Neuropeptides isotocin and arginine vasotocin in urophysis of three fish species

In this study, for the first time, both neuropeptides isotocin (IT) and arginine vasotocin (AVT) have been identified and measured in urophysis, the neurohaemal organ of the caudal neurosecretory system of teleost fish. So far, AVT, but not IT, was quantified by radioimmunoassay (RIA) in urophysis of several fish species. We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species. The efficiency of peptide’s SPE extraction was 79–85 %. In HPLC-FL method, the limits of detection (LOD) and quantification (LOQ) were estimated as 1.0 and 3.4 pmol/mL for IT and 0.25 and 2.20 pmol/mL for AVT. In LC–MS/MS method, LOD and LOQ were estimated as 0.4 and 1.2 pmol/mL for IT and 0.06 and 0.2 pmol/mL for AVT. The chromatographic methods are good alternative for RIA, because enable to measure both nonapeptides simultaneously in one sample. In round goby (Neogobius melanostomus), three-spined stickleback (Gasterosteus aculeatus) and sea bream (Sparus aurata), urophysial IT concentrations ranged between 0.056 and 0.678 pmol/mg tissue and AVT concentrations ranged between 0.0008 (or even below detection threshold) and 0.084 pmol/mg tissue

Neurohypophysial and paracrine vasopressinergic signaling regulates aquaporin trafficking to hydrate marine teleost oocytes

The dual aquaporin (Aqp1ab1/Aqp1ab2)-mediated hydration of marine teleost eggs, which occurs during oocyte meiosis resumption (maturation), is considered a key adaptation underpinning their evolutionary success in the oceans. However, the endocrine signals controlling this mechanism are almost unknown. Here, we investigated whether the nonapeptides arginine vasopressin (Avp, formerly vasotocin) and oxytocin (Oxt, formerly isotocin) are involved in marine teleost oocyte hydration using the gilthead seabream (Sparus aurata) as a model. We show that concomitant with an increased systemic production of Avp and Oxt, the nonapeptides are also produced and accumulated locally in the ovarian follicles during oocyte maturation and hydration. Functional characterization of representative Avp and Oxt receptor subtypes indicates that Avpr1aa and Oxtrb, expressed in the postvitellogenic oocyte, activate phospholipase C and protein kinase C pathways, while Avpr2aa, which is highly expressed in the oocyte and in the follicular theca and granulosa cells, activates the cAMP-protein kinase A (PKA) cascade. Using ex vivo, in vitro and mutagenesis approaches, we determined that Avpr2aa plays a major role in the PKA-mediated phosphorylation of the aquaporin subdomains driving membrane insertion of Aqp1ab2 in the theca and granulosa cells, and of Aqp1ab1 and Aqp1ab2 in the distal and proximal regions of the oocyte microvilli, respectively. The data further indicate that luteinizing hormone, which surges during oocyte maturation, induces the synthesis of Avp in the granulosa cells via progestin production and the nuclear progestin receptor. Collectively, our data suggest that both the neurohypophysial and paracrine vasopressinergic systems integrate to differentially regulate the trafficking of the Aqp1ab-type paralogs via a common Avp-Avpr2aa-PKA pathway to avoid competitive occupancy of the same plasma membrane space and maximize water influx during oocyte hydration

Brain levels of arginine–vasotocin and isotocin in dominant and subordinate males of a cichlid fish

The nonapeptides arginine–vasotocin (AVT) and isotocin (IT), which are the teleost homologues of arginine– vasopressin and oxytocin in mammals, have well established peripheral effects on osmoregulation and stress response, and central effects on social behavior. However, all studies that have looked so far into the relationship between these nonapeptides and social behavior have used indirectmeasures of AVT/IT activity (i.e. immunohistochemistry of AVT/IT immunoreactive neurons, or AVT/IT or their receptorsmRNA expressionwith in situ hybridization or qPCR) and therefore directmeasures of peptide levels in relation to social behavior are still lacking. Here we use a recently developed high-performance liquid chromatography analysis with fluorescence detection (HPLC-FL)method to quantify the levels of both AVT and IT inmacro-dissected brain areas [i.e. olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and hindbrain (= rhombencephalon minus cerebellum)] and pituitary of dominant and subordinate male cichlid fish (Oreochromis mossambicus). The pituitary shows higher levels of both peptides than any of the brain macroareas, and the olfactory bulbs have the highest AVT among all brain areas. Except for IT in the telencephalon there is a lack of correlations between central levels and pituitary peptide levels, suggesting an independent control of hypophysial and CNS nonapeptide secretion. There were also no correlations between AVT and IT levels either for each brain region or for the pituitary gland, suggesting a decoupled activity of the AVT and IT systems at the CNS level. Subordinate AVT pituitary levels are significantly higher than those of dominants, and dominant hindbrain IT levels are significantly higher than those of subordinates, suggesting a potential involvement of AVT in social stress in subordinate fish and of IT in the regulation of dominant behavior at the level of the hindbrain. Since in this species dominant males use urine to communicate social status and since AVT is known to have an antidiuretic effect,we have also investigated the effect of social status on urine storage. As predicted, dominantmales stored significantly more urine than subordinates. Given these results we suggest that AVT/IT play a key role in orchestrating social phenotypes, acting both as central neuromodulators that promote behavioral plasticity and as peripheral hormones that promote integrated physiological changes

Roles of neuropeptides arginine vasotocin and isotocin in endocrine regulation of physiological process in the gilthead sea bream (Sparus aurata)

International Symposium in Marine Science (ISMS) 2012 (Cádiz, 24-26 enero de 2012). III Simposio Internacional de Ciencias del MarLos neuropéptidos arginina-vasotocina (AVT) e isotocina (IT) de teleósteos, análogos respectivamente de la arginina-vasopresina (AVP) y oxitocina (OXY) de mamíferos, participan en diversos procesos fisiológicos de los organismos.Estudio financiado por los proyectos AGL2010-14876 otorgado por el MICINN (España) a JMM y 498/N-HISZP-JPR/2009/0 (Polonia) a EK. JAM-S está financiado por una beca predoctoral (FPU, Referencia AP2008-01194) del Ministerio de Educación (España).Peer Reviewe

Brain levels of nonapeptides in four labrid fish species with different levels of mutualistic behavior

a b s t r a c t There is strong evidence that brain nonapeptides are implicated as modulators of a wide array of social and reproductive behaviors in fishes. However, the question remains, as to whether there is a link between the distribution of active nonapeptides across brain regions and fishes specific behavioral phenotypes. To explore this link we compared the nonapeptides' profile across the brains of fishes representing different degrees of mutualistic behavior (here: cleaning behavior). Herein we studied the quantitative distribution of both nonapeptides, arginine vasotocin (AVT) and isotocin (IT), in the brains of four species of fish belonging to the family Labridae: two are obligatory cleaners throughout their entire life (Labroides dimidiatus and Labroides bicolor), one species is a facultative cleaner (Labropsis australis; juveniles are cleaners and adults are corallivorous), and one is a non-cleaner species, corallivorous throughout its entire life (Labrichthys unilineatus). The biologically available AVT and IT concentrations were measured simultaneously in distinct brain macro-areas: forebrain, optic tectum, cerebellum and brain stem, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We showed that the levels of both AVT and IT varied significantly across species, as measured in the whole brain or in the specific macro-areas. Significantly higher AVT concentrations in the cerebellum which were found in the obligate cleaners seemed to be related to expression of mutualistic behavior. On the other hand, the higher levels of brain IT in the non-cleaner L. unilineatus suggested that these might be linked to the development of sexual dimorphism, which occurs only in this non-cleaner species

Behavioural indicators of welfare in farmed fish

Behaviour represents a reaction to the environment as fish perceive it and is therefore a key element of fish welfare. This review summarises the main findings on how behavioural changes have been used to assess welfare in farmed fish, using both functional and feeling-based approaches. Changes in foraging behaviour, ventilatory activity, aggression, individual and group swimming behaviour, stereotypic and abnormal behaviour have been linked with acute and chronic stressors in aquaculture and can therefore be regarded as likely indicators of poor welfare. On the contrary, measurements of exploratory behaviour, feed anticipatory activity and reward-related operant behaviour are beginning to be considered as indicators of positive emotions and welfare in fish. Despite the lack of scientific agreement about the existence of sentience in fish, the possibility that they are capable of both positive and negative emotions may contribute to the development of new strategies (e. g. environmental enrichment) to promote good welfare. Numerous studies that use behavioural indicators of welfare show that behavioural changes can be interpreted as either good or poor welfare depending on the fish species. It is therefore essential to understand the species-specific biology before drawing any conclusions in relation to welfare. In addition, different individuals within the same species may exhibit divergent coping strategies towards stressors, and what is tolerated by some individuals may be detrimental to others. Therefore, the assessment of welfare in a few individuals may not represent the average welfare of a group and vice versa. This underlines the need to develop on-farm, operational behavioural welfare indicators that can be easily used to assess not only the individual welfare but also the welfare of the whole group (e. g. spatial distribution). With the ongoing development of video technology and image processing, the on-farm surveillance of behaviour may in the near future represent a low-cost, noninvasive tool to assess the welfare of farmed fish.Fundação para a Ciência e Tecnologia, Portugal [SFRH/BPD/42015/2007]info:eu-repo/semantics/publishedVersio

Annual reproductive cycle in two free living populations of three-spined stickleback (<i>Gasterosteus aculeatus</i> L.): patterns of ovarian and testicular development

The annual reproductive cycle in two wild populations of three-spinedstickleback was studied. Sticklebacks from the Dead Vistula River(Martwa Wis&#x0142;a) (brackish water) and the Oliva Stream (Potok Oliwski)(freshwater) were exposed to annual environmental changes in their naturalhabitats. Ovaries and livers (females), and testes and kidneys (males) werecollected during 1-2 years. The gonadosomatic <i>I</i>_G, hepatosomatic<i>I</i>_H, nephrosomatic <i>I</i>_N indices, kidney epithelium height(KEH) and size of oocytes were calculated.The number of mature oocytes andpercentage of ovulating females were determined during the spawning season.Histological changes in the ovaries and testes were described throughouta year. Annual reproductive cycles were similar in both populations ofsticklebacks. This is the first histological and morphological study carriedout throughout a year, simultaneously in two wild populations of three-spinedsticklebacks inhabiting different environments. An improved scale of gonadaldevelopment in conjunction with the determined indices and fecundity givea comprehensive description of the reproductive cycle. These new observations,in combination with previously reported features, provide a universal scalethat can be successfully used to distinguish all phases of gametogenesis insticklebacks in different habitats

Influence of exogenous thyroxine on plasma melatonin in juvenile Atlantic salmon (Salmo salar

Abstract One of the most clearly defined endocrine changes during the parr-smolt transformation of anadromous salmonids is an increase in plasma levels of thyroid hormones. The role of pineal hormone melatonin in timing and synchronisation of smoltification is widely discussed. The effect of administration of exogenous thyroxine (T4) on plasma melatonin was investigated in juvenile Atlantic salmon (Salmo salar) at the early stages of parr-smolt transformation. Fish were kept in fresh water under simulated-natural photoperiod and exposed to exogenous T4. Fish were sampled at 12.00 and 24.00 h from treatment and control tanks, 2 and 14 days after treatment started. Plasma melatonin and L-thyroxine were measured using RIA and competitive enzyme immunoassay, respectively. After 2 days of T4 treatment, marked difference in plasma melatonin concentration measured at 12.00 and 24.00 h was still observed in both groups. However, 2-week exposure to T4 caused a reduction in night-time plasma melatonin level and thus, probably, inhibited melatonin related time-keeping system in juvenile salmon. Additional studies are needed to clarify the mechanism of the described phenomenon.

Brain nonapeptide and gonadal steroid responses to deprivation of heterosexual contact in the black molly

Fish may respond to different social situations with changes in both physiology and behaviour. A unique feature of fish is that social interactions between males and females strongly affect the sexual characteristics of individuals. Here we provide the first insight into the endocrine background of two phenomena that occur in mono-sex groups of the black molly (Poecilia sphenops): masculinization in females and same-sex sexual behaviour, manifested by gonopodial displays towards same-sex tank mates and copulation attempts in males. In socially controlled situations, brain neurohormones impact phenotypic sex determination and sexual behaviour. Among these hormones are the nonapeptides arginine vasotocin (AVT) and isotocin (IT), counterparts of the well-known mammalian arginine vasopressin and oxytocin, respectively. To reveal potential hormone interactions, we measured the concentrations of bioactive AVT and IT in the brain, along with those of the sex steroids 17β-estradiol and 11-ketotestosterone in the gonads, of females, masculinized females, males displaying same-sex sexual behaviour and those who did not. These data were supplemented by morphological and histological analyses of the gonads. Correlations between brain nonapeptides and gonadal steroids strongly suggest a cross talk between hormonal systems. In the black molly, the masculinization process was associated with the production of brain AVT and gonadal steroids, whereas same-sex sexual behaviour involves both brain nonapeptides, but neither of the sex steroids. This study extends current knowledge of endocrine control of phenotypic sex and sexual behaviour in fish and for the first time links brain nonapeptides with the occurrence of male-male sexual behaviour in lower vertebrates