Pessimistic cognitive bias is associated with enhanced reproductive investment in female zebrafish.

Optimistic and pessimistic cognitive biases have been described in many animals and are related to the perceived valence of the environment. We, therefore, hypothesize that such cognitive bias can be adaptive depending on environmental conditions. In reward-rich environments, an optimistic bias would be favoured, whereas in harsh environments, a pessimistic one would thrive. Here, we empirically investigated the potential adaptive value of such bias using zebrafish as a model. We first phenotyped female zebrafish in an optimistic/pessimistic axis using a previously validated judgement bias assay. Optimistic and pessimistic females were then exposed to an unpredictable chronic stress protocol for 17 days, after which fish were euthanized and the sectional area of the different ovarian structures was quantified in both undisturbed and stressed groups. Our results show that zebrafish ovarian development responded to chronic stress, and that judgement bias impacted the relative area of the vitellogenic developmental stage, with pessimists showing higher vitellogenic areas as compared with optimists. These results suggest that pessimism maximizes reproductive investment, through increased vitellogenesis, indicating a relationship between cognitive bias and life-history organismal decisions.

A Behavioural Assay to Investigate Judgment Bias in Zebrafish.

In this protocol, we describe for the first time a judgment bias paradigm to phenotype the way zebrafish assess ambiguous stimuli. We have developed and validated a protocol for a judgment bias test based on a Go/No-go task, and performed using a half radial maze. After a habituation phase, fish are trained to discriminate between two reference arms [positive (P) and negative (N)]. For this purpose, they experience a positive event (food reward in P), when presented with a specific location/color cue, and a negative event (chasing with net in N), when presented with a different location/color cue. Acquisition of the discrimination learning between P and N is revealed by the latencies to enter the experimental arms of the behavioral maze being significantly lower for the P arm than for the N arm. Once zebrafish are able to discriminate between P and N arms, their latency to enter other maze arms spatially located between P and N [(Near Positive (NP), Ambiguous (A) = half-way between P and N, and Near Negative (NN)] is analyzed. Latencies (L) to enter NP, A and NN maze arms are interpreted as indicating the individual expectancy to experience a reward/punishment on each of them. A judgment bias score (JBS) is calculated from the latencies to enter the P, N, and A arms for each fish [JBS = (LA-LP)*100/(LN-LP)], based on which fish can be classified into an optimistic/pessimistic axis. A JBS below 50 indicates that fish perceive the ambiguous stimulus as a positive one (optimistic bias), while JBS above 50 indicates that fish perceive the ambiguous stimulus as a negative one (pessimistic bias). However, for classification criteria, it could be advantageous to use the method of selecting extreme phenotypes (e.g., upper and lower quartiles of the JBS), since JBS in zebrafish falls into a bimodal distribution (unpublished data). Therefore, this protocol provides a unique, inexpensive, and effective alternative to other methods of measuring affective states in zebrafish that might be of great interest to a broad target audience and have a large number of applications. Graphic abstract: Flow chart of the judgment bias protocol in zebrafish.

Kiss2 as a Regulator of Lh and Fsh Secretion via Paracrine/Autocrine Signaling in the Teleost Fish European Sea Bass (Dicentrarchus labrax).

Kisspeptins are key players in the neuroendocrine control of puberty and other reproductive processes in mammals. Several studies have demonstrated that the KISS/GPR54 system is expressed by gonadotrophs, but in vitro studies assessing the direct stimulatory effects of kisspeptin on gonadotropin secretion in the pituitary have provided conflicting results. In this study, we investigated whether kisspeptin directly influences the reproductive function of sea bass pituitary. First, the highly active peptides Kiss1-15 and Kiss2-12 were used to stimulate dispersed sea bass pituitary cells obtained from mature males. Our results show that, first, Kiss2-12 induced luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) release, whereas Kiss1-15 had no effect on gonadotropin secretion at full spermiation stage. Second, the distribution and nature of Kiss2 and its potential interactions with the gonadotropin-releasing hormone 1 (Gnrh1) system in the pituitary were analyzed using dual fluorescence immunohistochemistry. Kiss2 cells were found in the proximal pars distalis and colocalized with gonadotropin-immunoreactive cells. In summary, our results provide, for the first time in a teleost species, functional and neuroanatomical evidence that Kiss2 may act through different routes to directly modulate the activity of gonadotrophs, either as a hypophysiotropic neuropeptide or as an autocrine/paracrine factor.

Updating control of puberty in male European sea bass: A holistic approach.

Puberty is the process by which an immature animal acquires the ability to reproduce for the first time; its onset occurs soon after sexual differentiation and is characterized by the beginning of gametogenesis in both sexes. Here we present new insights on when and how the onset of puberty occurs in male European sea bass, its dependence on reaching a critical size, and how it can be controlled by photoperiod, revealing the existence of a photolabile period with important applications in aquaculture. Regarding size, apparently only European sea bass above a certain size threshold attain the ability to carry out gametogenesis during their first year of life, while their smaller counterparts fail to do so. This could imply that fish need to achieve an optimal threshold of hormone production, particularly from the kisspeptin/Gnrh/Gth systems, in order to initiate and conclude puberty. However, a long-term restricted feeding regime during the second year of life did not prevent the onset of puberty, thus suggesting that the fish are able to maintain the reproductive function, even at the expense of other functions. Finally, the study of daily hormonal rhythms under different photoperiod regimes revealed the equivalence between their core values and those of seasonal rhythms, in such a way that the daily rhythms could be considered as the functional units of the seasonal rhythms.

Differential activation of kiss receptors by Kiss1 and Kiss2 peptides in the sea bass.

Two forms of kiss gene (kiss1 and kiss2) have been described in the teleost sea bass. This study assesses the cloning and characterization of two Kiss receptor genes, namely kissr2 and kissr3 (known as gpr54-1b and gpr54-2b, respectively), and their signal transduction pathways in response to Kiss1 and Kiss2 peptides. Phylogenetic and synteny analyses indicate that these paralogs originated by duplication of an ancestral gene before teleost specific duplication. The kissr2 and kissr3 mRNAs encode proteins of 368 and 378 amino acids, respectively, and share 53.1% similarity in amino acid sequences. In silico analysis of the putative promoter regions of the sea bass Kiss receptor genes revealed conserved flanking regulatory sequences among teleosts. Both kissr2 and kissr3 are predominantly expressed in brain and gonads of sea bass, medaka and zebrafish. In the testis, the expression levels of sea bass kisspeptins and Kiss receptors point to a significant variation during the reproductive cycle. In vitro functional analyses revealed that sea bass Kiss receptor signals are transduced both via the protein kinase C and protein kinase A pathway. Synthetic sea bass Kiss1-15 and Kiss2-12 peptides activated Kiss receptors with different potencies, indicating a differential ligand selectivity. Our data suggest that Kissr2 and Kissr3 have a preference for Kiss1 and Kiss2 peptides, respectively, thus providing the basis for future studies aimed at establishing their physiologic roles in sea bass.

Gonadotropins in European sea bass: Endocrine roles and biotechnological applications.

Follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of the gonadal function in vertebrates. They act through specific receptors located in certain cell types found in the gonads. In fish, the differential roles of these hormones are being progressively elucidated due to the development of suitable tools for their study. In European sea bass (Dicentrarchus labrax), isolation of the genes coding for the gonadotropin subunits and receptors allowed in first instance to conduct expression studies. Later, to overcome the limitation of using native hormones, recombinant dimeric gonadotropins, which show different functional characteristics depending on the cell system and DNA construct, were generated. In addition, single gonadotropin beta-subunits have been produced and used as antigens for antibody production. This approach has allowed the development of detection methods for native gonadotropins, with European sea bass being one of the few species where both gonadotropins can be detected in their native form. By administering recombinant gonadotropins to gonad tissues in vitro, we were able to study their effects on steroidogenesis and intracellular pathways. Their administration in vivo has also been tested for use in basic studies and as a biotechnological approach for hormone therapy and assisted reproduction strategies. In addition to the production of recombinant hormones, gene-based therapies using somatic gene transfer have been offered as an alternative. This approach has been tested in sea bass for gonadotropin delivery in vivo. The hormones produced by the genes injected were functional and have allowed studies on the action of gonadotropins in spermatogenesis.

The forebrain-midbrain acts as functional endocrine signaling pathway of Kiss2/Gnrh1 system controlling the gonadotroph activity in the teleost fish European sea bass (Dicentrarchus labrax).

Some teleost species, including European sea bass, harbor two different kisspeptin coding genes: kiss1 and kiss2. Both genes are expressed in the brain, but their differential roles in the central control of fish reproduction are only beginning to be elucidated. In this study, we have examined the effects of intracerebroventricular injections of the highly active sea bass peptides Kiss1-15 and Kiss2-12 on spermiating male sea bass. Physiological saline, Kiss1-15, or Kiss2-12 was injected into the third ventricle. To establish the gene expression cascade involved in the action of kisspeptins, the expression of the two sea bass kisspeptin receptor genes (kiss1r and kiss2r) and the three sea bass Gnrh genes (gnrh1, gnrh2, and gnrh3) were analyzed in the forebrain-midbrain and the hypothalamus. In addition, the protein levels of hypothalamic and pituitary Gnrh1 were measured. Blood samples were collected at different times after injection to analyze the effects of kisspeptins on the release of gonadotropins (Lh and Fsh) and androgens (testosterone and 11-ketotestosterone). The present results provide the first evidence that the effects of Kiss2 on central regulation of reproductive function involve the neuroendocrine areas of the forebrain-midbrain in teleost fish. The marked effect of Kiss2 on kiss2r and gnrh1 expression in the forebrain-midbrain and on Gnrh1 release suggest that this neuronal system is involved in the neuroendocrine regulation of gonadotroph activity. This hypothesis was confirmed by a surge of plasma Lh in response to Kiss2, which presumably has a strong stimulatory effect on testosterone release, and thus on sperm quality parameters.

Expression of kisspeptins and kiss receptors suggests a large range of functions for kisspeptin systems in the brain of the European sea bass.

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.