Jorge Rafael Ronderos

Allatotropin-like peptide released by Malpighian tubules induces hindgutactivity associated with diuresis in the Chagas disease vector Triatomainfestans (Klug)

SUMMARY Haematophagous insects incorporate a large amount of blood with each meal, producing a big quantity of urine in a few hours to eliminate the excess water and Na+. Malpighian tubules (MTs) have traditionally been seen as a system that responds to neuroendocrine stimulus. In a related paper, we demonstrated that MTs of Triatoma infestans produce an autonomous endocrine secretion of an allatotropin-like (AT-like) peptide. In the present study, we report a myostimulatory activity of AT at the level of the hindgut (HG), associated with endocrine mechanisms regulating post-prandial diuresis. Allatotropin induced an increase in frequency and intensity of peristaltic contractions at the level of the HG. The release of the HG content in MTs–HG in vitro preparations undergoing an osmotic shock occurred at different times, depending on the number of MTs present, and there was no release in treatments without MTs. The application of an AT-antiserum to MTs–HG preparations undergoing osmotic shock produced a delay or a long-term blockade of diuresis, depending on the antiserum dilution applied. Similar results were obtained when AT-antiserum was applied in vivo prior to blood intake, decreasing the volume of urine eliminated during the first 2 h. Our results allow us to assign a specific endocrine function to the AT-like peptide released by MTs that is linked to the elimination of urine after blood meals.

Allatotropin: An Ancestral Myotropic Neuropeptide Involved in Feeding

Background Cell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Peptidic molecules secreted by neurons and epithelial cells play fundamental roles in cell-cell interactions, acting as local neuromodulators, neurohormones, as well as endocrine and paracrine messengers. Allatotropin (AT) is a neuropeptide originally described as a regulator of Juvenile Hormone synthesis, which plays multiple neural, endocrine and myoactive roles in insects and other organisms. Methods A combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT in Hydra plagiodesmica. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies. Conclusions AT antibodies immunolabeled nerve cells in the stalk, pedal disc, tentacles and hypostome. AT-Qdot conjugates recognized epithelial-muscular cell in the same tissues, suggesting the existence of anatomical and functional relationships between these two cell populations. Physiological assays indicated that the AT-like peptide is facilitating food ingestion. Significance Immunochemical, physiological and bioinformatics evidence advocates that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion.

Cardioacceleratory and myostimulatory activity of allatotropin in Triatoma infestans.

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

Allatotropin Modulates Myostimulatory and Cardioacceleratory Activities in Rhodnius prolixus (Stal).

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 excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation 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 circulation 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 contractions 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.

Allatotropin-like peptide in Malpighian tubules: insect renal tubules as an autonomous endocrine organ.

Malpighian tubules (MTs) are recognised as the main excretory organ in insects, ensuring water and mineral balance. Haematophagous insects incorporate with each meal a large quantity of blood, producing a particularly large volume of urine in a few hours. In the present study, we report the presence of an allatotropin-like (AT-like) peptide in MTs of Triatoma infestans (Klug). The AT-like content in MTs decreased during the first hours after blood-intake, correlating with the post-prandial diuresis. In vivo artificial dilution of haemolymph showed a similar effect. Isolated MTs challenged with a diluted saline solution resulted in an autonomous and reversible response of the organ regulating the quantity of peptide released to the medium, and suggesting that MTs synthesise the AT-like peptide. While MTs are recognised as the target for several hormones, our results corroborate that they also have the ability to produce and secrete a hormone in an autonomous way.

Daily variation of an allatotropin-like peptide in the Chagas disease vector Triatoma infestans (klug)

Triatominae insects exhibit circadian processes associated with the activity of neuropeptides, steroids hormones and their receptors. Allatotropin has proved to be multifunctional, acting as a myoregulator. During feeding, haematophagous insects incorporate a large quantity of blood, producing high volumes of urine. We have recently shown that an AT (Allatotropin)-like peptide released by the Malpighian tubules during post-prandial diuresis is responsible for the modulation of peristaltic contractions and the voiding of the hindgut. Feeding and post-prandial diuresis are critical processes in haematophagous insects. Regarding the role played after blood meal by the AT-like peptide, the existence of a daily rhythmic expression could have biological relevance preparing the insect for blood intake. In the present study we show the presence of a daily variation of this peptide at the level of Malpighian tubules and midgut. The content in these organs shows a peak previous to the beginning of the scotophase.

Expression of a neuropeptide similar to allatotropin in free living turbellaria (platyhelminthes)

Mechanisms coordinating cell-cell interaction have appeared early in evolution. Allatotropin (AT), a neuropeptide isolated based on its ability to stimulate the synthesis of juvenile hormones (JHs) in insects has also been found in other invertebrate phyla. Despite this function, AT has proved to be myotropic. In the present study we analyze its expression in two groups of Turbellaria (Catenulida, Macrostomida), and its probable relationship with muscle tissue. The results show the presence of an AT-like peptide in the free living turbellaria analyzed. The analysis of the expression of the peptide together with phalloidin, suggests a functional relationship between the peptide and muscle tissue, showing that it could be acting as a myoregulator. The finding of immunoreactive fibers associated with sensory organs like ciliated pits in Catenulida and eyes in Macrostomida makes probable that AT could play a role in the physiological mechanisms controlling circadian activities. Furthermore, the existence of AT in several phyla of Protostomata suggests that this peptide could be a synapomorphic feature of this group. Indeed, the presence in organisms that do not undergo metamorphosis, could be signaling that it was first involved in myotropic activities, being the stimulation of the synthesis of JHs a secondary function acquired by the phylum Arthropoda.

Allatotropin expression during the development of the fourth instar larvae of the kissing-bug Triatoma infestans (Klüg).

Allatotropin (AT) is a neuropeptide originally isolated from the brain of Manduca sexta and then characterized in several insect species. It acts as a neurohormone, as well as a neuromodulator. While it was primarily characterized on the basis of its ability to stimulate the secretion of juvenile hormones, it was also found that it acts as a cardioaccelerator and myostimulator. The presence of AT in IV instar larvae of T. infestans was previously described at the level of the Malpighian tubules. In the present study we report the presence of the peptide at the level of the brain, retrocerebral complex, as well as in the anterior midgut and aorta. The presence of AT at the corpora allata suggests that the peptide is acting on the gland during the first days of the moulting cycle. Neural processes at the level of the aorta and the anterior midgut suggests that, like in adults, the hormone is acting as a cardioaccelerator and myostimulator. The peptide was also found in open-type cells of the midgut. Finally the presence of allatotropic neurons in the optical lobe of the brain suggests that as in other species, the peptide is related with the control of circadian rhythms.

MOSQUITO LARVAE EXTRACT INDUCES MITOTIC RATE ALTERATIONS OF MICE TONGUE KERATINOCYTES ALONG A CIRCADIAN RHYTHM PERIOD

It has been demonstrated that the crude extract of mosquito larvae alters the mitotic rate of several mouse cell populations of young growing mice (25±1 days old). Furthermore, the dialysed fraction of the extract inhibited proliferation of hepatocytes from hepatectomized adult male mice (90 days old). Sampling during the period between 16 and 24 h after treatment (when mitotic peak normally occurs) shows an inhibiting effect on the G1/S interphase, whereas evaluation during the dark phase of the circadian rhythm period (i.e. 4 to 12 h after treatment) shows an increment of the mitotic rates suggesting a probable effect at G2/M restriction point. In the present paper we report the effect of the mosquito larvae crude extract on the proliferative activity of tongue keratinocytes along a circadian rhythm period. Treatments were intra‐peritoneally applied at 16/00 Time of Day/Time Post Injection and mice were killed at 00/08, 04/12, 08/16, 12/20 and 16/24 TD/TPI. As other cell populations previously analysed, the mitotic rate of tongue keratinocytes of extract receivers was significantly increased during night (when S phase normally occurs) and inhibited during the 08/16 to 16/24 period.

EARLY EFFECT OF MOSQUITO LARVAE EXTRACT ON MOUSE CELLS PROLIFERATIONIN VIVO

It has been demonstrated that mosquito larvae crude extract has an inhibiting effect on the mitotic rate of several mouse cell populations. The sampling period was 16–24h after treatment, when mitotic peak normally occurs. The present paper reports the effect of mosquito larvae crude extract on the proliferation of hepatocytes, renocytes, Lieberkhün crypt enterocytes, and sialocytes. In this case, the sampling period covered the dark phase of the day, during the first 12h after treatment. Colchicine‐arrested metaphases were controlled at 20/04, 00/08 and 04/12 (Time of Day/Time Post Injection). The mitotic rate was significantly increased in hepatocytes and renocytes and inhibited in duodenum enterocytes. In view of the time chosen to administer the treatments and the time elapsed until sampling, we conclude a probable effect of the extract at the G2‐M point of the cell cycle.