The conservation status of the species studied in this thesis (European eel and pufferfish) is currently frail, thus the main goal of this research was to develop, improve and apply several techniques and protocols with the aim of increasing the knowledge about their sperm biology, improving their reproductive performance and even helping future breeding in captivity. The reproductive performance of the males is often assessed through the sperm motion parameters analysed by the CASA system, so first we focused on how to standardize this technique in terms of procedural and biological settings. In this respect, we laid the foundations for applying a standard method to assess sperm quality in fish, making it possible for sperm studies to be compared both intra- and inter-laboratories using the proper CASA settings. Secondly, with the aim of improving the reproductive performance of European eel males, 3 thermal regimes (two of them variable: T10 and T15; and one of them constant: T20) and 3 hormonal treatments (hCG, hCGrec and PSMG) were assessed based on different sperm quality parameters. In the case of the thermal regimes, our results demonstrated that the onset and progression of spermiation are strongly influenced by water temperature, with treatment T20 showing the best results in all the sperm quality parameters. In the case of hormonal treatments, hCGrec produced the best results in all the sperm quality parameters, becoming an economically profitable/viable treatment and an effective alternative to the standard hCG treatment often used to induce spermiation in eel species. A preliminary physiological study regarding the changes to the main ions involved in the fish sperm activation process was carried out. Our results showed that intracellular concentrations of Ca2+ and K+ increased upon eel sperm activation, while pH gradually decreased over time, thus it is likely that all of them play an important role in the initiation of sperm motility in the European eel, as with other marine and freshwater teleosts. In the second part of this thesis, which focuses on the pufferfish, an in-depth study into the sperm of this species was carried out for future application in aquacultural matters. A short-term storage method for pufferfish sperm was developed, enabling us to preserve the sperm quality parameters for a relatively long time period (7 days) compared to fresh sperm samples. Moreover, the effects of both the osmolality and the ion composition of the activation media on the sperm motion parameters were evaluated, concluding that both factors play an essential role in the initiation of sperm motility of pufferfish sperm and probably, in marine fish sperm. Finally, in vitro fertilization trials were developed to assertain how the quantity and quality of male gametes affects the fertilization and hatching rates. We demonstrated that sperm/egg ratio and sperm quality are strongly related factors, suggesting that both should be taken into account as unique interrelated elements. In addition, coefficients of correlation among all the spermatozoa motion parameters provided by a CASA system and fertilization and hatching rates were estimated for the first time in a marine species. Spermatozoa velocities showed the highest coefficients (¿0.80), suggesting that the kinetics of the spermatozoa are a key factor in the fertilization process. / Gallego Albiach, V. (2013). Sperm physiology and quality in two marine teleosts: Anguilla anguilla & Takifugu niphobles [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34625 / TESIS / Premios Extraordinarios de tesis doctorales

Die Einschleppung gebietsfremder Parasiten durch den Menschen ist einer der Hauptgründe für das Auftreten neuer Krankheiten in the letzten Jahrzehnten. Neue Wirte sind oftmals anfälliger für diese invasiven Parasiten als die ursprünglichen Wirte. In schwerwiegenden Fällen können invasive Parasiten zu Massensterben und zum Aussterben ihrer neuen Wirte führen. Der ursprüngliche Wirt des Aalparasiten Anguillicola crassus ist der Japanische Aal. In den frühen 1980er Jahren wurde der Parasit in die Population des Europäischen Aals eingeschleppt. Er ist einer der Faktoren, die für den Populationsrückgang des Europäischen Aals verantwortlich sind. Die molekularen Prozesse, die zur stärkeren Anfälligkeit des Europäischen Aals im Vergleich zum Japanischen Aal führen, sind noch nicht zureichend bekannt. Die Analyse transkriptomweiter differenzieller Genexpression von Immungewebe ergab, dass im Europäischen Aal sowohl Immun- als auch Nichtimmungene differenziell exprimiert waren. Dies war im Japanischen Aal nicht der Fall und deutet darauf hin, dass der Europäische Aal eine ineffiziente und kostspielige Immunantwort auslöst. Die Resultate ensprechen jenen die schon in anderen Wirbeltierwirt-invasiven Parasiten-Systemen beobachtet wurden. Ausserdem stützen diese Resultate die Hypothese, dass neuen Wirten eine wirksame Immunantwort fehlt und sie deuten darauf hin, dass Nichtimmunprozesse wesentlich zur höheren Anfälligkeit von neuen Wirten beitragen. Als Reaktion of die negativen Fitnesseffekte können neue Wirte Abwehrmechanismen entwickeln. Im Europäischen Aal entspricht das der Einkapselung von A. crassus. Einkapselung führte zu eine niedrigere Abundanz adulter A. crassus. Dies deutet darauf hin, dass das Einkapseln sich positiv auf die Gesundheit infizierter Aale auswirkt. Jedoch war die Abundanz zweier nativer Parasiten höher in Aalen, die A. crassus einkapselten. Eine verbesserte Abwehr des eingeschleppten Parasiten könnte daher die Abwehr nativer Parasiten beeinträchtigen. / The human-mediated translocation of non-native parasites into foreign regions is one of the primary factors for the emergence of new diseases in recent decades. Novel hosts are often more susceptible to these invasive parasites than the native host. In severe cases, invasive parasites can lead to population declines and extinctions of their novel hosts. The eel parasite Anguillicola crassus is native to the Japanese eel. In the early 1980s it was introduced into the European eel population and is now considered to be one factor contributing to the population decline of its novel host. The underlying molecular processes determining higher susceptibility in the European eel compared to the Japanese eel are not well understood. Using whole-transcriptome differential gene expression analysis of immune organs, I found that genes involved in both immune and non-immune processes were differentially expressed in the European eel but not the Japanese eel, suggestive of an ineffective and costly immune response in the former. These results are in line with those observed between susceptible and resistant hosts in other vertebrate host-invasive parasite systems. Furthermore, the results support the hypothesis that novel hosts lack an effective immune response. The results also suggest that alteration of non-immune processes contributes substantially higher susceptibilities of novel hosts. In response to the negative fitness effects of invasive parasites, novel hosts can evolve coping mechanisms. The European eel has the capacity to encapsulate and kill A. crassus. Using natural infections, I found a lower abundance of adult A. crassus, the most costly parasitic stage in those eels encapsulating the parasite, suggesting that encapsulation can potentially improve health of infected eels. At the same time, the abundance of two native parasites was higher in those eels encapsulating A. crassus. Thus, coping with A. crassus may come at the expense of coping with native parasites.

Investigation of creatine- and adenylate phosphates involvement in fish spermatozoa metabolism is of high interest for fish spermatology. These compounds are necessary to support normal physiological state and motility of spermatozoa. The simultaneous changes in content of creatine- and adenylate phosphates in fish spermatozoa prior and during their motility are quite unclear. Therefore, studying and development of new methods for the quantification of creatine- and adenylate phosphates in spermatozoa of different fish species under such physiological conditions as maturation and in vitro manipulation are of high importance. One of the study outputs is the developed LC/HRPS (liquid chromatography coupled with high resolution product scan) method for the analysis of creatine- and adenylate phosphates content in fish spermatozoa (Chapter 2). Its main advantage is the possibility to detect and quantify several compounds (creatine, creatine phosphate (CP), AMP, ADP, ATP, and cAMP) simultaneously to obtain maximum information with minimum analytical effort. The method was validated taking into account such key parameters as limit of quantification, selectivity, recovery and repeatability. It represented an excellent performance allowing determination of target compounds in highly diluted fish sperm samples. Consequently, the method was applied for the quantification of aforementioned substances during sterlet (Acipenser ruthenus) spermatozoa maturation and in vitro manipulation with sperm of whitefish (Coregonus lavaretus maraena) and European eel (Anguilla Anguilla). The present study showed that immature sterlet spermatozoa are not able to initiate motility. Significant decrease of CP and stable levels of ATP and ADP content during their maturation were found. The critical importance of ATP regeneration system and oxidative phosphorilation for the maturation process of sterlet sperm as a prerequisite for successful fertilization was assumed (Chapter 3). Further experiments revealed that European eel spermatozoa were not able to initiate motility by activation medium (AM) at the start of the induced spermiation. They acquired the ability to be activated after the dilution with AM at the end of hormonal treatment. This accompanied by the increase of CP and cAMP levels in spermatozoa after activation. That allowed us to assume the involvement of ATP regenerating system and cAMP-dependent regulatory pathways in the process of hormonally induced spermiation (Chapter 4). Current study represents a first successful estimation of cAMP in fish spermatozoa during the motility period using the LC/HRPS. Important issues concerning the short-term storage of European eel sperm were rised. Storage at 4 °C was accompanied by higher marcoergic phosphates content and higher motility in comparison to the storage at 20 °C. It suggests the involvement of macroergic phosphates metabolism in short-term storage. (Chapter 4). Obtained results could contribute to the development of new effective methods for improving of spermiation and short-term sperm storage in European eel aquaculture. Various degrees of energy consumption in response to environment composition were found in whitefish spermatozoa. Energy consumption was significantly higher in motility activating conditions. No effect of osmolality was found on this process. The content of CP and ATP was significantly higher when cells were in motility inhibiting medium comparing to activation medium. No relationship between content of CP, ADP, and ATP and spermatozoa motility parameters in AM of different osmolality was found. Isotonic conditions favor the spermatozoa with longer motility period, higher linearity, and fast velocity without increase in ATP content (Chapter 5). This suggests that whitefish sperm energy management is more efficient after activation in isotonic conditions. Obtained results are of high interest for elaboration of new sperm motility activating media for fisheries practice.

Le déclin mondial des anguilles tempérées est lié aux effets synergiques de multiples pressions anthropiques. Cependant, la complexité du cycle de vie des anguilles et leurs incroyables capacités d’adaptation font qu’il est très difficile de connaître le poids relatif de chacune des pressions. Les anguilles tempérées sont trois espèces catadromes qui ont de très grandes aires de répartition pendant leurs phases de croissance continentales. Leurs panmixies et les longues dérives larvaires passives sont des freins aux adaptations locales, cependant on observe des patrons spatiaux de traits d’histoire de vie corrélés aux gradients environnementaux, à l’échelle du bassin versant et de son aire de répartition. Ce doctorat vise à (i) démontrer si ces patrons spatiaux d’histoire de vie sont le résultat de deux réponses adaptatives : le polymorphisme génétique et la plasticité phénotypique adaptative, et (ii) à réévaluer l’effet des différentes composantes du changement global en prenant en compte ces réponses adaptatives. Dans ce cadre, GenEve el, un modèle d’optimisation individu-centré a été développé. Ce modèle postule que la sélection de l’habitat dépendant du génotype et la plasticité phénotypique sont deux mécanismes permettant de faire face à l’hétérogénéité environnementale. Avec de telles hypothèses, le modèle permet de reproduire les patrons spatiaux observés concernant la longueur à l’argenture, le sexe-ratio et la distribution des écotypes. Par la suite, différents types des pressions anthropiques - les pêcheries de civelles et d’anguilles argentées, les obstacles à la migration de montaison et les mortalités dues aux turbines hydroélectriques - ont été intégrés dans le modèle.L’objectif a été d’évaluer leurs impacts sur l’échappement, à la fois en nombre, mais aussi sur différents attributs comme le sexe-ratio, la répartition entre génotypes, la longueur à l’argenture moyenne, et la production globale d’oeufs. Les résultats montrent que la pression qui induit la plus forte mortalité directe n’a pas forcément la plus forte influence sur la biomasse féconde et n’exerce pas nécessairement la pression sélective la plus forte sur les écotypes. Le modèle met aussi en évidence que la plasticité phénotypique peut être source de résilience pour la population et qu’elle atténue l’effet de certaines pressions, mais pas de toutes. Cela suggère également que la gestion ne doit pas seulement se concentrer sur les nombres de survivants et les mortalités directes, mais aussi sur la protection de la diversité au sein des populations. Finalement, un modèle démo-génétique est décrit pour résumer notre compréhension des populations d’anguilles. Un tel modèle pourra être utilisé à l’avenir pour explorer les conditions écologiques dans lesquelles le polymorphisme génétique et la plasticité phénotypique ont été sélectionnés à travers des générations et fournir de nouvelles recommandations pour la conservation des espèces d’anguilles en voie d’extinction. / The worldwide decline of temperate eels is due to a synergistic combination of several anthropogenic pressures. However, eels display very specific life-cycles and amazing adaptation capacities that impair our ability to assess the relative effects of each pressure. Temperate eels are three catadromous species with large spatially distribution area during their continental growth stage. Their panmixia and the passive larval drifts impair the possibility of local adaptation; however life history spatial patterns are correlated with environmental gradients at both river catchment and distribution area scales. ThisPhD aims (i) to explore whether these life history spatial patterns may result from two adaptive responses: genetic polymorphism and adaptive phenotypic plasticity, and (ii) to revisit the effect of different components of global change in consideration to these adaptive responses. In this context,GenEveel, an individual-based optimization model was developed. The model postulates that genetic dependent habitat selection and phenotypic plasticity are mechanisms to address environmental heterogeneity. With such assumptions, the model was able to mimic observed spatial patterns in length-at-silvering, sex ratio and distribution of ecotypes. Moreover, different types of anthropogenic pressures (glass eel fishery, silver eel fishery, obstacles to upstream migration, and mortality due to hydropower facilities) were integrated in the model. Then, the model was used to assess their impacts on the number of escapees and their attributes: sex ratio, repartition between genotypes, mean lengthat-silvering, and overall egg production. The results showed that the pressure that induces the highest direct mortality has not necessarily the greatest influence on the spawning biomass and does not necessarily exert the strongest selective pressure on the ecotypes. This demonstrates that phenotypic plasticity can be a source of resilience for the population and mitigates the effect of some but not all the pressures. It also suggests that management should not only focus on numbers and direct mortalitybut on the preservation of diversity within populations. Finally, a demo-genetic model is described summarizing our understanding of eel populations. Such model can be used in the future to explore the ecological conditions in which genetic polymorphism and phenotypic plasticity have been selected through generations and provide new insights for the conservation of endangered eel species.

In response to the drastic decline of the European eel (Anguilla anguilla (L.)) fisheries have been reduced and elvers are stocked in areas where natural abundances are low. Are these measures adequate? To answer different aspects of this question, we have analysed more than a century of eel tagging, using both traditional and more novel capture – recapture analyses. Based on these long-term data, we have evaluated the impact of the Swedish eel coastal fisheries using Survival analysis. Our analysis indicates that the fishing mortality just prior the 2009 fishing restrictions were in the order of 10%. More recent tagging programs have focused on issues related to the fate of stocked fish. If and how they migrate out of the Baltic Sea and further on towards the Atlantic Ocean. Both earlier and our new studies reveal that all eels recaptured on the Swedish East Coast, no matter of their origin, migrate at a reasonable speed and direction towards the outlets of the Baltic Sea. Even though it is sometimes difficult to determine their origin, our analyses indicate that stocked fish were scarce among the recaptures. In an experiment on the Swedish West Coast, we knew the individuals’ origin (stocked or wild) and they had similar migration patterns. In contrast, silver eel in Lake Mälaren – assumed to have been stocked as elvers or bootlace eels – seemed to have difficulties in finding the outlets. Instead they overwintered and lost weight. However, weight losses are also significant among non-stocked individuals in the Baltic Sea, both if they overwinter and if they appear to be on their way out from the area. It remains an open question whether eels from the Baltic region in general, and whether the overwintered fish in particular, manage to reach the spawning area in the Atlantic Ocean. Based on current knowledge, I advocate invoking the precautionary approach and to concentrate Swedish eel stockings to the West Coast and allow the young fish to spread out on their own. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p><p> </p>

[ES] Resumen Como pez de gran valor económico, procedente de una de las líneas de teleósteos más antiguas, con un ciclo de vida misterioso, un potencial de acuicultura excepcional, y con importancia cultural y actividades de pesca en casi todos los países de Europa, la anguila europea posee un enorme valor socioeconómico. Este valor se suma a la desgraciada situación actual en peligro crítico de población natural de anguilas europeas. Como el ciclo de vida de la anguila aún no se ha conseguido cerrar en cautiverio, si la especie se extingue en la naturaleza, no seremos capaces de recuperarla. El cierre del ciclo de vida de la anguila europea ha sido, por lo tanto, el objetivo final de varios estudios. Sin embargo, a pesar de una investigación científica sustancial, desde la década de 1930, varios aspectos de la maduración de la anguila, como el mecanismo que bloquea la maduración de la anguila en la etapa prepúber en cautiverio, aún no se conocen bien. Por lo tanto, es necesario ampliar nuestro conocimiento sobre la reproducción de la anguila para inducir mejores hipótesis y lograr un progreso sustancial. Para profundizar en este campo, esta tesis se realizó con el objetivo específico de desarrollar métodos innovadores para la inducción de la maduración de la anguila y aumentar el conjunto de conocimientos sobre los procesos europeos de maduración de la anguila. Los procedimientos hormonales utilizados actualmente para la maduración sexual de la anguila artificial probablemente no induzcan el proceso natural de maduración. Por lo tanto, esta tesis ha evaluado el potencial de las hormonas recombinantes específicas de la anguila para inducir un proceso de maduración más natural. Este estudio específico mostró que la espermatogénesis completa y la espermiación se pueden inducir con gonadotropinas específicas de anguila recombinante; sin embargo, la calidad del gameto resultante es aún inferior a los resultados de los protocolos establecidos. Sin embargo, la utilización de hormonas recombinantes tiene un gran potencial para futuras implementaciones. Además, el experimento de gonadotropina recombinante ha generado nuevos detalles sobre el efecto de las gonadotropinas homólogas en el eje BPG de las anguilas europeas. Trabajos previos han llevado a la hipótesis de que un tratamiento térmico adecuado puede reducir o reemplazar parcialmente los tratamientos hormonales estándar para la maduración sexual de la anguila europea, o puede mejorar la calidad y / o cantidad de gametos. En esta tesis, se probó el efecto de varios regímenes térmicos en el eje BPG de machos de anguila europeos prepúberes, sin administración de hormonas. Los resultados muestran claramente que un tratamiento de agua de mar fría durante 2 semanas (10 ° C) afecta el eje BPG de los machos de anguila europeas. Los resultados específicos incluyeron un aumento en la sincronización de espermatogonias, niveles elevados de testosterona y 11-ketotestosterona en plasma, agrupamiento de muestras de transcriptomas del eje BPG del grupo tratado con agua de mar fría y posiblemente niveles aumentados de la proteína subunidad ß de la hormona luteinizante de la hipófisis. Los genes transcritos diferencialmente incluyeron varios genes, procesos y vías interesantes, que parecen estar involucrados en la maduración "natural" temprana de la anguila y que pueden ser biomarcadores adecuados para las distintas etapas de este proceso. Para un análisis adecuado de los datos transcriptómicos, se creó un transcriptoma de anguila europea de novo. Se demostró que este transcriptoma de novo posee una superior integridad al genoma de anguila europea disponible y, por lo tanto, es una herramienta útil para el análisis adicional de genes específicos. Un análisis de este transcriptoma reveló un gran número de pares de genes parálogos, que mostraron una baja divergencia entre secuencias sinónimas. Entre las hipótesis potenciales sobre e / [CAT] Com a espècie de renom culinari que pertany a un dels llinatges teleostis més antics, amb un cicle vital misteriós, un potencial d'aqüicultura excepcional, i una tradició pesquera a gairebé tots els països d'Europa, l'anguila europea posseeix un enorme valor socioeconòmic. No obstant això, aquest valor només fa que augmentar la preocupació de la seva població, que actualment es troba catalogada com "en perill crític d'extinció". Atès que el cicle de vida de les anguiles encara no ha estat tancat en captivitat, l'espècie no serà salvable en el cas que s'extingeixi en estat natural, per la qual cosa tancar el cicle de vida d'aquesta espècie ha estat l'objectiu final de diversos grups d'investigació durant els últims anys.. No obstant això, i malgrat la investigació científica de qualitat duta a terme des de la dècada de 1930, encara hi han diversos aspectes de la maduració de les anguiles -com el mecanisme que bloqueja la maduració sexual de l'anguila a l'etapa pre-puberal en captivitat- que son poc coneguts en l'actualitat. Per tal d'ampliar els coneixements sobre la reproducció de les anguiles i aconseguir un progrés substancial, aquesta tesi es va dur a terme amb l'objectiu específic de desenvolupar mètodes innovadors per a la inducció de la maduració de l'anguila europea, a més de afegir-hi el coneixement en els processos de maduració bàsics d'aquesta espècie. Els procediments hormonals utilitzats actualment per a la maduració artificial de l'anguila europea no acaben d'induir el procés de maduració natural tal i com probablement es dóna a la natura. Doncs, en primer lloc, aquesta tesi va avaluar el potencial d'hormones recombinants específiques d'anguila europea per induir un procés de maduració molt més natural. Aquest estudi específic va mostrar que mitjançant estes gonadotropines específiques d'anguila europea és possible induir l'espermatogènesi i l'espermiació completes. Tot i que els resultats van mostrar que la qualitat dels gamets va ser inferior als resultats que generen els protocols establerts fins ara amb un altre tipus d'hormones (generalment d'origen humà), la utilització d'hormones recombinants específiques es presenta amb un gran potencial per a la seva implementació futura en la inducció de la maduració sexual de l'anguila europea, ja que l'estudi va generar noves idees sobre l'efecte de les gonadotropines l'eix BPG de l'anguila europea. En segon lloc, i treballant amb la hipòtesi que un tractament tèrmic adequat pot reduir o substituir parcialment els tractaments hormonals estàndards per a la maduració sexual de l'anguila europea, en aquesta tesi es va provar l'efecte de diversos règims tèrmics (sense administració d'hormones) en l'eix BPG dels mascles europeus pre-puberals amb l'objectiu de millorar la qualitat i / o quantitat dels gamets. Els resultats mostraren clarament que un tractament d'aigua de mar de 2 setmanes a baixa temperatura (10 °C) va afectar l'eix BPG dels mascles europeus d'anguila. Resultats més específics van mostrar un augment de la sincronització de les espermatogonies, elevats nivells plasmàtics de testosterona i 11-ketotestosterona, una agrupació de mostres de transcriptoma de l'eix BPG del grup tractat amb aigua de mar freda i, possiblement, un augment dels nivells de la proteïna de la subunitat ß de la hormona luteinitzant de la hipofisi. Els gens transcrits diferencials van al·ludir a diversos gens, processos i vies interessants, que semblen estar implicats en la maduració inicial de l'anguila "natural" i podrien resultar biomarcadors adequats per a les etapes d'aquest procés. No obstant això, es necessiten estudis addicionals per avaluar el potencial dels biomarcadors d'aquests gens i, de manera complementària, comprovar si un pre-tractament d'aigua de mar freda pot millorar la resposta de les anguiles europees a un tractament hormonal artificial, com suggereixen els resultats. Finalment, amb l'objectiu / [EN] As an expensive fish from one of the most ancient teleost lineages, with a mysterious life cycle, exceptional aquaculture potential, and cultural associations and fishing activity in almost every country in Europe, the European eel possess huge socioeconomic value. This value only adds to the misfortune of the current critically endangered state of the wild European eel population. As the eel lifecycle has not yet been closed in captivity, the species will not be salvable if it went extinct in the wild. Closing the life-cycle of the European eel has thus been the ultimate objective of several studies. However, despite the substantial scientific investigation, since the 1930s, several aspects of eel maturation, such as the mechanism which blocks eel sexual maturation at the pre-pubertal stage in captivity, is still poorly understood. Therefore, it is necessary to broaden our knowledge of eel reproduction to induce better hypotheses and therethrough achieve substantial progress. In order to further this field, this thesis was conducted with the specific objective of developing innovative methods for induction of eel maturation and add to the pool of knowledge of European eel maturation processes. The hormonal procedures currently used for artificial eel sexual maturation are probably not inducing the natural maturation process. Therefore, this thesis has evaluated the potential of eel specific recombinant hormones to induce a more natural maturation process. This specific study showed that full spermatogenesis and spermiation can be induced with recombinant eel specific gonadotropins; however, the resulting gamete quality is still inferior to the results of established protocols. Nevertheless, the utilization of recombinant hormones holds a large potential for future implementation. Furthermore, the recombinant gonadotropin experiment has generated novel insights into the effect of homologous gonadotropins on the BPG axis of European eels. Previous work has led to the hypothesis that the right thermal environmental treatment may reduce or partially replace the standard hormonal treatments for sexual maturation of European eel, or may improve gamete quality and/or quantity. In this thesis, the effect of various thermal regimes was tested on the BPG axis of pre-pubertal European eel males, without administration of hormones. The results clearly show that a 2 week cold (10 °C) seawater treatment effects the BPG-axis of European eel males. Specific results included an increase in the synchronization of spermatogonial cells, elevated testosterone and 11-ketotestosterone plasma levels, clustering of BPG-axis transcriptome samples from the cold seawater treated group and possibly increased levels of pituitary luteinizing hormone ß-subunit protein. Differentially transcribed genes alluded to several interesting genes, processes, and pathways, which appears to be involved in early "natural" eel maturation and may prove to be suitable biomarkers for the stages of this process. In order for proper analysis of the transcriptomic data, a de novo European eel transcriptome was assembled. This de novo transcriptome was proven to have superior completeness to the available European eel genome and is thus a useful tool for further analysis of specific genes. An analysis of this transcriptome revealed a large number of paralog gene pairs, which showed low synonymous sequence divergence. Among the potential hypothesis regarding the origin of these paralog gene pairs, the hypothesis of a 4R whole genome duplication is among the most parsimonious. Several of these duplicated genes were involved in reproduction and the onset of puberty. Regardless of the origin, further analysis of these genes may reveal eel specific adaptations, which could help to better understand the exceptional reproductive system of eels. / Rozenfeld, C. (2019). Development of innovative methods for induction of European eel (Anguilla anguilla) spermatogenesis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125697 / TESIS

Många djurarter väljer någon gång att vandra under sin livshistoria, denna vandring innebär såväl fördelar som nackdelar. Den europeiska ålen (Anguilla anguilla) är en av dessa arter. Ålen är idag akut hotad och antalet ålar som idag försöker nå sina uppväxtplatser i europeiska sötvatten är en bråkdel av vad de en gång var. På sin vandring möter ålen olika faror bla. vandringshinder i form av dammar och andra mänskligt byggda hinder. Vissa av dessa är utrustade med fiskvägar för att underlätta vandring för olika fiskarter. Syftet med denna studie är att undersöka om dessa fiskvägar kan utgöra en konstgjord selektion, där ålens individuella beteenden kan spela roll för dess benägenhet att klättra upp för en konstgjord fiskväg. Min hypotes är att individer som uppvisar ett explorativt beteende i högre grad kommer att klättra än de som inte uppvisar ett explorativt beteende. För att testa detta fick 269 glasålar med en genomsnittlig vikt på 0,36 g möjlighet att välja att klättra eller inte klättra uppför en konstgjord ramp. Efter klättringsförsöket filmades ålarna och deras aktivitet registrerades. Resultatet visar att de ålar som valde att klättra, i genomsnitt hade signifikant lägre aktivet, samt var signifikant tyngre än de ålar som inte klättrade. Dock fanns ingen korrelation mellan vikt och aktivitet inom respektive grupp. Resultatet stämmer med tidigare forskning, där lägre simaktivitet visat sig ha ett samband med ett ökat explorativt beteende. Studien visar att fiskvägarna kan utgöra ett konstgjort selektionstryck och påverka lokala bestånd, men med tanke på ålens panmiktiska livshistoria utgör specifika hinder troligen ingen selektion som kommer påverka populationen som helhet. / Many animal species choose to migrate at some point during their life history, and this migration involves both advantages and disadvantages. The European eel (Anguilla anguilla) is one of these species. The eel is today acutely threatened and the number of eels trying to reach their breeding grounds in European freshwater today is a fraction of what they once were. On its migration, the eel encounters among other things, barriers in the form of dams and other human-built obstacles, some of which are equipped with fishways to facilitate the migration of different fish species. The purpose of this study is to investigate whether these fishways can constitute an artificial selection, where the individual behaviours of eels can play a role in its propensity to climb an artificial fishway. My hypothesis is that individuals who exhibit exploratory behaviour will climb to a greater extent than those who do not exhibit exploratory behaviour. To test this, 269 glass eels with an average weight of 0.36 g were given the opportunity to choose to climb or not climb an artificial ramp. After the climbing attempt, the eels were filmed, and their activity recorded. The result shows that the eels that chose to climb had, on average, significantly lower activity and were significantly heavier than the eels that did not climb. However, there was no correlation between weight and activity within each group. The result is consistent with previous research, where lower swimming activity has been shown to have a connection with increased exploratory behavior. The study shows that the fishways can constitute an artificial selection and affect local populations, but given the eel's panmictic life history, specific obstacles probably do not constitute a selection that will affect the entire population. / Optimerade passagelösningar för ål

Den europeiska blankålen (Anguilla anguilla) har genomgått en stor populationsminskning de senaste decennierna på grund av antropogena strukturer i form av vattenkraftverk som orsakar fragmentering. Vid vattenkraftverks intagskanaler finns intagsgaller installerade för att förhindra drivgods från att skada turbinerna. Forskning har utvecklat lösningar i form av anpassade galler, med låg lutning, som leder fisken till en flyktöppning vid sidan av kraftverket. Det har observerats att utformningen av gallret påverkar fiskars beteenden och beteendeobservationer kan därför ge viktig kunskap om hur väl åtgärder funkar och vilka förbättringar som krävs. Syftet med denna studie var att undersöka beteenderespons och passageframgång för europeisk blankål (Anguilla anguilla) vid tre olika typer av låglutande ß-galler med 30 mm spaltvidd. Man ville också undersöka om det finns någon koppling mellan ålarnas beteenderespons och gallrens vägledande funktion.  Experimentet utfördes vid Vattenfalls Research and Development laboratorium i Älvkarleby. En artificiell miljö i form av en ränna användes för att simulera ett turbinintag. Här testades blankålars (n=69) beteende och passage av tre olika typer av ß-galler med (i) horisontella järn orienterade i linje med vattnets strömriktning (H0), (ii) vertikala järn orienterade i linje med vattnets strömriktning (V0) och (iii) vertikala järn med 60° vinkel mot strömmen (V60). Resultatet visar att det inte finns någon signifikant skillnad i ålarnas beteenderespons mellan de tre olika gallren. Däremot såg man att det fanns en koppling mellan ålarnas beteenderespons och gallrens vägledande förmåga. De ålar som vägleddes av gallren hade huvud som första fysiska kontakt med gallret. Vidare forskning på ålbeteenden vid intagsgaller är nödvändigt för att öka migrationsframgången hos europeisk ål. / The European silver eel (Anguilla anguilla) has undergone a large population decline in recent decades due to anthropogenic factors such as hydropower plant river fragmentation. At the intake channel of hydropower plants there are trash racks for preventing drifting obstacles to damage the turbines. Research has developed solutions such as angled bar racks, which guide fish to a bypass. The design of the racks has been observed has an effect on fish behavior and its therefore important to provide actions and improvements. The aim of this study was to investigate behavioral and passage success of the European silver eel (Anguilla anguilla) of three different types of low-sloping angled bar racks with 30 mm clear spacing and whether there is any connection between the behavioral response of the eels and the guiding function of the racks. The experiment took place at Vattenfall's Research and Development laboratory located in Älvkarleby. A flume resembling an intake channel was used. Here (n=69) eels were tested on three different types of low-sloping angled bar racks: (i) horizontally bars oriented in line with the water current streamwise (H0), (ii) vertical bars oriented in line with the water current streamwise (V0) and (iii) vertical bars with a 60° angle to the current (V60). The result shows that there is no significant difference in the eels behavioral response between the three different racks. However, it was seen that there was a connection between the behavioral response of the eels and the guiding ability of the racks. Al eels that were guided by the racks, only had head as first physical contact with the rack. Further research on eel behaviors at intake racks is necessary to increase the migration success of European eel.

To assess anthropogenic impacts of point-source and diffuse discharges on fish populations of the Waikato River, compare responses to different discharges and identify potential sentinel fish species, we sampled wild populations of brown bullhead catfish (Ameiurus nebulosus, (LeSueur, 1819)), shortfin eel (Anguilla australis Richardson, 1848), and common bully (Gobiomorphus cotidianus McDowall, 1975) in the Waikato River. Sites upstream and downstream of: geothermal; bleached kraft mill effluent (BKME); sewage and thermal point-source discharges were sampled. At each site, the population parameters, relative abundance, age structure and individual indices such as: condition factor; and organ (gonad, liver, and spleen) somatic weight ratios; and number and size of follicles per female were assessed. Indicators of fish residence and in some cases exposure to contaminants in discharges were analyzed. Bile chemistry of brown bullhead and shortfin eel was assayed, liver and muscle metal levels were analyzed for brown bullhead and shortfin eel respectively, and stable isotopes of C and N in common bully were measured. Bile, metal and isotopic signatures gave strong evidence that fish had been resident at sites for some time before sampling. Signatures of bile and metal contaminants showed contamination was localised to discharge areas. Gradients in stable isotopes in common bully showed evidence of changes in water sources and anthropogenic effects along the river. Biochemical variables, hepatic ethoxyresorufin-O-deethylase (EROD) and plasma steroids indicated exposure and response of brown bullhead and shortfin eel to pulp and paper contaminants at the BKME site. Physiological (blood) variables showed fish largely responded in a predictable way to elevated water temperatures at discharge sites at time of sampling, however total haemoglobin of brown bullhead and common bully blood failed to increase at the BKME site despite elevated temperatures and low dissolved oxygen. Growth rates, condition factor, age structure, and gonadosomatic index (GSI) suggest that discharges with significant heat or nutrients benefit brown bullhead despite physiological impairment at the BKME site. Shortfin eel individuals also benefited from heated water discharges. No consistent impacts on common bully health were obvious at individual discharge sites, or cumulatively along the river due to the gradual deterioration in water quality downstream. Common bully individuals also benefited from heat in discharges but lack of juveniles at sites where numerous juvenile brown bullhead were found, suggest that unlike brown bullhead populations, common bully populations were not responding with significant recruitment. Although I found little evidence of toxic effects of discharges on shortfin eel, caution is required in assessing the potential of contaminants to impact eel populations due to the life history of shortfin eel, and exploited nature of populations. For example, reproductive damage suffered by adult eels may not immediately manifest itself in the effected population due to temporal delays in gonadal maturation, and recruitment, and single panmictic populations supplementing recruitment of impacted populations. Distinct changes in population parameters at each of the paired sites and changes in individual variables showed that fish responded to discharges. The range of responses in species suggests different sensitivity to contaminants and amount of benefit which each species receives from heat in discharges. In these terms shortfin eel would be the most resistant, then brown bullhead and lastly common bully. Interpretation of population-level impacts at the geothermal and BKME discharge sites is made difficult due to benefits of additional heat. There is also the possibility that detection of sub-lethal or chronic effects on sensitive juvenile life-stages may be being hidden by compensatory density population responses. Responses and life history of common bully made them the preferred indicator species of the three species sampled, and supported overseas examples using small-bodied fish species as sentinels.

Notre objectif a été d'étudier l'origine et l'évolution du système kisspeptine, un nouvel acteur du contrôle de la reproduction. L'analyse de génomes d'espèces occupant des positions clés dans la classification des vertébrés nous a permis de réévaluer la diversité des kisspeptines (Kiss) ainsi que celle de leurs récepteurs (Kissr). A partir des données obtenues, nous avons pu proposer une nouvelle classification des familles Kiss et Kissr, basée sur des critères phylogénétiques et synténiques. De plus, nous avons montré que la diversité des Kiss, d'une part, et celle des Kissr, d'autre part, résultent des deux événements de duplication génomique (1R et 2R) survenus à la base des vertébrés. Des pertes de gènes ont ensuite diminué le nombre de Kiss et Kissr chez les vertébrés, notamment en annulant l'impact de la 3R, propre aux téléostéens. Nos résultats ont révélé des histoires évolutives complexes ayant conduit à la perte ou à la conservation de Kiss et Kissr différents selon les lignées de vertébrés. Afin d'aborder les mécanismes évolutifs qui ont pu mener à la conservation de plusieurs Kiss et Kissr chez un même organisme, nous nous sommes intéressés à l'anguille européenne qui possède, comme nous l'avons montré, deux Kiss et trois Kissr. L'étude de ce modèle a apporté un éclairage nouveau sur la fonctionnalité de ce système chez les vertébrés, avec la découverte d'un effet inhibiteur sur l'expression de la LHβ hypophysaire, alors que certains composants du système pourraient avoir un rôle stimulateur classique sur la GnRH cérébrale. Cette thèse a permis d'illustrer la complexité du système kisspeptine, tout en clarifiant sa classification et son histoire évolutive