Tesis - Universidad Autonoma de Baja California. / Includes bibliographical references (leaves [44-47]).

Genetic differentiation among juvenile samples of the European seabass (Dicentrarchus labrax) from the coast of Portugal is reported by means of two types of genetic markers: allozymes and microsatellites. Repeat samples were taken from 5 different nursery grounds (Aveiro, Foz, Obidos, Milfontes and Faro) along the coast of Portugal between November 1992 and February 1994. Starch-gel electrophoresis was used to assess the level and distribution of genetic variability of 38 loci. Six of these were found to be polymorphic at the 99% level and were used in population surveys: AAT-3*, ADA *, GPI-I *, GPI-2*, G3PDH-2*, SOD*. Statistical analysis revealed low but statistically significant multilocus F.'I (0.0108, p<O.OOI) values suggesting that population structuring exists along the Portuguese coast line. The results indicate that there is some restriction in gene flow between the more southerly population at Faro and all other sites to the North. Five microsatellite loci were screened in over 300 individuals. High levels of polymorphism (number of alleles observed per locus ranged from 20 to 41) and observed heterozygosities, ranging from 0.45 to 0.89 (mean over all loci = 0.71) were detected. Two loci displayed heterozygosity deficits (Dla6 and Labrax-9) and were not used in population comparisons. Statistical analysis revealed low but statistically significant multilocus FST (0.0025, p<O.OOl) at the other three loci (Dlall, Labrax-3 and Labrax-8). No clear geographic patterns emerged from these results. Overall allozymes performed well when compared to microsatellites, in detecting microgeographic genetic structure in this species. Microsatellites revealed high levels of polymorphism that should prove useful as markers in the management of wild and farmed seabass stocks in the future. The level of differentiation, low values of F ST , detected among the sites is low but is typical of marine species which have a much greater chance of mixing.

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Commercial aquaculture for marine species is gaining importance in todays industry. Over fishing of the natural recource has sadly meant that many of the higher predators halibut, turbot, seabass, sea bream and tuna are now financially suitable for the industry. This thesis covers a wide range of topics involving two of the most valuable marine species, namely the Atlantic halibut and European seabas, from broodstock to larval first-feeding. Broodstock husbandry and especially nutrition are often the last to receive attention. This study shows that careful management of the broodstock, firstly by the close timing of stripping, secondly enhancement of the broodstock diet with n-3 and n-6 HUFA can increase both fecundity and egg quality right up to the point where the larvae switch from endogenous to exogenous feeding. The implications for the industry for these two species are twofold. Firstly, The identification that halibut eggs can be fertilised within 6 hours of ovulation with no detrimental effects on fertilisation rates and subsequent egg performance means that eggs could be transported to specialist egg and larval rearing units. Thus the potential exists for the industry to be divided into separate units dealing with different stages of the life cycle much like the salmon industry. Secondly, the development of an artificial pelleted brood stock diet would mean that problems of consistency and quality and also the dangers of disease infection via the feed can be removed from broodstock management. The stage of first-feeding for halibut is still considered to be the main bottleneck in the production of this species. The current work has shown that small systems of 100-1 can be used to successfully rear halibut larvae. Although careful consideration must be made of the system design to ensure that predator (larvae) and prey (Artemia) remain homogeneously dispersed. Also, the need to use rotifers at first feed has been shown to be unecessary. However, nutrition of first-feeding larvae still requires the use of wild zooplankton to ensure successful pigmentation. Yet, Artemia supplied with commercial enrichments still perform adequately in terms of their overall growth.

Aquaculture is an essential developing sector for world food production however one of the major bottlenecks for the sustainability of the aquaculture industry is the ability to control fish reproduction in captivity and to produce high quality seeds. European sea bass is a one of most commercially important species for the European fish farming industry. If broodstock management under captivity is well established, problems remain in hatcheries where survival can be low and deformity prevalence high as well as in on growing sites where fish reach puberty early especially with skewed sex ratio towards males. Sea bass displays strong seasonality in its physiology and is therefore an excellent candidate for the study of the photo-neuroendocrine control of reproduction and growth. The overall aims of this thesis were to better understand the molecular and endocrine drivers that control the Brain-Pituitary-Gonad axis in repeat spawner sea bass, and expand our knowledge of sea bass light and temperature regulation of melatonin production. First, this PhD project investigated the seasonal expression of kisspeptin, GnRH and gonadotropin genes in relation to the gonadal development throughout a reproductive cycle in male repeat spawning sea bass (Chapter 3). A partial sequence for the receptor kissr4 was isolated and described showing similarity to all other teleost species sequences available to date. QPCR molecular assays were validated to mesure the expression of a suite of genes along the BPG axis including kisspeptin related genes (Kiss1 and Kiss2 and its receptor kissr4) over a full reproductive cycle (12 months) in adult male European sea bass. Brain Kisspeptin mRNA expression levels (kiss1, 2 and kissr4) showed clear seasonal profiles and correlated well to other BPG markers (GnRHs, fshβ and lhβ), supporting a possible involvement of kisspeptin genes in the seasonal control of reproduction in repeat spawning sea bass. Moreover, clear seasonal patterns were observed for expression of the genes encoding for pituitary mRNA expression of lhβ and fshβ, with a significant correlation between expression of both subunits and GSI and steroids levels. However, no clear seasonal profiles in brain GnRHs gene expression were observed with the exception to some peaks in GnRH1 and GnRH2. The second part of this PhD project investigated the potential direct effect of the two kisspeptin core peptides (kiss1 and kiss2) on the pituitary gonadotropin gene expression (Chapter 4). The aim of this work was to better understand the mechanism by which kisspeptin acts on the BPG axis. This was done by testing the kisspeptin decapeptide core sequences on the lhβ and fshβ transcript expression in primary culture of sea bass pituitary cells using QPCR technique. The findings, as a whole, provided evidence that kisspeptin can act directly on the pituitary gonadotroph cells and modulate fshß and lhß mRNA expression in sea bass although effects were limited and not uniform. Of note, kissr4 gene expression was also detected in the sea bass pituitary. The third part of this PhD project focused on the effects of environmental signals (photoperiod and temperature) on melatonin production (Chapter 5). Environmental manipulation is routinely used in the aquaculture industry with the purpose of enhancing growth and manipulating the timing of reproduction in seasonal fish species like sea bass. Melatonin, known as the light perception and time keeping hormone, has been suggested to play key roles in the synchronisation of most physiological functions in vertebrates, although the mechanisms by which melatonin controls reproduction, growth and behaviour are still not fully understood in fish. The studies performed aimed .to determine the synergistic effects of both temperature and photoperiod on the daily phase and amplitudinal changes in melatonin production through both in vivo and in vitro trials. The results confirmed the diel melatonin rhythm in sea bass as previously reported in many teleost species with “high at night” and “low at day” melatonin profiles. Temperature showed clear effects on the amplitude of the melatonin production under both in vivo and in vitro conditions for both long day and short day photoperiods. Furthermore, no endogenous melatonin production was found under constant darkness in both in vivo and in vitro conditions. These results suggested a lack of intrapineal (or located elsewhere such as retina and/or deep brain) oscillators in sea bass, contrasting with previous reports. These results further enhance our knowledge of light perception and circadian rhythmicity in sea bass, while the circadian system remains to be characterised in sea bass and teleosts as a whole. Overall, this doctoral work broadened our understanding on the photoneuroendocrine control of reproduction in a seasonal fish species, sea bass. New knowledge gained and tools developed from this work should help to develop/optimise husbandry techniques for the sea bass farming industry with the view to increase production and profitability and thus promoting the sustainable expansion of the sea bass aquaculture in Europe. It has also the potential to help the fishery sector in the modelling of wild sea bass populations.

The current general economic and food crises have generated an unsettled future for food and feed production and prices in general. Increasing demand, prices and fluctuations in supply in world markets for fishmeal, fish oil, soybean meal, maize and wheat meal emphasize the need to reduce the dependence of the fish feed industry on these ingredients by increasing choices among a wider range of raw materials. Legume seed such as peas, chickpeas and faba beans are promising ingredients for aquafeeds due to their high protein compared to cereals but also for their energy content. The objective of the present study was to investigate the use of these legumes in both high and low inclusion level in diets for the two main species farmed in the Mediterranean countries namely, European seabass (Dicentrarchus labrax L.) and gilthead seabream (Sparus aurata L.). In Chapter 3 the effects of different processing conditions were examined on whole seed flours of tested ingredients with respect to both nutritional and antinutritional factors and physical characteristics of the experimental diet pellets including high and low levels of each legume. Extrusion of raw material resulted in a clear reduction in trypsin inhibitors (TI) with chickpeas showing a decrease of up to 90% and complete inactivation for wheat flour, while for peas and faba beans reduction was less than 50% in most cases. Extrusion processing was less effective in the reduction of phytic acid and total tannins, occasionally reaching 22% and 18% respectively. Oligosaccharides and total NSP were not drastically affected by processing, however faba bean NSP showed greater reduction. A redistribution of soluble and insoluble NSP fractions was noted for chickpea and pea flours after extrusion. Physical characteristics of the pellets were not negatively affected for any of the tested diets. In Experiment I the effects of total or partial wheat substitution by legumes were investigated on nutrient digestibility, gastrointestinal evacuation rate and serum glucose response of European seabass. Use of legumes in seabass and seabream diets resulted in an overall increase in gastrointestinal evacuation time and a delay in glucose load. Specifically, gastric evacuation time was greatly delayed when seabass was fed a diet with high levels (30%) of chickpeas, while foregut evacuation time was mostly delayed by the diet including a high level (30%) of faba beans. In addition, glucose levels in seabass serum were also affected by the type of carbohydrates ingested with wheat starch showing more rapid increase and decrease of glucose compared to fish fed pea and chickpea diets, while faba bean starch resulted in a lower glucose peak. In Experiment II growth, digestibility, hematological parameters, histological effects and fillet organoleptic characteristics and the interaction between starch inclusion level (8% and 16% or 17% and 35% of legumes respectively) and legume type were estimated when tested legumes replaced wheat in European seabass diets. Digestibility coefficients were satisfactory for all nutrients (&gt;93%) while legume diets at a low level had beneficial effects on growth parameters when compared to the control diet, with chickpeas showing a significant improvement in SGR (P&lt;0.05). High level legume diets did not result in any negative effect on growth. HSI was increased with increasing starch/legume inclusion in the feed and serum glucose also increased for fish fed high levels of faba beans and chickpeas. Carcass proximate composition was not affected by replacement of wheat in the diets, excluding the increase of fat content in fish fed chickpeas. Sensory analysis showed no differences between fish fed the control and high legume inclusion diets. Lastly in Experiment III growth, hematological parameters, histological effects and the interaction between starch inclusion level (low and high) and legume type were evaluated when tested legumes were included in gilthead seabream diets. Decreased, but not significantly so, growth was observed for all diets including legumes compared to the control. Poorer SGR were observed for pea and faba bean diets when these legumes were included at high levels. Liver glycogen increased with increasing starch level, but HSI did not differ significantly for any of the diet treatments. Histological examination of hindgut did not show pathological effects, such as enteritis, for in either species or for any of the diets. Increased absorptive vacuoles were found for control and pea diets (high level) only for seabass. The findings of this thesis showed that the two important species cultivated in Mediterranean countries responded differently to the same raw materials used at high levels in the diets. Overall legumes had a strong effect on gastrointestinal evacuation reducing the rate of feed or digesta passage. Peas, chickpeas and faba beans successfully replaced wheat in seabass diets resulting in improved growth coefficients. However, when the same legumes included in seabream diets growth performance was not improved compared to the wheat based diet.

The aim of the current thesis was the analysis of the genetics of sex determination of farmed fish with sexual dimorphism, using Next Generation Sequencing. Three different species of farmed fish with sex-determining systems of varying complexity were studied. Both full-sibs and more distantly related specimens of Atlantic halibut (Hippoglossus hippoglossus), Nile tilapia (Oreochromis niloticus) and European sea bass (Dicentrarchus labrax) were used for this study. Application of Restriction-site Associated DNA sequencing (RAD-seq) and double digest Restriction-site Associated DNA sequencing (ddRAD-seq), two related techniques based on next generation sequencing, allowed the identification of thousands of Single Nucleotide Polymorphisms (SNPs; &gt; 3,000) for each of the above species. The first SNP-based genetic maps for the above species were constructed during the current study. The first evidence concerning the location of the sex-determining region of Atlantic halibut is provided in this study. In the case of Nile tilapia both novel sex-determining regions and fine mapping of the major sex-determining region are presented. In the study of European sea bass evidence concerning the absence of a major sex-determining gene was provided. Indications of putative sex-determining regions in this species are also provided. The results of the current thesis help to broaden current knowledge concerning sex determination in three important farmed fish. In addition the results of the current thesis have practical applications as well, towards the production of mono-sex stocks of those species for the aquaculture industry.

Isogenic clonal fish lines are a powerful resource for aquaculture-related research. Fully inbred individuals, clone founders, can be produced either through mitotic gynogenesis or androgenesis and a further generation from those propagates fully inbred clonal lines. Despite rapid generation, as opposed to successive generation of sibling mating as in mice, the production of such lines may be hampered due to (i) potential residual contribution from irradiated gametes associated with poorly optimised protocols, (ii) reduced survival of clone founders and (iii) spontaneous arisal of meiotic gynogenetics with varying degree of heterozygosity, contaminating fully homozygous progenies. This research set out to address challenges and gain insights into isogenic clonal fish lines development by using double-digest RADseq (ddRADseq) to generate large numbers of genetic markers covering the genome of interest. Analysis of potential contribution from irradiated sperm indicated successful uniparental inheritance in meiotic and mitotic gynogenetics European seabass. Exclusive transmission of maternal alleles was detected in G1 progeny of Atlantic salmon (with a duplicated genome), while G2 progenies presented varying levels of sire contribution suggesting sub-optimal UV irradiation which was undetected previously with 27 microsatellite markers. Identification of telomeric markers in European seabass, with higher recombination frequencies for efficient differentiation of meiotic and mitotic gynogenetics was successful, and a genetic linkage map was generated from this data. One clear case of a spontaneous meiotic gynogenetic fish was detected among 18 putative DH fish in European seabass, despite earlier screening for isogenicity using 11 microsatellite markers. An unidentified larval DNA restriction digestion inhibition mechanism observed in Nile tilapia prevented the construction of SNP-based genetic linkage map. In summary, this study provides strong evidence on efficacy of NGS technologies for the development and verification of isogenic clonal fish lines. Reliable establishment of isogenic clonal fish lines is critical for their utility as a research tool.

The gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) require n-3 long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for optimal growth and health. Due to the rapid growth of global aquaculture the quantity of marine oils used in aquafeeds has been limited, yet the overall quantity of oil in an aquafeed has increased by the addition of vegetable oil (VO) to supply dietary energy. For aquaculture to continue to grow more fish must be produced with less marine ingredients, yet EPA and DHA must be maintained at levels above fish requirements. This project set out to re-evaluate the requirement for EPA and DHA in gilthead seabream and European seabass. Two dose-response studies were designed and executed where juvenile seabream and seabass were fed one of six levels of EPA+DHA (0.2 – 3.2 % as fed). Biometric data were collected and analysed to determine new requirement estimates for EPA+DHA for fish of two weight ranges (24 – 80 g and 80 – 200 g). The effects of the dietary LC-PUFA gradient on lipid composition and metabolism were also considered.

Tesis por compendio de publicaciones / La luz y los patrones de alimentación influyeron sobre el comportamiento, fisiología y reloj molecular de dos peces teleósteos. Se clonaron dos criptocromos de lubina (cry1 y cry2), expresándose en todos los tejidos estudiados y mostrando ritmos diarios de expresión en cerebro, hígado y corazón (excepto cry2). La fase (día/noche) de alimentación a demanda en lubina afecta a los patrones diarios de actividad amilasa intestinal y expresión de period1 en tejidos periféricos (i.e. hígado), pero no en cerebro. Durante las inversiones estacionales de alimentación de la lubina, mostraron mayores valores de glucosa sanguínea durante la fase de alimentación, revelando también los mayores valores medios en lubinas nocturnas invierno. El pez cebra utilizó un nuevo comedero a demanda, mostrando independencia de fase entre ritmos circadianos de alimentación (nocturno) y locomoción (diurno), con diferentes periodicidades. Además la actividad y posición del pez cebra resultó espectro-dependiente, provocando mayores efectos las longitudes de onda cortas. / Thesis summary in English Light and feeding patterns influence on the behaviour, physiology and molecular clock of two teleost fish. Two cryptochromes (cry1 and cry2) were cloned, being expressed in all studied tissues and showing daily rhythm of expression in brain, liver and heart (except cry2). The self-seabass feeding phase (day/night) affected on daily patterns of intestinal amylase activity and on the period1 expression in peripheral tissues (i.e. liver), but not in brain. During the seasonal feeding inversions in seabass, the highest blood glucose levels were shown during their feeding phase, revealing also the highest values in nocturnal seabass in winter. Zebrafish used a new self-feeding system, displaying phase-independence between feeding (nocturnal) and locomotor (diurnal) circadian rhythms, with different periodicities. Moreover, the zebrafish activity and vertical position were spectrum-dependent, being the main effects provoked by short-wavelengths.