Herve Migaud

Current knowledge on the melatonin system in teleost fish.

Melatonin is a much conserved feature in vertebrates that plays a central role in the entrainment of daily and annual physiological rhythms. Investigations aiming at understanding how melatonin mediates the effects of photoperiod on crucial functions and behaviors have been very active in the last decades, particularly in mammals. In fish a clear-cut picture is still missing. Here we review the available data on (i) the sites of melatonin production in fish, (ii) the mechanisms that control its daily and annual rhythms of production and (iii) the characterization of its different receptor subtypes, their location and regulation. The in vivo and in vitro data on melatonin effects on crucial neuroendocrine regulations, including reproduction, growth, feeding and behavioral responses, are also reviewed. Finally we discuss how manipulation of the photic cues impact on fish circannual clock and annual cycle of reproduction, and how this can be used for aquaculture purposes.

Current knowledge on the photoneuroendocrine regulation of reproduction in temperate fish species

Seasonality is an important adaptive trait in temperate fish species as it entrains or regulates most physiological events such as reproductive cycle, growth profile, locomotor activity and key life-stage transitions. Photoperiod is undoubtedly one of the most predictable environmental signals that can be used by most living organisms including fishes in temperate areas. This said, however, understanding of how such a simple signal can dictate the time of gonadal recruitment and spawning, for example, is a complex task. Over the past few decades, many scientists attempted to unravel the roots of photoperiodic signalling in teleosts by investigating the role of melatonin in reproduction, but without great success. In fact, the hormone melatonin is recognized as the biological time-keeping hormone in fishes mainly due to the fact that it reflects the seasonal variation in daylength across the whole animal kingdom rather than the existence of direct evidences of its role in the entrainment of reproduction in fishes. Recently, however, some new studies clearly suggested that melatonin interacts with the reproductive cascade at a number of key steps such as through the dopaminergic system in the brain or the synchronization of the final oocyte maturation in the gonad. Interestingly, in the past few years, additional pathways have become apparent in the search for a fish photoneuroendocrine system including the clock-gene network and kisspeptin signalling and although research on these topics are still in their infancy, it is moving at great pace. This review thus aims to bring together the current knowledge on the photic control of reproduction mainly focusing on seasonal temperate fish species and shape the current working hypotheses supported by recent findings obtained in teleosts or based on knowledge gathered in mammalian and avian species. Four of the main potential regulatory systems (light perception, melatonin, clock genes and kisspeptin) in fish reproduction are reviewed.

A comparative ex vivo and in vivo study of day and night perception in teleosts species using the melatonin rhythm

Abstract:  The purpose of this study was to determine and compare the light sensitivity of two commercially important, phylogenetically different teleost species in terms of melatonin production. Three series of experiments were performed on both Atlantic salmon and European sea bass. First, a range of light intensities were tested ex vivo on pineal melatonin production in culture during the dark phase. Then, light transmission through the skull was investigated, and finally short‐term in vivo light sensitivity trials were performed. Results showed that sea bass pineal gland ex vivo are at least 10 times more sensitive to light than that of the salmon. Light intensity threshold in sea bass appeared to be between 3.8 × 10−5 and 3.8 × 10−6 W/m2 in contrast to 3.8 × 10−4 and 3.8 × 10−5 W/m2 in salmon. These highlighted species‐specific light sensitivities of pineal melatonin production that are likely to be the result of adaptation to particular photic niches. Light transmission results showed that a significantly higher percentage of light penetrates the sea bass pineal window relative to salmon, and confirmed that penetration is directly related to wavelength with higher penetration towards the red end of the visible spectrum. Although results obtained in vivo were comparable, large differences between ex vivo and in vivo were observed in both species. The pineal gland in isolation thus appeared to have different sensitivities as the whole animal, suggesting that retinal and/or deep brain photoreception may contribute, in vivo, to the control of melatonin production.

Mapping the sex determination locus in the Atlantic halibut (Hippoglossus hippoglossus) using RAD sequencing

BackgroundAtlantic halibut (Hippoglossus hippoglossus) is a high-value, niche market species for cold-water marine aquaculture. Production of monosex female stocks is desirable in commercial production since females grow faster and mature later than males. Understanding the sex determination mechanism and developing sex-associated markers will shorten the time for the development of monosex female production, thus decreasing the costs of farming.ResultsHalibut juveniles were masculinised with 17 α-methyldihydrotestosterone (MDHT) and grown to maturity. Progeny groups from four treated males were reared and sexed. Two of these groups (n = 26 and 70) consisted of only females, while the other two (n = 30 and 71) contained balanced sex ratios (50% and 48% females respectively). DNA from parents and offspring from the two mixed-sex families were used as a template for Restriction-site Associated DNA (RAD) sequencing. The 648 million raw reads produced 90,105 unique RAD-tags. A linkage map was constructed based on 5703 Single Nucleotide Polymorphism (SNP) markers and 7 microsatellites consisting of 24 linkage groups, which corresponds to the number of chromosome pairs in this species. A major sex determining locus was mapped to linkage group 13 in both families. Assays for 10 SNPs with significant association with phenotypic sex were tested in both population data and in 3 additional families. Using a variety of machine-learning algorithms 97% correct classification could be obtained with the 3% of errors being phenotypic males predicted to be females.ConclusionAltogether our findings support the hypothesis that the Atlantic halibut has an XX/XY sex determination system. Assays are described for sex-associated DNA markers developed from the RAD sequencing analysis to fast track progeny testing and implement monosex female halibut production for an immediate improvement in productivity. These should also help to speed up the inclusion of neomales derived from many families to maintain a larger effective population size and ensure long-term improvement through selective breeding.

Evidence for differential photic regulation of pineal melatonin synthesis in teleosts

Abstract:  The aim of this study was to compare the circadian control of melatonin production in teleosts. To do so, the effects of ophthalmectomy on circulating melatonin rhythms were studied along with ex vivo pineal culture in six different teleosts. Results strongly suggested that the circadian control of melatonin production could have dramatically changed with at least three different systems being present in teleosts when one considers the photic regulation of pineal melatonin production. First, salmonids presented a decentralized system in which the pineal gland responds directly to light independently of the eyes. Then, in seabass and cod both the eyes and the pineal gland are required to sustain full night‐time melatonin production. Finally, a third type of circadian control of melatonin production is proposed in tilapia and catfish in which the pineal gland would not be light sensitive (or only slightly) and required the eyes to perceive light and inhibit melatonin synthesis. Further studies (anatomical, ultrastructural, retinal projections) are needed to confirm these results. Ex vivo experiments indirectly confirmed these results, as while the pineal gland responded normally to day–night rhythms in salmonids, seabass and cod, only very low levels were obtained at night in tilapia and no melatonin could be measured from isolated pineal glands in catfish. Together, these findings suggest that mechanisms involved in the perception of light and the transduction of this signal through the circadian axis has changed in teleosts possibly as a reflection of the photic environment in which they have evolved in.

Induction of out-of-season spawning in Eurasian perch Perca fluviatilis: effects of rates of cooling and cooling durations on female gametogenesis and spawning

Abstract This study was designed to determine the influence of different thermal conditions during an out-of-season reproductive cycle on gonad recrudescence, plasma steroid levels (testosterone and estradiol), vitellogenin (VTG) concentrations and spawning in Eurasian perch ( Perca fluviatilis ) females. The experiment was performed in 450-l square polyester indoor tanks located in two rooms equipped with controlled light and temperature devices. Four thermal regimes were tested with two different rates of cooling from 21 to 6 °C, 3 weeks (3w) or 6 weeks (6w), and two different durations at 6 °C, 3 months (3m) or 5 months (5m). The photoperiod was fixed at LD 12:12. A long cooling period (6w) resulted in greater gonadosomatic index (GSI) (3.6±0.5%) correlated with a larger oocyte diameter (787.9±25.1 μm). The plasma levels of testosterone (13.63±1.18 ng ml −1 ) in the former groups were higher than in the short cooling period groups (6.82±0.9 ng ml −1 ). A longer duration at 6 °C (5m) resulted in higher GSI (14.8±1%), plasma testosterone levels (26.2±0.4 ng ml −1 ) and plasma protein phosphorus (PPP, 1.33±0.3 μg ml −1 ). The fish from the 6w 5m batch showed the highest plasma estradiol and testosterone levels, whereas the 3w 5m group showed the largest GSI. At the end of the experiment, several spontaneous out-of-season spawnings were collected in batch 6w 5m. Biopsy showed that most oocytes from groups 3w 5m and 6w 5m females were mature (migration of the germinal vesicle from central position), whereas few females showed a beginning of migration in groups 3w 3m and 6w 3m, suggesting that the gonad development and reproductive success of Eurasian perch mainly depends on the chilling duration (long cold period) rather than on the cooling one, in order to obtain out-of-season spawning.

Seasonal Variations in Clock‐Gene Expression in Atlantic Salmon (Salmo salar)

In homeothermic vertebrates inhabiting temperate latitudes, it is clear that the seasonal changes in daylength are decoded by the master circadian clock, which through secondary messengers (like pineal melatonin secretion) entrains rhythmic physiology to local conditions. In contrast, the entrainment and neuroendocrine regulation of rhythmic physiology in temperate teleosts is not as clear, primarily due to the lack of understanding of the clock gene system in these species. In this study, we analyzed the diel expression of the clock‐genes in brains of Atlantic salmon, a species that is both highly photoperiodic and displays robust clock‐controlled behavior. Atlantic salmon parr were acclimated to either long‐day (LD) or short‐day (SD) photoperiods for one month and thereafter sampled at 4 h intervals over a 24 h cycle. Clock, Bmal1, Per2, and Cry2 were all actively expressed in salmon brain homogenates and, with the exception of Per2, all displayed rhythmic expression under SD photoperiods that parallels that reported in zebrafish. Interestingly, daylength significantly altered the mRNA expression of all clock genes studied, with Clock, Bmal1, and Per2 all becoming arrhythmic under the LD compared to SD photoperiod, while Cry2 expression was phase delayed under LD. It is thus proposed that the clock‐gene system is actively expressed in Atlantic salmon, and, furthermore, as has been reported in homeothermic vertebrates, it appears that clock expression is daylength‐dependent.

Comparative seawater performance and deformity prevalence in out-of-season diploid and triploid Atlantic salmon (Salmo salar) post-smolts.

The use of sterile triploid stock in the Atlantic salmon (Salmo salar, L) farming industry is the only commercially available means to prevent the ecological impact of domesticated escapees. This study compared the seawater (SW) performance and deformity prevalence of diploid and triploid post-smolts from 2 full-sib families produced out-of-season. Triploids completed smoltification 4 weeks earlier and at a significantly higher body-weight. Growth and survival in SW were not significantly affected by ploidy. The incidence of external deformities, dominated by jaw malformation, was ~12% in triploids and below 5% in diploids. Vertebral deformities were more prevalent in the fastest growing triploid family only. Heart morphometry differed between ploidies which may relate to a higher cardiac workload in triploids. No clear alteration of the gill apparatus was detected. The most significant detrimental effect of triploidy was on the rate and severity of cataract that were observed from August onward (50% and 92% of diploids and triploids respectively affected after 1-year in SW). At that time, cataracts were diagnosed by histological examinations as irreversible with a probable osmotic origin which could arise from factors such as water quality, nutritional deficiencies or thermal variations. This study warrants further research aiming at adapting rearing practices to the needs of triploid stocks as to improve their performance and welfare.

Differential light intensity and spectral sensitivities of Atlantic salmon, European sea bass and Atlantic cod pineal glands ex vivo

Photoperiod is perceived by pineal photoreceptors and transduced into rhythmic melatonin signals. These rhythms can be influenced by light intensity and spectral content. In this study we compared the light sensitivity of Atlantic salmon, European sea bass and Atlantic cod by testing ex vivo the effect of different intensities and narrow bandwidth lights on nocturnal melatonin suppression by isolated pineal glands in a flow-through culture system. Using combinations of neutral density and bandpass interference filters we tested a range of light intensities (ranging from 1.22x10(13) to 3.85x10(6) photons s(-1) cm(-2)) and three wavelengths of 80 nm width (472, 555 and 661 nm corresponding to blue, green and red, respectively). Results showed clear species specific light intensity and spectral sensitivities, with cod being from 100 to 1000 times more sensitive than sea bass and salmon. Regarding the influence of spectrum, red light was less efficient on suppressing melatonin than blue and green in salmon but results were not as clear in the two other species studied. Finally, the first evidence of relative photoreception in teleosts was obtained in cod suggesting that the definition of illuminance thresholds (day/night perception) would depend on the day intensity. Indeed, a single order of magnitude increase or decrease in day intensity was shown to elicit a significant shift in the intensity response curve of night-time melatonin suppression. Taken together, this study demonstrated species specific light intensity and spectral sensitivities within temperate teleosts.

GPR54 and rGnRH I gene expression during the onset of puberty in Nile tilapia

The Kiss1/GPR54 system has recently been shown to play a key role in the onset of puberty in mammals. Growing evidence suggests that this system is also conserved across vertebrates although very few studies so far have been performed in lower vertebrates. The aims of this study were firstly in the teleost Nile tilapia to screen tissues for GPR54 expression levels, secondly to measure the expression patterns of GPR54 and GnRH I receptor (rGnRH I) in whole brains during the onset of puberty and finally to determine the effects of continuous illumination (LL) on receptor expression levels. Results confirmed that GPR54 was predominantly expressed in the brain and pituitary of adult tilapia. Furthermore, a significant increase of GPR54 gene expression was found in tilapia brains at 11 weeks post hatch (wph) followed by rGnRH I at 13 wph just prior to the histological observation of vitellogenic oocytes and active spermatogenesis in ova and testes at 17 wph. These results suggest a correlation between the increase of GPR54 expression in the brain and the onset of puberty. Finally, a significant effect of LL was observed on GPR54 expression levels which were characterized by a delayed surge with significantly lower levels than those of control fish. The current study not only suggests a link between the Kiss1/GPR54 system and the onset of puberty in a tropical batch spawning teleost that would be a highly conserved feature across vertebrates but also that the transcriptional mechanisms regulating GPR54 expression could be directly or indirectly influenced by light.