Alicia Felip

Control of puberty in farmed fish

Puberty comprises the transition from an immature juvenile to a mature adult state of the reproductive system, i.e. the individual becomes capable of reproducing sexually for the first time, which implies functional competence of the brain-pituitary-gonad (BPG) axis. Early puberty is a major problem in many farmed fish species due to negative effects on growth performance, flesh composition, external appearance, behaviour, health, welfare and survival, as well as possible genetic impact on wild populations. Late puberty can also be a problem for broodstock management in some species, while some species completely fail to enter puberty under farming conditions. Age and size at puberty varies between and within species and strains, and are modulated by genetic and environmental factors. Puberty onset is controlled by activation of the BPG axis, and a range of internal and external factors are hypothesised to stimulate and/or modulate this activation such as growth, adiposity, feed intake, photoperiod, temperature and social factors. For example, there is a positive correlation between rapid growth and early puberty in fish. Age at puberty can be controlled by selective breeding or control of photoperiod, feeding or temperature. Monosex stocks can exploit sex dimorphic growth patterns and sterility can be achieved by triploidisation. However, all these techniques have limitations under commercial farming conditions. Further knowledge is needed on both basic and applied aspects of puberty control to refine existing methods and to develop new methods that are efficient in terms of production and acceptable in terms of fish welfare and sustainability.

Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals.

Kisspeptins, the products of KiSS-1 gene, have recently emerged as fundamental regulators of reproductive function in different mammalian and, presumably, non-mammalian species. To date, a single form of KiSS-1 has been described in mammals, and recently, in several fish species and Xenopus. We report herein the cloning and characterization of two distinct KiSS-like genes, namely, KiSS-1 and KiSS-2, in the teleost sea bass. While KiSS-1 encodes a peptide identical to rodent kisspeptin-10, the predicted KiSS-2 decapeptide diverges at 4 amino acids (FNFNPFGLRF). Genome database searches showed that both genes are present in non-placental vertebrate genomes. Indeed, phylogenetic and genome mapping analyses suggest that KiSS-1 and KiSS-2 are paralogous genes that originated by duplication of an ancestral gene, although KiSS-2 is lost in placental mammals. KiSS-1 and KiSS-2 mRNAs are present in brain and gonads of sea bass, medaka and zebrafish. Comparative functional studies demonstrated that KiSS-2 decapeptide was significantly more potent than KiSS-1 peptide in inducing LH and FSH secretion in sea bass. In contrast, KiSS-2 decapeptide only weakly elicited LH secretion in rats, whereas KiSS-1 peptide was maximally effective. Our data are the first to provide conclusive evidence for the existence of a second KiSS gene, KiSS-2, in non-placental vertebrates, whose product is likely to play a dominant stimulatory role in the regulation of the gonadotropic axis at least in teleosts.

Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species

The induction of triploidy and gynogenesis by chromosome set manipulation has traditionally been studied more intensively in freshwater than in marine fish. In the last years, however, several studies have applied these manipulations in about a dozen marine species, including mainly sparids, moronids and flatfishes. This paper focuses on the methodologies used to induce, verify, and assess performance of both triploids and gynogenetics of these marine species. Since many of them are batch spawners and have small and fragile eggs and larvae, peculiarities relating to broodstock management, gamete quality and mortality assessment during early larval stages are also taken into account. However, data show that if handling is correct and the treatments are optimized, triploid and gynogenetic rates of 100% can be easily achieved. Survival of triploids with respect to the controls is about 70–80%, whereas in gynogenetics it is generally low and more variable, depending on the species considered. In the marine fish investigated so far, triploidy has not resulted in significantly higher growth rates. On the other hand, the induction of gynogenesis has resulted in the production of both all-female and mix-sex stocks. Throughout the paper, special reference is made to the European sea bass (Dicentrarchus labrax L.), a species of both basic and applied interest, for which a comprehensive study has been carried out on the induction, verification and performance of triploids and gynogenetics.

Comparative insights of the kisspeptin/kisspeptin receptor system: Lessons from non-mammalian vertebrates

Kisspeptins, the peptide products of the Kiss1 gene, were initially identified in mammals as ligands of the G protein-coupled receptor 54 (GPR54; also termed Kiss1R) with ability to suppress tumor metastasis. In late 2003, the indispensable role of kisspeptins in the control of reproductive function was disclosed by the seminal observations that humans and mice carrying inactivating mutations of GPR54 displayed hypogonadotropic hypogonadism. Since then, numerous experimental studies, conducted initially in several mammalian species, have substantiated the roles of kisspeptins as essential players in the physiologic regulation of key aspects of reproductive maturation and function, including the timing of puberty onset, the dynamic control of gonadotropin secretion via stimulation of GnRH neurons, the transmission of the negative and positive feedback effects of sex steroids, the metabolic regulation of fertility and the control of reproductive function by environmental (photoperiodic) cues. Notably, while studies about kisspeptins in non-mammals appeared initially to lag behind, significant efforts have been devoted recently to define the genomic organization and functional characteristics of kiss/kisspeptins and gpr54 in different non-mammalian species, including fish, reptiles and amphibians. These analyses, which will be comprehensively revised herein, have not only substantiated the conserved, essential roles of kisspeptins in the control of reproduction, but have also disclosed intriguing evolutionary aspects of kisspeptins and their receptors. Such comparative approaches will be instrumental to fuel further studies on the molecular regulation and physiological roles of kisspeptins, thus helping to unveil the complex biology of this system as indispensable regulator of the reproductive axis in a wide diversity of animal species.

Identification of the Major Sex-Determining Region of Turbot ( Scophthalmus maximus )

Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.

Genetic, hormonal and environmental approaches for the control of reproduction in the European sea bass (Dicentrarchus labrax L.)

The European sea bass Dicentrarchus labrax is a very important commercial fish in Europe, the production of which by aquaculture has increased several fold during the last decades. Studies on biology, control of reproduction, broodstock management, offspring quality and rearing techniques, as well as large investments in the aquaculture sector, have made possible this rapid increase in production. However, under culture conditions, there are still important problems that negatively influence its industrial production. These are: (1) high percentage of males (70–90%), some of which are precocious at the first year of life; (2) in males, puberty typically appears in the second year of life (at commercialization), 1 year before females; and (3) males grow less than females, being 40% lower in body weight at harvest time. Consequently, reduction of the number of males and/or production of all-female stocks and sterile fish and prevention of puberty are highly desirable goals. The present work reviews the latest scientific advances on the genetic and physiological control of the sex and the process of puberty in the sea bass to provide tools to alleviate some shortcuts in its industrial production.

Optimal conditions for the induction of triploidy in the sea bass (Dicentrarchus labrax L.)

Abstract The optimal conditions for the induction of triploidy in the European sea bass ( Dicentrarchus labrax L.) using cold shocks were investigated. Three treatment variables were considered: the time after fertilization when the shock was applied, the temperature of the shock and the duration of the shock. Ploidy was determined by counting the nuclear organizer regions, karyotype analysis and measures of erythrocyte size. A first series of experiments performed at what was subsequently shown to be a sub-optimal shock duration (5 min), demonstrated that the best time after fertilization for cold shock application was 5 min, regardless of temperature of shock (0 or 2 °C). The maximum percent of triploids obtained was 87% at survival rates approximately 90% of that of the untreated controls. In a second series of experiments, this timing (5 min after fertilization) was used to determine the best combination of temperature (0, 2 or 4 °C) and duration of shock (5, 10, 15 or 20 min) for optimal triploid induction. In these experiments, triploidy reached 100%, with survival approximately 80% of controls. However, when the shock temperature was above 0 °C and the duration shorter than 10 min, triploidy induction became highly variable. The optimum treatment parameters for the induction of triploidy in the sea bass were established to be as follows: time after fertilization, 5 min; duration of shock, 10 min; temperature of shock, 0 °C. Pre-shock incubation temperature was 12–13 °C in all cases. These conditions were used to mass-produce triploid sea bass. Results showed that under these conditions triploidy induction could reach 100%, although it was essential to reproduce optimum shock conditions, especially temperature, when dealing with large volumes of eggs. At 8 months of age, triploid sea bass had similar growth than their diploid counterparts. In conclusion, this paper presents the optimal conditions for triploidy induction in the sea bass using cold shock and indicates that it is possible to mass-produce triploids of this commercially important species.

Hormonal and Environmental Control of Puberty in Perciform Fish The Case of Sea Bass

A specific chronology for puberty and changes at the brain–pituitary–gonad axis for sea bass are reviewed. Recent findings demonstrate that the Kisspeptin system, gonadotropin releasing hormones, follicle stimulating hormone, 11‐ketotestosterone, and leptin are potential candidates for the onset of puberty of this fish species, stressing the importance of the daily and annual rhythms of some of these hormones. Environmental control of puberty is also reviewed, specifically the manipulations of constant photoperiods for altering or even suppressing the onset of puberty in sea bass. Recently, a possible narrow sensitive period for suppressing gonadogenesis in sea bass has been identified.

Growth and gonadal development in triploid sea bass (Dicentrarchus labrax L.) during the first two years of age

Abstract The aquaculture of the sea bass is now well established in Mediterranean countries. However, under culture conditions the sexual maturation is an undesirable process that reduces growth. In addition, spermiation around the time of marketing of fish is another inconvenience. In an attempt to solve these problems, triploidy was induced in the sea bass according to a previously published protocol. A cold shock applied starting 5 min after fertilization for 10 min at 0°C was used for the triploidisation of a large volume of sea bass eggs. Control and shocked fish were reared in duplicate groups under natural conditions of temperature and photoperiod using standard procedures for sea bass aquaculture. Triploidy was determined in a sample of fish at 3 months of age by karyotyping (2n=48; 3n=72), and individually checked at 15 months of age by measurement of the major axis of erythrocytes (1.2–1.3 times larger in shocked fish than in controls; P

Expression of kisspeptins in the brain and pituitary of the european sea bass (Dicentrarchus labrax)

Kisspeptins are now considered key players in the neuroendocrine control of puberty and reproduction, at least in mammals. Most teleosts have two kiss genes, kiss1 and kiss2, but their sites of expression are still poorly documented. As a first step in investigating the role of kisspeptins in the European sea bass, a perciform fish, we studied the distribution of kiss1 and kiss2‐expressing cells in the brain of males and females undergoing their first sexual maturation. Animals were examined at early and late in the reproductive season. We also examined the putative expression of estrogen receptors in kiss‐expressing cells and, finally, we investigated whether kisspeptins are expressed in the pituitary gland. We show that kiss1‐expressing cells were consistently detected in the habenula and, in mature males and females, in the rostral mediobasal hypothalamus. In both sexes, kiss2‐expressing cells were consistently detected at the level of the preoptic area, but the main kiss2 mRNA‐positive population was observed in the dorsal hypothalamus, above and under the lateral recess. No obvious sexual differences in kiss1 and kiss2 mRNA expression were detected. Additional studies based on confocal imaging clearly showed that most kiss1 mRNA‐containing cells of the mediobasal hypothalamus strongly express ERα and slightly express ERβ2. At the pituitary level, both sexes exhibited kiss1 mRNA expression in most FSHβ‐positive cells and never in LHβ‐positive cells. J. Comp. Neurol. 521:933–948, 2013.