezo proteins characterized in vertebrates.

Piezo proteins are a highly conserved family, present in most eukaryotes including plants and protists (Coste et al., 2010). The protein predicted by the R. prolixus transcriptome shares two recognizable domains including the one associated with the C terminal, which in fact contains pore properties (Coste et al., 2015). We found other conserved motifs, such as FLYRSPET, which might be considered as a signature for Metazoa. The use of two agonists such as Jedi1 and Jedi2 induced a statistically significant increase of the frequency of contractions at both the aorta and the anterior midgut, showing the involvement of Piezo proteins. Indeed, owing to the specificity of these agonists, it would be assumed that the Piezo protein present in R. prolixus is a Piezo1 protein (Wang et al., 2018).

Regarding the existence of a reflex response in the anterior midgut triggering peristaltic contractions of this organ under resting conditions, the existence of allatotropic open-type cells located in the epithelial sheet at the anterior region of the crop in T. infestans unfed individuals was shown (Riccillo and Ronderos, 2010; Sterkel et al., 2010). As it is known that AT stimulates visceral muscle contraction, it was originally proposed that the allatotropic open-type cells could sense changes in the content of the lumen of the organ when the insect begins to feed (Sterkel et al., 2010). This signal would initiate the secretion of the peptide locally to modulate muscle contractions in a paracrine way. In view of these new findings, Piezo proteins located on the muscle walls of the crop and/or at allatotropic cells sensing local pressure changes could be responsible for the peristaltic activity of the crop under resting conditions. The implication of Piezo proteins as secretory modulators has already been proposed; indeed, it was shown that these proteins modulate the secretion of adrenomedulin by endothelial cells (Iring et al., 2019) and 5-HT at the level of the gut (Zhu et al., 2023) in vertebrates.

Besides the relevance of mechanosensitive proteins to the situation under study, Piezo proteins might explain other mechanisms associated with physiological changes during and after feeding, such as those related to growth and/or reproduction. It is largely known that the diuresis as well as the plasticization of the abdominal cuticle is caused by 5-HT, which is secreted during feeding. It is also known that this secretory mechanism is caused by the distension of the abdomen (Maddrell, 1964b). Taking into account the results presented, it is highly probable that Piezo proteins could be also relevant to regulating these processes. In fact, the finding of the existence of these new mechanosensitive proteins in R. prolixus could shed light on this and another numerous physiological mechanisms in Triatominae and other insect species.

Our results show that under resting conditions, the mechanisms controlling haemolymph circulation are highly complex. Indeed, they depend on different factors including automatism and nervous, neuroendocrine and paracrine pathways. Certainly, to ensure the maintenance of the haemolymph flow in resting conditions, a reflex mechanism driven by mechanosensitive channels such as Piezo proteins is also present. As was shown, the resting situation would be the main physiological state throughout the life of the Triatominae. Thus, the knowledge of this complex system could be relevant for developing new alternative methods of control of natural populations of Chagas disease vectors.

We thank Dr F. G. Noriega for generously supplying allatotropin and allatostatin-C. We also thank Dr F. Bolognani for the critical reading of the manuscript, and the reviewers for their valuable comments to improve it.