Immunohistochemical localisation of vasopressin/oxytocin-type, corazonin-type and luqin-type neuropeptide expression in the starfish Asterias rubens using antibodies to the C-terminal region of precursor proteins.

Neuropeptides derived from larger precursor proteins are secreted as signalling molecules by neurons and regulate diverse physiological and behavioural processes in animals. Recently, we reported the discovery of ArCRZ (HNTFTMGGQNRWKAG-NH2) and ArLQ (EEKTRFPKFMRW-NH2)-novel neuropeptides in the starfish Asterias rubens that are orthologs of arthropod corazonins and molluscan luqins, respectively. However, our efforts to generate antibodies to ArCRZ and ArLQ have been unsuccessful, precluding immunohistochemical analysis of their expression. Here, we investigated an alternative experimental approach for neuropeptide immunohistochemistry by generating antibodies to peptides corresponding to the C-terminal region of the precursor proteins. As proof of principle, we generated antibodies to the C-terminal region of the precursor of the vasopressin/oxytocin-type neuropeptide asterotocin and show that these reveal immunostaining in A. rubens that is very similar to that observed with asterotocin antibodies. Furthermore, antibodies to the C-terminal region of the ArCRZ precursor (ArCRZP) and the ArLQ precursor (ArLQP) produced patterns of immunostaining consistent, respectively, with the distribution of ArCRZP and ArLQP transcripts revealed by mRNA in situ hybridisation. Detailed immunohistochemical analysis revealed widespread expression of ArCRZP and ArLQP in A. rubens, including the central nervous system, digestive system and the body wall and its associated appendages (e.g. tube feet), providing a neuroanatomical framework for investigation and interpretation of the pharmacological actions of ArCRZ and ArLQ in A. rubens. Furthermore, our findings provide a basis for use of antibodies to the C-terminal region of neuropeptide precursor proteins in other species where the production of antibodies to the bioactive neuropeptides is unsuccessful.

Somatostatin-type and allatostatin-C-type neuropeptides are paralogous and have opposing myoregulatory roles in an echinoderm.

Somatostatin (SS) and allatostatin-C (ASTC) are inhibitory neuropeptides in chordates and protostomes, respectively, which hitherto were identified as orthologs. However, echinoderms have two SS/ASTC-type neuropeptides (SS1 and SS2), and here, our analysis of sequence data indicates that SS1 is an ortholog of ASTC and SS2 is an ortholog of SS. The occurrence of both SS-type and ASTC-type neuropeptides in echinoderms provides a unique context to compare their physiological roles. Investigation of the expression and actions of the ASTC-type neuropeptide ArSS1 in the starfish Asterias rubens revealed that it causes muscle contraction (myoexcitation), contrasting with myoinhibitory effects of the SS-type neuropeptide ArSS2. Our findings suggest that SS-type and ASTC-type neuropeptides are paralogous and originated by gene duplication in a common ancestor of the Bilateria, with only one type being retained in chordates (SS) and protostomes (ASTC) but with both types being retained in echinoderms. Loss of ASTC-type and SS-type neuropeptides in chordates and protostomes, respectively, may have been due to their functional redundancy as inhibitory regulators of physiological processes. Conversely, the retention of both neuropeptide types in echinoderms may be a consequence of the evolution of a myoexcitatory role for ASTC-type neuropeptides mediated by as yet unknown signaling mechanisms.

Ancient role of sulfakinin/cholecystokinin-type signalling in inhibitory regulation of feeding processes revealed in an echinoderm.

Sulfakinin (SK)/cholecystokinin (CCK)-type neuropeptides regulate feeding and digestion in protostomes (e.g. insects) and chordates. Here, we characterised SK/CCK-type signalling for the first time in a non-chordate deuterostome - the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArSK/CCK1, ArSK/CCK2) derived from the precursor protein ArSK/CCKP act as ligands for an SK/CCK-type receptor (ArSK/CCKR) and these peptides/proteins are expressed in the nervous system, digestive system, tube feet, and body wall. Furthermore, ArSK/CCK1 and ArSK/CCK2 cause dose-dependent contraction of cardiac stomach, tube foot, and apical muscle preparations in vitro, and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of SK/CCK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals.

Characterization of NGFFYamide Signaling in Starfish Reveals Roles in Regulation of Feeding Behavior and Locomotory Systems.

Neuropeptides in deuterostomian invertebrates that have an Asn-Gly motif (NG peptides) have been identified as orthologs of vertebrate neuropeptide-S (NPS)-type peptides and protostomian crustacean cardioactive peptide (CCAP)-type neuropeptides. To obtain new insights into the physiological roles of NG peptides in deuterostomian invertebrates, here we have characterized the NG peptide signaling system in an echinoderm-the starfish Asterias rubens. The neuropeptide NGFFYamide was identified as the ligand for an A. rubens NPS/CCAP-type receptor, providing further confirmation that NG peptides are orthologs of NPS/CCAP-type neuropeptides. Using mRNA in situ hybridization, cells expressing the NGFFYamide precursor transcript were revealed in the radial nerve cords, circumoral nerve ring, coelomic epithelium, apical muscle, body wall, stomach, and tube feet of A. rubens, indicating that NGFFYamide may have a variety of physiological roles in starfish. One of the most remarkable aspects of starfish biology is their feeding behavior, where the stomach is everted out of the mouth over the soft tissue of prey. Previously, we reported that NGFFYamide triggers retraction of the everted stomach in A. rubens and here we show that in vivo injection of NGFFYamide causes a significant delay in the onset of feeding on prey. To investigate roles in regulating other aspects of starfish physiology, we examined the in vitro effects of NGFFYamide and found that it causes relaxation of acetylcholine-contracted apical muscle preparations and induction of tonic and phasic contraction of tube feet. Furthermore, analysis of the effects of in vivo injection of NGFFYamide on starfish locomotor activity revealed that it causes a significant reduction in mean velocity and distance traveled. Interestingly, experimental studies on mammals have revealed that NPS is an anxiolytic that suppresses appetite and induces hyperactivity in mammals. Our characterization of the actions of NGFFYamide in starfish indicates that NPS/NG peptide/CCAP-type signaling is an evolutionarily ancient regulator of feeding and locomotion.

Leptin expression is rhythmic in brain and liver of goldfish (Carassius auratus). Role of feeding time.

Daily rhythms of feeding regulators are currently arousing research interest due to the relevance of the temporal harmony of endocrine regulators for growth and welfare in vertebrates. However, it is unknown the leptin circadian pattern in fish. The aim of this study is to investigate if leptin (gLep-aI and gLep-aII) expression is rhythmic in goldfish (Carassius auratus) liver and brain, and if such rhythms are driven by feeding time through a food entrainable oscillator. Fish maintained under 12-h light:12-h dark photoperiod and a scheduled feeding time showed 24-h locomotor activity and glycaemia rhythms. Moreover, hepatic gLep-aI and brain gLep-aI and gLep-aII expression were rhythmic with different daily profiles, showing a postprandial increase of leptin expression in the liver but not in the brain. Under constant light and different feeding regimes (scheduled fed at 10:00, 22:00 or randomly fed), feeding time synchronized daily rhythms in locomotor activity, glycaemia and clock gene expression (gPer1a, gPer3 and gCry3), but the rhythmic expression of hepatic gLep-aI and brain gLep-aII only remained in fed fish at 10:00. In summary, daily rhythms of leptin expression in goldfish are differently regulated at central and peripheral level, and they are not directly driven by clock genes. The role of food entrained oscillators on leptin expression rhythms in fish remains to be demonstrated.

Role of oleoylethanolamide as a feeding regulator in goldfish.

Oleoylethanolamide (OEA) is a bioactive lipid mediator, produced in the intestine and other tissues, which is involved in energy balance regulation in mammals, modulating feeding and lipid metabolism. The purpose of the present study was to investigate the presence and possible role of OEA in feeding regulation in goldfish (Carassius auratus). We assessed whether goldfish peripheral tissues and brain contain OEA and their regulation by nutritional status. OEA was detected in all studied tissues (liver, intestinal bulb, proximal intestine, muscle, hypothalamus, telencephalon and brainstem). Food deprivation (48 h) reduced intestinal OEA levels and levels increased upon re-feeding, suggesting that this compound may be involved in the short-term regulation of food intake in goldfish, as a satiety factor. Next, the effects of acute intraperitoneal administration of OEA on feeding, swimming and plasma levels of glucose and triglycerides were analysed. Food intake, swimming activity and circulating triglyceride levels were reduced by OEA 2 h post-injection. Finally, the possible interplay among OEA and other feeding regulators (leptin, cholecystokinin, ghrelin, neuropeptide Y, orexin and monoamines) was investigated. OEA actions on energy homeostasis in goldfish could be mediated, at least in part, through interactions with ghrelin and the serotonergic system, as OEA treatment reduced ghrelin expression in the intestinal bulb, and increased serotonergic activity in the telencephalon. In summary, our results indicate for the first time in fish that OEA could be involved in the regulation of feeding, swimming and lipid metabolism, suggesting a high conservation of OEA actions in energy balance throughout vertebrate evolution.

Ghrelin increases food intake, swimming activity and growth in juvenile brown trout (Salmo trutta).

Several key functions of ghrelin are well conserved through vertebrate phylogeny. However, some of ghrelin's effects are contradictory and among teleosts only a limited number of species have been used in functional studies on food intake and foraging-related behaviors. Here we investigated the long-term effects of ghrelin on food intake, growth, swimming activity and aggressive contest behavior in one year old wild brown trout (Salmo trutta) using intraperitoneal implants. Food intake and swimming activity were individually recorded starting from day 1, and aggressive behavior was tested at day 11, after ghrelin implantation. Body weight and growth rate were measured from the beginning to the end of the experiment. Triglycerides and lipase activity in muscle and liver; monoaminergic activity in the telencephalon and brainstem; and neuropeptide Y (NPY) mRNA levels in the hypothalamus were analyzed. Ghrelin treatment was found to increase food intake and growth without modifying lipid deposition or lipid metabolism in liver and muscle. Ghrelin treatment led to an increased foraging activity and a trend towards a higher swimming activity. Moreover, ghrelin-treated fish showed a tendency to initiate more conflicts, but this motivation was not reflected in a higher ability to win the conflicts. No changes were observed in monoaminergic activity and NPY mRNA levels in the brain. Ghrelin is therefore suggested to act as an orexigenic hormone regulating behavior in juvenile wild brown trout. These actions are accompanied with an increased growth without the alteration of liver and muscle lipid metabolism and they do not seem to be mediated by changes in brain monoaminergic activity or hypothalamic expression of NPY.

Light-dark cycle and feeding time differentially entrains the gut molecular clock of the goldfish (Carassius auratus).

The aim of the present study was to investigate how photocycle and feeding-time cues regulate the daily expression of Per1a, Per2a, Per3, and Cry3 in the goldfish hindgut. For this purpose, we studied the daily rhythmicity of these genes in fish maintained under different lighting conditions and under different feeding regimes (scheduled or not). We also studied whether the timing of just one meal is able to reset the hindgut molecular clock. In a first experiment, randomly fed fish were divided into four groups and kept under different light conditions for 30 d: 12 h light and 12 h dark (12L:12D), an inverted photoperiod (12D:12L), constant darkness (24D), and constant light (24L). In a second study, fish maintained under 24L were divided into four groups fed at different time points for 35 d: (1) fish scheduled-fed once a day (at 10:00 h); (2) fish fed with a 12-h shifted schedule (at 22:00 h), (3) fish fed at 10:00 h throughout the experiment, except the last day when fed at 22:00 h; and (4) a randomly fed group of fish. Fish were sacrificed every 6 h throughout a 24-h cycle. In both experiments, gPer1a, gPer2a, gPer3, and gCry3 transcripts were quantified using Real Time-qPCR in the hindgut. Results show the clock genes gPer1a, gPer2a, and gCry3 are synchronized by both zeitgebers, the photocycle and feeding regime, in goldfish hindgut. Moreover, such clock genes anticipate light-on and food delivery, when these cues appear in a cyclic manner. In the absence of both zeitgebers, gCry3 and gPer2a rhythmicity disappeared. In contrast, the gPer1 rhythm was maintained under 24L and random feeding conditions, but not always, suggesting that food when randomly supplied is able to reset the clock depending on other factors, such as the energetic and metabolic conditions of the fish. The expression of gPer2a was not activated during the light phase of the cycle, suggesting the hindgut of goldfish is a non-direct photosensitive organ. In contrast to the other three genes, gPer3 expression in the goldfish hindgut seemed to be dependent on the timing of the last food delivery, even in the presence of a photocycle. This gene was the only one that maintained daily rhythms under both constant lighting conditions (24D and 24L), although with lower amplitude than when a photocycle was present. This indicates that, although the acrophase (peak time) of the gPer3 expression rhythm seems to be driven by feeding time, there is an interaction of both zeitgebers, food and light, to regulate its expression. In conclusion, present data indicate: (1) the hindgut of goldfish can be synchronized in vivo by both the photocycle and feeding time; (2) food is a potent signal that entrains this peripheral oscillator; and (3) both environmental cues seems to target different elements of the molecular clock.