Tesi sul tema "Anguilla"

La producción de anguilas se basa en la captura de angulas, pero la captura de éstas no se ha podido incrementar al mismo ritmo que su demanda para el engorde en piscifactorías. La caída de las capturas de angulas llevó a la búsqueda por todo el mundo de nuevos suministros, planteándose la reproducción en cautividad como una de las alternativas de mayor importancia. Debido a que las anguilas no maduran sexualmente en cautividad se han desarrollado métodos de inducción hormonal que logran la producción de volúmenes importantes de esperma de buena calidad (Pérez et al. 2000; Asturiano et al. 2004; Müller et al. 2004). Este hecho ha motivado el interés por desarrollar técnicas para la recolección y manejo apropiado del esperma de esta especie. En este sentido se decidió realizar una serie de experimentos con la finalidad de desarrollar técnicas de congelación y estudios sobre la calidad del esperma de anguila europea. El primero de ellos trató de optimizar los tratamientos de inducción hormonal en machos de anguila europea (procedentes de piscifactoría) para obtención de esperma. Fueron probados cinco tratamientos hormonales (A-E) con gonadotropina coriónica humana (hCG) para inducir la maduración gonadal y la espermiación en machos de anguilas procedentes de piscifactoría. Se vio que una dosis semanal de 1.5 UI hCG/g pez, es la que permite obtener un mayor porcentaje de machos espermiantes, un periodo de espermiación más largo y un mayor volumen de esperma. A continuación se estudiaron protocolos de criopreservación utilizando como base medios diluyentes descritos para anguila japonesa (K15, K30, TNK) y dos descritos anteriormente para anguila europea (P1 y P2). A los espermatozoides congelados en estos medios se les realizaron análisis de movilidad (pre y postcongelación): la velocidad angular (VAP), velocidad curvilínea (VCL), velocidad de línea directa (VSL) y frecuencia de batido del flagelo (BCF). Estos parámetros se analizaron con un programa inform
Garzón Cortés, VDL. (2007). Inducción hormonal de la espermiación y criopreservación de esperma en anguila europea (Anguilla anguilla) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1944
Palancia

L’anguilla europea (Anguilla anguilla) è una specie eurialina catadroma, la cui area di riproduzione si trova a grande distanza dall’areale di distribuzione. Presenta un ciclo biologico piuttosto complesso caratterizzato da due metamorfosi: la prima, tipicamente larvale permette al “leptocefalo”, forma larvale marina tipica degli anguilliformi, di trasformarsi in anguilla “ceca”; la seconda, invece, trasforma l’anguilla “gialla” in “argentina” attraverso un processo definito di “argentinizzazione” nel quale si delineano preadattamenti alla maturazione sessuale che sarà completa durante la migrazione riproduttiva verso il Mar dei Sargassi. L’”argentinizzazione” innesca modificazioni fisiologiche (regressione del tratto digestivo), e morfologiche (aumento della massa corporea, ispessimento della pelle, modificazione dell’occhio e nuova pigmentazione). Il colore bruno verdastro con ventre giallo è prerogativa delle anguille in fase trofica, cioè quelle gialle, sessualmente immature. Quelle argentine presentano, invece, una colorazione più scura sul dorso e argenteo sul ventre. Inoltre le anguille argentine vanno incontro a cambiamenti ormonali per adattarsi all’ambiente marino, compiere la lunga migrazione e riprodursi. Per avere a disposizione risorse energetiche anche senza nutrirsi per molti mesi, devono accumulare grandi riserve di cui il 60% è destinato per lo sviluppo delle gonadi. Tuttavia i processi metabolici che consentono alle anguille argentine di effettuare lunghe migrazioni mantenendo un’alta efficienza del nuoto e nel contempo utilizzando limitate risorse, non sono sufficientemente conosciuti. Per fornire nuove informazioni, questo lavoro di tesi ha indagato se vi sia una modificazione del quadro proteico del siero delle anguille argentine rispetto alle gialle, e quali proteine eventualmente risultino sovra- o sotto- espresse in ciascuna delle due fasi del ciclo biologico. A tale fine si è applicata una tecnica innovativa, quale l’analisi proteomica, che permette di identificare in modo sistematico le proteine all’interno di un substrato biologico come iin questo caso il siero. Le anguille gialle ed argentine prese in esame sono state prelevate nelle Valli di Comacchio (FE). La valutazione quantitativa delle proteine, separate tramite elettroforesi bidimensionale su gel di poliacrilamide, si è fondata sull’analisi delle immagini attraverso specifici programmi (Proteomweaver). A tale proposito, si sono confrontati i gel di anguilla gialla ed argentina individuando gli spot corrispondenti alle proteine e quantificandoli. Gli spot differenzialmente espressi sono stati prelevati per essere poi identificati tramite spettrometria di massa (MS/MS), arrivando al riconoscimento di diverse proteine. Tra queste, sono state identificate due diverse isoforme di apolipoproteina, l’una più espressa nell’anguilla gialla, l’altra nell’argentina; tali proteine svolgono un ruolo importante nella facilitazione del trasporto lipidico e nell’assorbimento dei lipidi associato anche con la crescita degli ovociti. Il treno di spot relativo alla transferrina è decisamente più evidente nelle anguille argentine, il che potrebbe essere correlato al ruolo del ferro nell’emoglobina, mirato ad un maggiore apporto di ossigeno ai tessuti durante la lunga migrazione riproduttiva di questi animali. Lo studio è del tutto nuovo e al momento non sono disponibili sufficienti informazioni al contorno per trarre più ampie conclusioni; tuttavia esso merita di essere proseguito per contribuire alla comprensione degli eventi di metamorfosi in questa specie dal ciclo vitale complesso e in gran parte sconosciuto.

L'anguille européenne (Anguilla anguilla) présente un cycle de vie complexe avec un stade civelle qui remonte les estuaires pour atteindre les rivières et entamer une phase de croissance. Cependant, cette migration estuarienne est connue pour être facultative, certains individus s'installant en mer, en estuaire ou alternant les séjours en rivière et en estuaire. Les civelles ne se nourrissent pas ou peu pendant leur migration et leurs schémas migratoires pourraient donc être étroitement associés aux réserves énergétiques des individus. La migration facultative des civelles pourrait donc reposer sur une stratégie conditionnelle, les individus présentant d’importantes réserves d'énergie ayant une capacité migratoire élevée. Cependant, certaines études s’avèrent contradictoires et l’objectif de cette thèse était d'étudier la stratégie conditionnelle chez les civelles européennes en se basant non seulement sur les réserves d'énergie mais également sur la mobilisation de l'énergie (métabolisme et expression des gènes liés à l'énergie). L’essentiel des travaux a été mené à la fois sur des civelles d'automne et de printemps, car elles présentent d’importantes différences de réserves énergétiques. Nous avons tout d'abord caractérisé le statut énergétique individuel des civelles marines échantillonnées en automne et au printemps et l’avons relié à leur comportement migratoire évalué dans des installations expérimentales. Les civelles d'automne présentaient des réserves énergétiques plus élevées et une plus grande capacité à produire de l'énergie que les civelles de printemps. Ces résultats confirment que les civelles d'automne et de printemps présentent de fortes différences dans leur statut énergétique et qu'elles doivent être étudiées séparément. Nous émettons l'hypothèse qu'il pourrait exister un seuil énergétique en dessous duquel la migration pourrait être conditionnée par ce facteur. Afin de mieux comprendre les mécanismes de sédentarisation dans les estuaires, la relation entre le statut énergétique et le comportement migratoire des civelles marines et estuariennes a ensuite été étudié parallèlement en automne et au printemps. Nos résultats soutiennent l’hypothèse d’une stratégie conditionnelle basée sur l'énergie lorsque les réserves énergétiques deviennent un facteur limitant (civelles de printemps). La perte de poids, la mesure du métabolisme et l'expression de gènes liés au métabolisme suggèrent également que les civelles estuariennes étaient plus stressées et avaient une capacité de production d'énergie plus faible que les marines. Les civelles non synchronisées au courant (considérées comme ayant une faible probabilité de migration) présentaient une dépense énergétique plus élevée que les individus synchronisés, ce qui pourrait refléter un stress et/ou une vulnérabilité au stress plus élevés chez les premières. Afin de tester cette hypothèse, des civelles ont été exposées à un facteur de stress potentiel dans les estuaires, à savoir le méthylmercure (MeHg), et les effets de ce contaminant sur le comportement migratoire et le statut énergétique des individus a été étudié. Nos résultats mettent en évidence une diminution de l’activité de nage chez les civelles non synchronisées mais pas chez les synchronisés, suggérant que les premières pourraient être plus sensibles au MeHg. Puis, les civelles non synchronisées présentaient un niveau d'expression des gènes du métabolisme inférieur à celui de leurs homologues synchronisées dans la tête et pas dans le muscle ni dans les viscères. Les travaux réalisés dans le cadre de cette thèse suggèrent que le statut énergétique et la sensibilité au stress pourraient conditionner la migration estuarienne des civelles mais les mécanismes sous-jacents et les relations entre ces facteurs ainsi qu’avec les horloges endogènes qui contrôlent la migration restent à élucider
Résumé en anglais : The European eel (Anguilla anguilla) present a complex life cycle with a glass eel stage migrating up estuary to reach river for growth. However, this estuarine migration is known to be facultative, with some individuals settling at sea, in estuaries or alternating stays in rivers and estuaries. As glass eels feed little or not at all during their migration, their divergent migration patterns may be closely associated with individual’s body condition. To date, one major theory of conditional strategy proposed that the facultative migration in European glass eels is based on energy stores, the individuals with a high migratory capacity presenting high energy stores. However, this theory has been proved controversial and the aim of this thesis was to investigate the conditional strategy in European glass eels based on more comprehensive measures of energetic status, including not only energy stores but also energy mobilization (metabolism and energy-related genes expression). We also focused on both autumn and spring glass eels, which present dramatic difference in energy stores. We first characterized the individual energetic status of marine glass eels sampled in autumn and spring and related this status to their migration behavior assessed in experimental facilities. Autumn glass eels presented higher energy stores and a higher ability to produce energy than spring ones. This confirmed that autumn and spring glass eels present strong differences in their energetic status and that they have to be studied separately. We hypothesized that a potential threshold in energetic status may exist below which migration could be conditioned by energetics. Then, to unveil the underlying mechanisms of settlement processes in estuaries in relation to energy-based conditional strategy, we investigated the relationship between energetic status and migration behavior in both marine and estuarine glass eels. Estuarine individuals displayed lower weight than marine ones in autumn but not in spring supporting the idea that a conditional strategy based on energy may explain facultative migration when energy reserves become a limiting factor. Weight loss, standard metabolism and the expression of metabolism-related genes suggested that estuarine glass eels were more stressed and had a lower capacity of energy production than marine fish. The non-synchronized glass eels also presented a higher energy expenditure than synchronized individuals possibly reflecting a higher stress and/or vulnerability to stress in the former. We further exposed glass eels to a potential stressor in estuaries - methylmercury (MeHg) in order to investigate the effects of this contaminant on glass eels’ migratory behavior and energetic status. Our results first suggested that non-synchronized glass eels were more vulnerable to MeHg reflected by a decrease in swimming activity. MeHg also affected the relation between individual metabolism-related genes expression level and swimming activity, supporting our hypothesis that stress factors may influence the settlement processes in glass eels. Finally, it is noteworthy that non-synchronized glass eels displayed lower expression level of metabolism genes than their synchronized counterpart in the head but not in muscle nor in viscera. Altogether, these results provided evidences that the energetic status and sensitivity to stress may condition estuarine migration in glass eels but the underlying mechanisms and relationships between these factors but also with the endogenous clocks driving migration remain to be elucidated

Européenne, (Anguilla anguilla) a conduit à une diminution du nombre d’anguille jaune dans les bassins versants. En 2007, le règlement (CE) instaure que 60% des civelles pêchées (<12 cm de long) doivent être dédiés à des opérations de repeuplement. Cependant, pour migrer et coloniser un nouvel environnement, les civelles doivent avoir une bonne condition énergétique. Or, les civelles ne s’alimentent pas ou peu durant leur migration estuarienne et la vitesse d’utilisation de leurs réserves dépend de divers facteurs (température, contaminants etc.). Dans l’estuaire de l’Adour, différents travaux ont mis en évidence de fortes concentrations en methylmercure (MeHg) dans les sédiments et une bioaccumulation de ce composé chez l’anguille jaune. L’exposition au MeHg entraîne la mise en place de processus de détoxication qui pourraient être couteux en énergie ce qui, chez des organismes jeûnant comme les civelles, conduirait à une perte de poids pouvant affecter leurs capacités de migration. Pour étudier l’impact du méthylmercure sur le potentiel de migration des civelles, nous avons tout d’abord (1) effectuer un état des lieux des concentrations en MeHg des civelles de l’estuaire de l’Adour, (2) évaluer l’effet du MeHg sur deux synchroniseurs de la migration estuarienne: le signal crépusculaire et l’alternance des marées chez les civelles grâce à l’utilisation de traceurs isotopiques et (3) étudier dans le milieu naturel (Adour) le comportement de migration, les taux de contamination au mercure et les mécanismes de détoxication des civelles. Nos résultats montrent des fluctuations temporelles et saisonnières de la concentration en MeHg des civelles prélevées en entrée et en milieu de l’estuaire. Par ailleurs, l’exposition au MeHg, en milieu contrôlé, a augmenté l'activité des civelles mais pas leur comportement de migration. Cependant, suite à la contamination, la structure mitochondriale et le métabolisme ont été modifiés suggérant un plus fort stress oxydant et une activation des défenses antioxydantes notamment des civelles ayant un faible potentiel de migration (non migrantes).En complément, l’étude en milieu naturel a mis en évidence une plus forte demande énergétique pour les civelles non migrantes entrainant une augmentation du fonctionnement des mitochondries et pouvant conduire à un plus fort stress oxydant
Over the last three decades, the arrival of Anguilla anguilla glass eels has markedly declined which has lead to a decrease in the number of yellow eels in the watersheds. In 2007, the Regulation (EC) indicated that 60% of glass eels (<12cm long) caught by fisheries should be used for restocking. To migrate and colonize a new environment, glass eels must have a good energetic condition. However, during their estuarine migration, glass eels either feed very little or more likely not at all. The speed at which they expend their energy stores depends on various factors (temperature, contaminants etc.). In the Adour estuary, many studies have reported relatively high methylmercury (MeHg) concentrations in the sediments, and bioaccumulation in yellow eels. Exposure to MeHg requires energy for detoxification which, in starving organisms such as glass eels, might lead to weight loss, decreasing their ability to migrate up the estuary. To study the impact of MeHg on migratory behaviour in glass eels, we first conducted an inventory of MeHg concentrations of glass eels in the estuary of the Adour, (2) evaluate the effect of MeHg on two synchronizers of estuarine migration: dusk and tide on glass eels through the use of isotopic tracers and (3) to study the natural environment (Adour) migratory behavior, mercury contamination levels and detoxification mechanisms of glass eels. Our results show temporal and seasonal fluctuations in the concentration of MeHg glass eels collected input and middle of the estuary. Moreover, MeHg exposure, in a controlled environment, increased the activity of glass eels but not in response to migratory behavior. However, due to contamination, mitochondrial structure and metabolism have been modified suggesting a stronger oxidative stress and activation of antioxidant defences of glass eels, particularly those with low migration potential (non-migrant). In addition, the wild study showed a higher energy demand for non-migrating glass eels causing an increase in mitochondrial function which may lead to higher oxidative stress

Le conoscenze relative al controllo ormonale del metabolismo epatico dei pesci sono ancora piuttosto limitate e per molti anni sono state controverse. Per lungo tempo si è ritenuto che le catecolamine, adrenalina e noradrenalina, agissero nel fegato dei pesci soltanto attraverso i recettori adrenergici di tipo β. Quindi l’assetto recettoriale dei mammiferi, che comprende recettori α e β, era considerato frutto di un processo evolutivo che non aveva ancora avuto luogo nei pesci. Successivamente, nel fegato di vari teleostei è stata dimostrata la presenza di recettori sia α che β. Tuttavia il ruolo fisiologico dei due tipi di recettori non è ancora chiaro. Per esempio, in acciughe e sgombri non è stato fatto alcuno studio sulla risposta alle catecolamine ottenuta attraverso i recettori α e β, nel fegato di trota i recettori α non sono accoppiati alla cascata fisiologica che porta al rilascio di glucosio, e in anguilla e pesce gatto l’azione delle catecolamine attraverso recettori β è predominante rispetto a quella attraverso recettori α. L’utilizzo di ligandi farmacologici non ha portato a chiarimenti significativi, perché la loro specificità per i recettori di mammifero non trova sempre riscontro nei pesci. In questo studio, quindi, abbiamo studiato l’espressione dei geni codificanti per i recettori α e β adrenergici attraverso la tecnica della PCR real time, ottenendo i primi dati in letteratura per quanto riguarda la loro quantificazione assoluta. L’organismo modello utilizzato è stata l’anguilla, teleosteo caratterizzato da un ciclo biologico molto particolare in cui si distinguono nettamente una fase gialla ed una argentina. Le anguille argentine non sono mai state studiate a tale proposito, e date le estreme differenze nella disponibilità e nell’uso delle risorse energetiche in questi due stadi di crescita, il presente studio ha mirato a valutare la differente sensibilità alle catecolamine da parte degli epatociti isolati da anguille gialle ed argentine. I nostri dati hanno confermato quanto solo ipotizzato nei vari studi pubblicati negli ultimi due decenni, ma mai avvalorato da risultati sperimentali, cioè che i recettori α e β sono contemporaneamente espressi negli epatociti dell’anguilla, sia gialla che argentina, e la proporzione tra loro giustifica il ruolo significativamente maggiore giocato dai recettori β. Nelle anguille argentine infatti, come nelle gialle, l’effetto dell’adrenalina sul rilascio di glucosio ottenuto attraverso recettori β è chiaramente predominante. Inoltre, i nostri dati indicano che in due diverse fasi del ciclo vitale dell’anguilla, così come si osserva nell’ontogenesi dei mammiferi, i recettori adrenergici sono espressi in quantità differente.

Staigus natūraliai migruojančių ungurių jauniklių skaičiaus mažėjimas didžiojoje paplitimo arealo dalyje stebimas nuo 1980 m. ICES (angl.: The International Council for the Exploration of the Sea) 2001 m. ungurių išteklių būklę įvardino kaip “esančią už saugių biologinio minimumo ribų”. Kokios priežastys lemia šitokį drastišką ungurių išteklių mažėjimą, aiškaus atsakymo nėra, tačiau mokslininkai kelia eilę hipotezių. Greičiausiai tokį išteklių mažėjimą lemia ne viena, o visas kompleksas priežasčių, tai besaikė žvejyba, klimato kaita, tarša, buveinių pakenkimas. Lietuvoje, ir kaimyninėse šalyse europinių ungurių ištekliai sumažėjo kaip ir visame jų paplitimo areale. Lyginant su 1982 metų sugavimais vidaus vandenyse jų sumažėjo dvigubai, o Kuršių mariose trigubai. Taip pat sumažėjo ir ungurių valstybiniai įžuvinimo mastai. Per pastarąjį dešimtmetį ungurių sugavimai Estijoje ir Latvijoje vidaus vandenyse sumažėjo beveik du kartus, o Lenkijoje 0,5 karto. Šiuo metu Lietuvoje ungurių žvejyba reguliuojama pagal kvotų, žvejybos įrankių, žvejybos sezonų, verslinio laimikio dydžio kontroliavimo sistemas. Kaimyninėse šalyse taikomi labai panašūs ungurių išteklių apsaugos būdai. Europos Bendrijų Komisija išleido paskutinį tarybos reglamentą (KOM (2005) 572), nustatantį priemones ungurių ištekliams atkurti. Pagrindinis reglamento elementas – parengti nacionalinius europinių ungurių išteklių valdymo planus, kuriuos taikydamos visos valstybės narės užtikrintų, kad 40 % užaugusių sidabrinių... [toliau žr. visą tekstą]
SUMMARY A steep decline in recruitment has occurred over most of the continent since the early 1980. In 2001, ICES announced European eel to be outside safe biological limits. Although several hypotheses have been brought forward as possible explanations of causes of this widespread decline, no unambiguous cause has been identified. This is mostly due to fishing at unsustainable levels, climate changes, pollution or habitat loss. Stock of European reduced in all Baltic countries, like in all European eel spread territory Eel landings in Lithuania is reduced in inland and territorial waters. Eel landings since 1982, deduced double in inland waters, and triple in Coronian lagoon. El stocking with glass eels or elvers reduced too. During the last decade eel landings from inland waters reduced double in Estonia and Latvia, while in Poland landings reduced 0,5 times. Eel commercial fishery in Lithuania is controlled by quotas, fishing gear, fishing seasons, commercial size system. In neighbour countries similar fishery control system is being used. Commission of the European communities presented proposal for a council regulation establishing measures for the recovery of the stock of European Eel. The main aspect is that each member country has to prepare management plan to ensure the escapement to sea for at least 40 % of the biomass of silver eel. For Lithuania it would be useful to maintain fishermen’s initiative to stock inland waters by silver eels or elvers, and in this way... [to full text]

Doutoramento em Biologia
No contexto dos contaminantes aquáticos, os herbicidas são considerados como um dos grupos mais perigosos. Uma vez aplicados, estes são facilmente transportados para cursos de água, quer devido a uma pulverização pouco cuidada ou devido a fenómenos de escorrência superficial e/ou subterrânea. A presença destes agroquímicos no ambiente tem vindo a ser associada a efeitos nefastos em organismos não-alvo, como é o caso dos peixes. Contudo, existe ainda uma grande lacuna no que diz respeito à informação científica relacionada com o seu impacto genotóxico. Deste modo, a presente tese foi delineada com o intuito de avaliar o risco genotóxico em peixes de duas formulações de herbicidas: o Roundup®, que tem como princípio activo o glifosato, e o Garlon®, que apresenta o triclopir na base da sua constituição, produtos estes largamente utilizados na limpeza de campos agrícolas, assim como em florestas. Foi ainda planeado desenvolver uma base de conhecimento no que diz respeito aos mecanismos de dano do ADN. Como último objectivo, pretendeu-se contribuir para a mitigação dos efeitos dos agroquímicos no biota aquático, nomeadamente em peixes, fornecendo dados científicos no sentido de melhorar as práticas agrícolas e florestais. Este estudo foi realizado adoptando a enguia europeia (Anguilla anguilla L.) como organismo-teste, e submetendo-a a exposições de curta duração (1 e 3 dias) dos produtos comerciais mencionados, em concentrações consideradas ambientalmente realistas. Para a avaliação da genotoxicidade foram aplicadas duas metodologias: o ensaio do cometa e o teste das anomalias nucleares eritrocíticas (ANE). Enquanto o ensaio do cometa detecta quebras na cadeia do ADN, um dano passível de ser reparado, o aparecimento das ANE revela lesões cromossomais, sinalizando um tipo de dano de difícil reparação. O ensaio do cometa foi ainda melhorado com uma nova etapa que incluiu a incubação com enzimas de reparação (FPG e EndoIII), permitindo perceber a ocorrência de dano oxidativo no ADN. No que diz respeito ao Roundup®, o envolvimento do sistema antioxidante como indicador de um estado próoxidante foi também alvo de estudo. Uma vez que as referidas formulações se apresentam sob a forma de misturas, o potencial genotóxico dos seus princípios activos foi também avaliado individualmente. No caso particular do Roundup®, também foram estudados o seu surfactante (amina polietoxilada; POEA) e o principal metabolito ambiental (ácido aminometilfosfórico; AMPA). Os resultados obtidos mostraram a capacidade do Roundup® em induzir tanto dano no ADN (em células de sangue, guelras e fígado) como dano cromossómico (em células de sangue). A investigação sobre o possível envolvimento do stresse oxidativo demonstrou que o tipo de dano no ADN varia com as concentrações testadas e com a duração da exposição. Deste modo, com o aumento do tempo de exposição, os processos relacionados com o envolvimento de espécies reactivas de oxigénio (ERO) ganharam preponderância como mecanismo de dano no ADN, facto que é corroborado pela activação do sistema antioxidante observado nas guelras, assim como pelo aumento dos sítios sensíveis a FPG em hepatócitos. O glifosato e o POEA foram também considerados genotóxicos. O POEA mostrou induzir uma maior extensão de dano no ADN, tanto comparado com o glifosato como com a mistura comercial. Apesar de ambos os componentes contribuirem para a genotoxicidade da formulação, a soma dos seus efeitos individuais nunca foi observada, apontando para um antagonismo entre eles e indicando que o POEA não aumenta o risco associado ao princípio activo. Deste modo, realça-se a necessidade de regulamentar limiares de segurança para todos os componentes da formulação, recomendando, em particular, a revisão da classificação do risco do POEA (actualmente classificado com “inerte”). Uma vez confirmada a capacidade do principal metabolito do glifosato – AMPA – em exercer dano no ADN assim como dano cromossómico, os produtos da degradação ambiental dos princípios activos assumem-se como um problema silencioso, realçando assim a importância de incluir o AMPA na avaliação do risco relacionado com herbicidas com base no glifosato. A formulação Garlon® e o seu princípio activo triclopir mostraram um claro potencial genotóxico. Adicionalmente, o Garlon® mostrou possuir um potencial genotóxico mais elevado do que o seu princípio activo. No entanto, a capacidade de infligir dano oxidativo no ADN não foi demonstrada para nenhum dos agentes. No que concerne à avaliação da progressão do dano após a remoção da fonte de contaminação, nem os peixes expostos a Roundup® nem os expostos a Garlon® conseguiram restaurar completamente a integridade do seu ADN ao fim de 14 dias. No que concerne ao Roundup®, o uso de enzimas de reparação de lesões específicas do ADN associado ao teste do cometa permitiu detectar um aparecimento tardio de dano oxidativo, indicando deste modo um decaimento progressivo da protecção antioxidante e ainda uma incapacidade de reparar este tipo de dano. O período de pós-exposição correspondente ao Garlon® revelou uma tendência de diminuição dos níveis de dano, apesar de nunca se observar uma completa recuperação. Ainda assim, foi evidente uma intervenção eficiente das enzimas de reparação do ADN, mais concretamente as direccionadas às purinas oxidadas. A avaliação das metodologias adoptadas tornou evidente que o procedimento base do ensaio do cometa, que detecta apenas o dano nãoespecífico no ADN, possui algumas limitações quando comparado com a metodologia que incluiu a incubação com as enzimas de reparação, uma vez que a última mostrou reduzir a possibilidade de ocorrência de resultados falsos negativos. Os dois parâmetros adoptados (ensaio do cometa e teste das ANE) demonstraram possuir aptidões complementares, sendo assim recomendado a sua utilização conjunta com vista a efectuar uma avaliação mais adequada do risco genotóxico. Globalmente, os resultados obtidos forneceram indicações de grande utilidade para as entidades reguladoras, contribuindo ainda para a (re)formulação de medidas de conservação do ambiente aquático. Neste sentido, os dados obtidos apontam para a importância da avaliação de risco dos herbicidas incluir testes de genotoxicidade. A magnitude de risco detectada para ambas as formulações adverte para a necessidade de adopção de medidas restritivas em relação à sua aplicação na proximidade de cursos de água. Como medidas mitigadoras de impactos ambientais, aponta-se o desenvolvimento de formulações que incorporem adjuvantes selecionados com base na sua baixa toxicidade.
Herbicides are considered among the most hazardous contaminants of water bodies, since they easily reach these ecosystems through aerial spray drift, artificial drainage systems and surface or sub-surface runoff. The occurrence of these agrochemicals in the aquatic environment has been associated to deleterious effects in non-target organisms, namely fish. However, a considerable gap is evident regarding the scientific information on their genotoxic impact. Therefore, the present thesis was designed with the intention to evaluate the genotoxic risk to fish of the herbicide formulations Roundup® (glyphosate-based) and Garlon® (triclopyr-based), representing broadly used products worldwide to manage unwanted vegetation in agriculture and forestry. It was also planned to develop of a biologically base knowledge on DNA damage mechanisms. As ultimate goal, it was intended to contribute to mitigate the effects of agrochemicals in aquatic biota, namely fish, providing scientific data able to improve forestry and agriculture managing practices. The study was carried out adopting the European eel (Anguilla anguilla L.) as test organism and performing short-term exposures (1 and 3 days) to environmentally realistic concentrations of the mentioned commercial products. Two different genotoxic endpoints were adopted: comet and erythrocytic nuclear abnormalities (ENA) assays. The comet assay measures DNA stand breaks, a repairable type of damage, whereas the ENA assay identifies chromosomal lesions, signalizing a type of damage hardly repairable. The comet assay was also upgraded with an extra-step involving incubation with repair enzymes (FPG and EndoIII), in order to detect oxidative DNA damage. In what concerns to Roundup®, the involvement of the antioxidant system as indication of a pro-oxidant status was also assessed. Once the aforementioned formulations are presented as mixtures of chemicals, the genotoxic potential of their active ingredients individually was also assessed. In the case of Roundup®, the evaluation of the risk associated to the surfactant (polyethoxylated amine; POEA) and to the major environmental breakdown product of the active principle (aminomethylphosphonic acid; AMPA) was carried out as well. The results obtained showed the Roundup® ability to induce both DNA (in blood, gills and liver cells) and chromosomal damage (in blood cells). The investigation on the causative involvement of oxidative stress demonstrated that the type of DNA damage varies with tested concentrations and exposure duration. Thus, ROS-dependent processes gained preponderance as a mechanism of DNA damage with the increase of exposure length, which was corroborated by the antioxidant activation observed in gills as well as the net FPG-sensitive sites elevation detected in liver. Glyphosate and the surfactant POEA were also found to be genotoxic. Moreover, POEA induced the highest extent of DNA damage, when compared to glyphosate and the commercial mixture. Though both components showed to contribute to the overall genotoxicity of the herbicide formulation, the sum of their individual effects was never observed, pointing out an antagonistic interaction between them, indicating that POEA does not increase the risk associated to the active ingredient. These findings also emphasized the need to define regulatory thresholds for all the formulation components, recommending, in particular, the revision of the hazard classification of POEA (classified as “inert” until date). Since the ability of the main environmental metabolite of glyphosate - AMPA - in exert DNA and chromosomal damage was also confirmed, it was pointed out the silent problem that the products of environmental degradation of the active ingredients can constitute. In addition, the importance to include AMPA in risk assessment studies concerning the glyphosate-based herbicides was highlighted. The formulation Garlon® and its active ingredient triclopyr also showed a clear genotoxic potential. In addition, it was demonstrated the higher genotoxicity of the formulation, in comparison to the active ingredient. However, their ability in exert oxidative DNA damage could not be demonstrated. In what concerns to the evolution of the damage progression after removal of the contamination source, neither fish exposed to Roundup® nor Garlon® achieved a complete restoration of DNA integrity in 14 days. In relation to Roundup®, the use of the DNA lesion-specific repair enzymes allowed understanding the occurrence of a late oxidative DNA damage, indicating a progressive decay of cell antioxidant protection as well as the incapacity to repair this particular type of damage. The Garlon® post-exposure period revealed a tendency to decrease damage levels, although not enough to be regarded as an effective recovery. However, an efficient intervention of DNA repair enzymes specifically directed to oxidized purines became evident. Evaluating the performance of the adopted genotoxic endpoints, it was evident that the standard comet procedure, detecting only non-specific DNA damage, displayed some limitations when compared to the methodology that includes the incubation with the repair enzymes, since the latter reduced the possibility of false negative results. The two adopted endpoints (comet and ENA assays) demonstrated complementary aptitudes, being recommended their jointly application since it allows a more effective genotoxic risk assessment. Overall, the results obtained provided useful recommendations for policymaking, contributing to (re)formulate regulatory procedures for protecting the health of aquatic environment. In this direction, the data gathered in this work point to the importance of performing a genotoxic evaluation in order to actually determine the hazard posed by herbicides and their by-products. The magnitude of risk detected for both formulations strongly advise the adoption of restrictive measures in relation to their application in the proximity of watercourses. As mitigation measures, the development of formulations incorporating adjuvants selected on the basis of their lower toxicity emerged as a recommended path.

Contrairement à la majorité des mammifères, la plupart des poissons ne possèdent pas de chromosomes sexuels hétéromorphes (type XY/XX). Chez un grand nombre de poisson la labilité du sexe est extrêmement prononcée. Cela se traduit par le fait que le déterminisme du sexe peut être sous l’influence de variables environnementales. C’est le cas chez l’anguille où la proportion de mâle augmente avec le nombre d’individus présents en un lieu donné. Cette thèse a pour but de mieux comprendre les facteurs qui régissent la destiné sexuelle des individus. Ainsi, les relations inter-individuelles et la croissance ont été étudiées afin d’évaluer leur possible rôle dans le déterminisme du sexe. Les résultats acquis au cours de cette thèse suggèrent que la densité per se ne serait pas le facteur primordial influençant le déterminisme du sexe, mais plutôt l’estimation faite par les individus de la qualité du milieu
Contrarily to most mammals, the majority of fish doesn’t have heteromorphic sexual chromosomes (type XY/XX). In most fish, the gonad development is extremely labile and for some of them, the sex can be influenced/determined by environmental factors. This is observed in eel, where the proportion of males increases with the number of individuals at a given location. The goal of this thesis is to better understand processes that act upon sex determination in eel. Thus, inter-individual relationships as well as the growing potential of each fish were studied to evaluate their possible role in sex determination. The findings gathered throughout this thesis support the hypothesis of a metagametic (environmental) sex determination and also suggests that the estimation of the quality of the environment, made by juveniles’ eels, is one of the key factors influencing sex determination

Une étude parasitaire de l'anguille d'Europe a été menée dans les lagunes de Biguglia et d'Urbino en Corse. La composition des communautés de parasites a été décrite. Treize espèces parasites ont été identifiées parmi lesquelles: trois digènes, Bucephalus anguillae, Deropristis inflata, Lecithochirium musculus; un monogène, Pseudodactylogyrus anguillae; trois cestodes, Bothriocephalus claviceps, Proteocephalus macrocephalus, Myzophyllobothrium sp. (larve); trois nématodes, Anguillicoloides crassus, Contracaecum sp. (larve enkystée), Goezia anguillae; un acanthocéphale, Acanthocephaloides incrassatus; un copépode, Ergasilus gibbus; et un myxozoaire, Myxobolus portucalensis. La présence d'espèces invasives, notamment le parasite branchial P. anguillae et le nématode parasite A. crassus, dans les lagunes corses est confirmée. Ces espèces, et particulièrement le monogène, présentent des valeurs épidémiologiques croissantes depuis les dernières études menées. Plusieurs espèces présentent des différences de prévalence significatives entre les deux lagunes. Des différences au niveau de la richesse spécifique et des valeurs de diversité, plus élevées pour les parasites des anguilles de la lagune d'Urbino au niveau intestinal métacommunautaire et infracommunautaire, ont été démontrées. Cependant les valeurs les plus élevées de diversité spécifique et les valeurs de dominance les plus basses ont été calculées pour les communautés parasitaires des anguilles de la lagune de Biguglia. Nous avons également mis en avant une diversité parasitaire spécifique plutôt faible chez les anguilles des lagunes corses par rapport aux autres lagunes d'Europe. Les communautés parasitaires de l'anguille d'Europe dans les lagunes de Biguglia et d'Urbino en Corse sont marquées par l'environnement de leur hôte. Une dépendance vis-à-vis de la salinité de la lagune a ainsi été démontrée. Les valeurs d'infestation les plus élevées ont été observées durant les saisons les plus chaudes de l'année pour la majorité des espèces parasites observées (B. anguillae, D. inflata, L. musculus, P. anguillae, P. macrocephalus, A. crassus, les kystes de Contracaecum sp., A. incrassatus et E. gibbus). Nous avons également démontré que l'état d'argenture et la taille ont une influence significative sur les taux d'infestation de sept espèces parasites (D. inflata, L. musculus, P. anguillae, P. macrocephalus, les kystes de Contracaecum sp., A. incrassatus et E. gibbus). La méthode de l'espèce indicatrice a confirmé que le site d'étude, la saison, l'état d'argenture ou la taille de l'anguille pouvait influer sur la présence de certaines espèces parasites. Le tégument de trois digènes parasites de l'anguille d'Europe, B. anguillae, L. musculus et D. inflata, a été étudié en microscopie électronique à balayage et à transmission. Nous avons démontré la présence de structures caractéristiques de l'organisation tégumentaire des digènes ainsi que de formations spécifiques, notamment au niveau de la structure des récepteurs sensoriels et des écailles.

L’objectif de cette thèse est d’étudier le rôle de neuropeptides cérébraux, telle que la Neurokinin B codée par le gène tac3, dans le contrôle de la reproduction d’une espèce en danger, l’anguille Européenne, Anguilla anguilla. La maturation sexuelle de l’anguille est bloquée à un stade prépubertaire avant la migration océanique. Etant donnée sa position phylogénétique basale parmi les téléostéens, l’anguille est un modèle pertinent pour étudier l’évolution moléculaire et fonctionnelle de neuropeptides d’intérêt. Deux gènes paralogues tachykinine 3 (tac3) ont été identifiés dans le génome de l’anguille, chacun codant pour deux peptides. Ces gènes paralogues résultent de la duplication complète du génome spécifique aux téléostéens, comme le montrent les analyses phylogénétiques et synténiques. Les analyses de qPCR montrent que les deux gènes sont exprimés dans le cerveau. Les quatre peptides d’anguille ont été synthétisés et testés sur des cultures primaires de cellules hypophysaires d’anguille. Les quatre peptides inhibent l’expression de l’hormone lutéinisante et d’un récepteur à la gonadolibérine, révélant un double rôle inhibiteur dans le contrôle de la reproduction
The aim of this PhD is to investigate the role of brain neuropeptides, such as neurokinin B, encoded by tac3 gene, in the control of reproduction of an endangered species, the European eel, Anguilla anguilla. The sexual maturation of the eel is blocked at a prepubertal stage before the oceanic migration. Due to its basal phylogenetic position among teleosts, the eel is also a relevant model for studying molecular and functional evolution of key neuropeptides. Two tachykinin 3 (tac3) paralogous genes were identified in the eel genome, each encoding two peptides. These paralogs result from the teleost-specific whole genome duplication, as shown by phylogeny and synteny analyses. Both genes are expressed in the brain as shown by qPCR. The four eel peptides were synthesized and tested on primary cultures of eel pituitary cells. The four peptides inhibited the expression of luteinizing hormone and gonadotropin-releasing hormone receptor, revealing a dual inhibitory role in the control of reproduction

1. Groups of eels, Anguilla anguilla, were adapted from freshwater (FW) to seawater, (SW) for periods of 90 - 300 mins. maximum (acute transfer), 0 - 7 days (chronic transfer), or for more than 14 days (longterm seawater transfer). 2. Acute SW transfer led to a decline in blood pressure, an elevation in plasma osmolality and chloride concentration, an immediate "reflex" drinking response and a non-significant increase in plasma angiotensin II (AH) concentration. 3. Administration of papaverine to FW adapted eel caused hypotension, with subsequent recovery of blood pressure, elevation in the drinking rate and plasma All concentration, and a decline in plasma osmolality. Captopril alone had no effect on blood pressure, drinking rate, osmolality or All concentration, but was successful in partially blocking the papaverine-induced blood pressure recovery and increase in AH concentration, with complete inhibition of the drinking. 4. Administration of papaverine to SW adapted eel caused hypotension, with partial recovery of blood pressure, increased drinking rate, plasma All concentration and plasma osmolality. Captopril alone caused a sustained decrease in blood pressure, inhibition of basal SW drinking and a reduction in plasma All concentration, with change in plasma osmolality. Administration of captopril prior to papaverine was successful in partially blocking the papaverine-induced recovery in blood pressure, increase in drinking, plasma All concentration, and plasma osmolality. 5. Chronic SW transfer led to a general decline in blood pressure, increase in plasma electrolyte concentration, elevation in drinking rate after 4-5 days, an increase in plasma All concentration, and a rise in Na+-K+-ATPase, all leading to long term SW values. 6. Plasma arginine vasotocin concentrations were unchanged in long term-FW and SW adapted fish, with a small transitory rise after 4 days in SW. 7. Cortisol plasma concentrations were similar in both long term- FW and SW- adapted fish, with a rise observed 1 day after transfer to SW. 8. Metabolic clearance rates (MCR) and blood production rates (BPR) were significantly elevated in long term SW adapted fish and during chronic SW adaptation, compared to the FW levels. Binding of 125I-AII to gill (filaments and lamellae), brain (cerebellum and medulla oblongata), kidney (head and caudal), and liver was observed in long term FW and SW adapted fish and 6 day SW transfer animals, with significant increase observed in binding in the caudal kidney and cerebellum and medulla oblongata between the FW group and 6 day SW transfer group.

The European eel is critically endangered (IUCN 2021), only 1% of the population remains and the recruitment has been estimated to be 5-10% of their historical values. The eel is in dire need of remedial efforts and many studies suggest that these efforts are best applied to elvers when these have ended their migration. To satisfy the need for suitable habitats of elvers, we need to understand which habitat is best suited to increase survival and growth. This study focuses on how habitat use of elvers, specifically use of gravel and sand bottom substrates, influences behaviour and activity. In a laboratory experiment, eels in aquaria with gravel substrate were 33 % less visible and had 50 % lower levels of observed activity than eels in aquaria with sand substrate. This difference in visibility and activity has implications for survival by affecting the ability to avoiding visual predators. If we can increase the chances of survival of elvers by strategical placement of gravel substrate as a remedial measure, we could be adding to the recovery of the population.
Europeisk ål är kritiskt hotad (IUCN 2021), endast 1% kvarstår av populationen och rekryteringen antas ligga runt 5–10% av historiska värden. Ålen är i stort behov av åtgärdsinsatser och många studier antyder att det är vid juvenil ålder då ålen avslutat sin vandring som insatser bäst kan tillämpas. För att tillgodose juvenila ålars behov av lämpligt habitat behöver vi kartlägga vilken typ av habitat som är bäst lämpad för överlevnad och tillväxt. Vid restaurering av vattendrag är det en relativt enkel insats att strategiskt placera substrat som förstärker ålens överlevnad. Denna studie har därför undersökt hur ålars habitatanvändning (grus- vs. sandsubstrat) påverkar beteende och aktivitet i ett laboratorieexperiment. De ålar som befann sig i akvarier med grussubstrat var 33 % mindre synlighet och observerades ha 50 % lägre aktivitet än de som befann sig i akvarier med sand. Dessa skillnader i synlighet och aktivitet kan ha en inverkan på överlevnadschanserna genom att det påverkar sannolikheten att kunna undvika visuella predatorer. Om vi kan öka chansen till överlevnad i juvenil ålder genom strategisk placering av grussubstrat vid åtgärdsinsatser bör detta bidra till att stärka populationen.

Glass eels of the European eel migrate from coastal waters inland to freshwater as part of the catadromous lifecycle. The osmotic challenge faced at this time is augmented by their large surface area to volume ratio, and by the fact that the migration may only be completed after several attempts, due to the effects of tide and river flow. Glass eels and elvers developed normally when maintained in waters of differing salinity over a six month period. Drinking rates increased with environmental acclimation salinity (from 0.072 +/- 0.023 mul/g/h to 0.698 +/- 0.099 mul/g/h in FW and SW respectively), and freshwater acclimated fish exhibited a rapid drinking response upon contact with seawater. These accounts of dipsogenic behaviour are similar to those previously reported for adult eels. Results obtained from determinations of branchial Na+K+ATPase activities were more equivocal. Only after nearly five months were activities higher in SW (508.52 +/- 99.76 nmoles/Spairs gills/h) as compared to FW fish (151.65 +/- 8.9 nmoles/5pairs gills/h). Following the transfer of FW acclimated fish to SW there was a trend towards increased Na+K+ATPase activity after seven days post-transfer, which reached a significant peak after two months post-transfer. A transient increase in whole body cortisol content was noted following the transfer of fish from freshwater (388.02 + 90.38 pg/g) to seawater (6268.44 +/- 773.14 pg/g). However, it was not possible to ascertain that this was due to a direct effect of environmental salinity change. There were no clear changes in interrenal cell morphology between salinity groups, although the cells did appear reduced in size with time, regardless of environmental salinity. Total body Na+ content increased with time, and was higher in SW (58.66 +/- 1.66 mumoles/g) as compared to FW reared fish (44.85 +/-1.01 mumoles/g).

[EN] The European eel (Anguilla anguilla, L., 1758) population is in dramatic decline, so much so that this species has been listed as "Critically Endangered" on the Red List of Threatened Species, by the International Union for Conservation of Nature (IUCN). The European eel has a complex life cycle, with sexual maturation blocked in the absence of the reproductive oceanic migration, and an inability to mature in captivity without the administration of hormonal treatments. Even though experimental maturation induces gamete production of both sexes, the fertilization results in infertile eggs, unviable embryos and larvae, which die within a few days of hatching. Therefore, understanding the eel reproductive physiology during maturation is very important if we want to recover the wild eel population. Furthermore, due to its phylogenetic position, representative of a basal group of teleosts, the Elopomorphs, the Anguilla species may provide insights into ancestral regulatory physiology processes of reproduction in teleosts, the largest group of vertebrates. In this thesis, characterization, phylogeny and synteny analyses have given us new insight into the evolutionary history of the reproductive process in vertebrates. The European eel possesses five membrane (mPRs) and two nuclear (nPR or pgrs) progestin receptors. Eel mPRs clustered in two major monophyletic groups. Phylogeny analysis of vertebrate nPRs and PLCz1 (sperm specific protein) places both eel PLCz1 and nPR sequences at the base of the teleost clade, which is consistent with the basal position of elopomorphs in the phylogeny of teleosts. To further resolve the origin of the duplicated eel nPRs, synteny analyses of the nPR neighboring genes in several vertebrate genomes were performed. Phylogeny and synteny analyses allowed us to propose the hypothesis that eel duplicated nPRs originated from the 3R. In order to gain a better understanding of the role of the genes implicated in eel reproduction, analyses of their regulation during experimental maturation were carried out. The change in salinity induced parallel increases in E2 plasma and nuclear estrogen receptor expression levels, revealing a stimulatory effect of salinity on the E2 signalling pathway along the BPG axis, leading to a control of spermatogonial stem cell renewal. Brain and pituitary estrogen receptors may then mediate the stimulation of androgens and steroidogenic enzymes linked to androgen synthesis. Androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This is consistent with our studies on estrogen and progestin receptors. In the testis, progestin seems to regulate meiosis through membrane and nuclear progestin receptors, and final sperm maturation seems to be controlled by both estrogen and progestin through the estrogen and progestin membrane receptors. Finally, eel sperm-specific PLCz1 seems to have an important function in spermatozoa by inducing egg activation and temperature may play a role in its regulation, especially during the process of spermiogenesis. This thesis attempts to evaluate the physiological function of the genes involved in eel reproduction during spermatogenesis, and demonstrates that salinity and temperature play crucial roles in the sexual maturation of the male European eel.
[ES] La anguila europea (Anguilla anguilla, L., 1758) está sufriendo un declive dramático y ha sido incluida en la categoría de especies "En peligro crítico" en la Lista Roja de Especies Amenazadas, por la International Union for Conservation of Nature (IUCN). La anguila europea tiene un ciclo de vida complejo, con un bloqueo de la maduración sexual hasta que se produce la migración reproductiva, y no madura en cautividad sin la aplicación de tratamientos hormonales. La inducción de la maduración sexual conlleva la producción de gametos de ambos sexos, pero los resultados de la fertilización son huevos no fértiles, embriones no viables, o larvas que mueren pocos días después de la eclosión. Por tanto, la comprensión de la fisiología reproductiva de la anguila durante la maduración es imprescindible para recuperar sus poblaciones naturales. Además, dada su posición filogenética, como representantes de un grupo basal de los teleósteos, los elopomorfos, las especies del género Anguilla podrían proporcionar nuevas perspectivas sobre los procesos ancestrales de regulación de la fisiología de la reproducción de los teleósteos, el mayor grupo de vertebrados. En esta tesis, los resultados de caracterización, análisis de filogenia y sintenia ofrecen nuevas perspectivas de la historia evolutiva del proceso reproductivo de los vertebrados. La anguila europea posee cinco receptores de progestágenos de membrana (mPRs) y dos nucleares (nPR o pgrs). Los mPRs de la anguila se engloban en dos grandes grupos monofiléticos. Las filogenias de los nPRs y de la PLCz1 (una proteína específica del esperma) sitúan a las secuencias de la anguila de PLCz1 y de nPRs en la base del grupo de los teleósteos, lo que coincide con la posición basal de los elopomorfos en la filogenia de los teleósteos. Para resolver el origen de la duplicidad de los nPRs de anguila, se realizaron análisis de sintenia de los genes próximos a los nPRs. Los análisis de filogenia y sintenia nos permitieron formular la hipótesis de que los nPRs duplicados de la anguila se originaron en la 3° duplicación del genoma que se produjo en teleósteos. Para entender mejor el papel de los genes implicados en la reproducción de la anguila, se hicieron análisis de su regulación durante la maduración experimental. El cambio de salinidad indujo aumentos paralelos del nivel plasmático de E2 y de la expresión de los receptores nucleares de estrógenos, que refleja un efecto estimulador de la salinidad sobre la ruta de señalización del E2 dentro del eje cerebro-hipófisis-gónada, que conlleva el control de la renovación de las espermatogonias indiferenciadas. Los receptores de estrógeno en el eje cerebro-hipófisis-gónada podrían mediar la estimulación de la síntesis de andrógenos y de los enzimas esteroidogénicos unidos a ella. Esa síntesis de andrógenos no depende de la temperatura, pero la continuación del proceso de maduración requiere de temperaturas más altas para inducir un cambio en las rutas esteroidogénicas hacia la síntesis de estrógenos y progestágenos. Esto coincide con nuestros estudios sobre receptores de estrógenos y de progestágenos. En el testículo, los progestágenos parecen regular la meiosis mediante la participación de dos mPRs y un nPR, y la maduración final del esperma parece estar controlada tanto por estrógenos como por progestágenos mediante los receptores de estrógenos y de progestágenos de membrana. Finalmente, la PLCz1 de anguila podría tener una importante función en la activación del huevo inducida por el espermatozoide, y la temperatura podría jugar un papel en su regulación, especialmente durante el proceso de espermiogénesis. Esta tesis intentó evaluar la función fisiológica de los genes implicados en la reproducción de la anguila durante la espermatogénesis, y demuestra que la salinidad y la temperatura juegan papeles cruciales en la maduración sexual de los machos de anguila
[CAT] La població d'anguila europea (Anguilla anguilla, L., 1758) està sofrint un declivi dramàtic, i aquesta espècie ha estat inclosa en la categoria d'espècies "En perill crític" en la Llista Roja d'Espècies Amenaçades per la International Union for Conservation of Nature (IUCN). L'anguila europea té un cicle de vida complex, amb un bloqueig de la maduració sexual que es manté fins que es produeix la migració reproductiva, i no madura en captivitat sense l'aplicació de tractaments hormonals. Però, fins i tot quan la inducció de la maduració sexual comporta la producció de gàmetes d'ambdós sexes, els resultats de la fertilització son ous no fèrtils, embrions no viables o larves que moren pocs dies després de l'eclosió. Per això, la comprensió de la fisiologia reproductiva de l'anguila durant la maduració és imprescindible per aconseguir la recuperació de les poblacions naturals d'anguila. A més, donada la seua posició filogenètica com a representant d'un grup basal de teleostis, els elopomorfos, les espècies del gènere Anguilla podrien proporcionar noves perspectives al voltant dels processos ancestrals de regulació de la fisiologia de la reproducció dels teleostis, el grup més nombrós dels vertebrats. En aquesta tesi, els resultats de caracterització i l'anàlisi de la filogènia i la sintènia ofereixen noves perspectives de la història evolutiva del procés reproductiu dels vertebrats. L'anguila europea posseeix cinc receptors de progestàgens de membrana (mPRs) i dos nuclears (nPR o pgrs). Els mPRs de l'anguila s'engloben en dos grans grups monofilètics. L'anàlisi filogenètic dels nPRs i de la PLCz1 (una proteïna específica de l'esperma) de l'anguila respecte a les de la resta de vertebrats situa a les seqüències d'aquestes proteïnes en la base dels grups dels teleostis, la qual cosa coincideix amb la posició basals dels elopomorfos en la filogènia dels teleostis. Per tal de resoldre l'origen de la duplicitat dels nPRs de l'anguila, es realitzaren anàlisis de sintènia dels gens pròxims als dels nPRs en els genomes de diversos vertebrats. Aquests anàlisis ens permeteren formular la hipòtesi de que els nPRS duplicats de l'anguila es van originar en la tercera duplicació del genoma que es va produir en teleostis. Per arribar a entendre millor el paper dels gens implicats en la reproducció de l'anguila, s'analitzà la seua regulació durant la maduració experimental. Els canvis en la salinitat induïren augments en paral·lel del nivell plasmàtic d'E2 i de l'expressió dels receptors nuclears d'estrògens, reflectint un efecte estimulador de la salinitat sobre la ruta de senyalització d'E2 en l'eix cervell-hipòfisi-gònada, que comportaria el control de la renovació dels espermatogonis indiferenciats. Els receptors d'estrògens en l'eix cervell-hipòfisi-gònada podrien, d'aquesta forma, intervindre en l'estimulació de la síntesi d'andrògens i dels enzims esteroidogènics units a la síntesi d'andrògens. Aquesta síntesi d'andrògens no depén de la temperatura, però la continuació del procés de maduració requereix de temperatures més altes per induir un canvi en les rutes esteroidogènics cap a la síntesi d'estrògens i progestàgens. En els testicles, els progestàgens pareixen regular la meiosi mitjançant la participació dels receptors de progestàgens de membrana i nuclears, i la maduració final de l'esperma sembla estar controlada tant pels estrògens com per progestàgens de membrana. Finalment, la PLCz1 específica de l'esperma de l'anguila podria tindre una funció de rellevància en l'activació dels ous induïda pels espermatozoides, i la temperatura podria tindre el seu paper en la regulació d'aquesta, especialment durant el procés de l'espermiogènesi. Aquesta tesi ha avaluat la funció fisiològica dels gens implicats en al reproducció de l'anguila durant l'espermatogènesi, i ha demostrat que la salinitat i la temperatura tenen papers clau
Morini, MAM. (2016). Molecular approaches related to the European eel (Anguilla anguilla) reproductive process [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68513
TESIS
Premiado

Mestrado em Biologia Aplicada
The presence of pesticides in water bodies is a worrying environmental issue, occurring mainly due to spray-drift, surface runoff or inadvertent applications. This vast class of agrochemicals is known to induce several pernicious effects in non-target aquatic organisms, namely fish. However, data concerning the genotoxic impact of these compounds are scarce. Hence, the present thesis aimed to fill the knowledge lacuna on pesticides capacity to induce genotoxicity to fish, addressing two commercial formulations: Decis®, a deltamethrin-based insecticide, and Roundup®, a glyphosate-based herbicide, each representing a widely used class of biocides. The present work aimed to assess: (i) the chromosomal damaging potential of Decis®, (ii) the DNA damage induction by Roundup®, (iii) the involvement of oxidative processes on the DNA integrity loss as well as (iv) the involvement of DNA repair system in the progression of the DNA damage induced by Roundup®. European eel (Anguilla anguilla L.) was adopted as test organism, performing short-term exposures of 3 days to environmentally realistic concentrations of Decis® and Roundup® and post-exposure evaluations of 1, 7 and 14 days to assess the damage evolution in pesticide-free water. In order to assess the genotoxic damage induced by Decis®, the erythrocytic nuclear abnormalities (ENA) assay was performed, evaluating chromosomal damage, a hardly repairable type of lesion. In what concerns to Roundup® experiment, the comet assay was adopted as genotoxic endpoint, measuring DNA damage as strand breaks, able to be repaired. In order to assess the involvement of oxidative damage in Roundup®-induced genotoxicity, the comet assay was performed with an extra step, where nucleoids were digested with the endonucleases formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (EndoIII), measuring oxidized purines and pyrimidines, respectively. The base excision repair (BER) assay was adopted to evaluate the oxidative DNA damage repair ability of eels exposed in vivo to Roundup®. In general, Decis® and Roundup® demonstrated their genotoxic induction to A. anguilla, since they induced cytogenetic and DNA damage, respectively. In both cases, the damage showed to be of transient nature, since fish were able to completely reverse the previously induced damage. Decis® demonstrated its genotoxic potential by the increase of erythrocytic nuclear abnormalities frequency as a result of its clastogenicity and/or aneugenicity properties. Cytogenetic damage values of exposed eels returned to control levels after 7 days in insecticide-free water. This recovery appeared to be mainly a result from a preferential removal of cells with abnormal nuclear morphology, since no alterations were noticed at the erythropoiesis dynamics. Roundup® was capable of inducing DNA damage in hepatic cells and, though not directly reflected in an increase of breaks at FPG- and EndoIIIsensitive sites, the involvement of oxidative processes in Roundup® genotoxicity was confirmed. The DNA damage values of exposed eels returned to control levels after 1 day in herbicide-free water, mainly due to the repairable condition of this type of damage and the cessation of the exposure. DNA repair enzymes seem to be susceptible to inhibitory actions associated to higher levels of Roundup® constituents/metabolites and/or ROS likely to occur in liver during the exposure period. However, in the postexposure period, an increased capacity to repair oxidative DNA damage emerged, being a crucial pathway for the complete recovery from the genetic damage induced. Evaluating the performance of the genotoxic endpoints adopted in the present study, ENA and comet assays revealed to be suitable to detect the described types of damage. In addition, it became clear that both should be used as complementary tools, since each one measures specific damage types that might be detected at different moments concerning exposure/postexposure periods. In what concerns the management/monitoring practices of pesticides use, the present thesis demonstrated that aquatic biota is under substantial threat and provided useful data to develop stricter regulatory directives. Further studies on this subject should be encouraged, contributing to the increased knowledge and the assessment of the environmental risk to fish populations. In addition, it should be investigated the real impact of pesticides, considering the fish ability to rapidly reverse the short-term effects. This might help the establishment of stricter regulatory procedures aiming the reduction of pesticides overuse and/or inadvertent applications as well as the choice of less toxic compounds.
A ocorrência de pesticidas no meio aquático tornou-se uma questão ambiental de elevada preocupação, considerando que esta vasta classe de compostos pode induzir um leque variado de efeitos nefastos em organismos aquáticos, nomeadamente em peixes. A sua presença em cursos de água é devida, principalmente, a aplicações negligentes, como pulverizações perto dos mesmos e à escorrência dos solos. No entanto, verifica-se uma grave lacuna no que diz respeito à informação científica referente ao seu impacte genotóxico. Deste modo, a presente tese visou avaliar o risco genotóxico em peixes de duas formulações comerciais com capacidade biocida: Decis®, um inseticida que tem a deltametrina como base da sua composição química, e Roundup®, um herbicida, com glifosato como princípio ativo, representando desta forma duas classes de pesticidas com vasta utilização. Concretamente, o presente estudo delineou-se de forma a responder aos seguintes objetivos: (i) a avaliação do potencial de indução de lesões cromossómicas pelo Decis®, (ii) a capacidade do Roundup® na indução de dano no ADN, (iii) o envolvimento de processos de dano oxidativo na perda de integridade do ADN assim como (iv) a participação dos processos de reparação do ADN na progressão do dano induzido pelo Roundup®. Adotou-se a enguia europeia (Anguilla Anguilla L.) como organismo-teste, submetendo-a a concentrações ambientalmente realistas de Decis® e Roundup® durante exposições de curta duração (3 dias), seguidas por avaliações de pós-exposição de 1, 7 e 14 dias como forma de avaliar a evolução do dano após a cessação da exposição. O dano genotóxico induzido pelo Decis® foi avaliado adotando o teste das anomalias nucleares eritrocíticas (ANE), através do qual se podem observar lesões cromossómicas sinalizando um tipo de dano de difícil reparação. A genotoxicidade do Roundup® foi determinada utilizando o ensaio do cometa que deteta quebras no ADN, sendo este tipo de dano suscetível de ser reparado. Com o intuito de esclarecer o envolvimento de processos oxidativos na genotoxicidade deste herbicida, o ensaio do cometa foi melhorado com uma etapa adicional, através da qual a incubação com enzimas de reparação, as endonucleases FPG e EndoIII, permite a deteção de purinas e pirimidinas oxidadas, respetivamente. A aplicação do ensaio da reparação por excisão de bases (REB) permitiu avaliar a capacidade de reparação de dano oxidativo no ADN em enguias expostas in vivo a Roundup®. No geral, os pesticidas Decis® e Roundup® mostraram ser genotóxicos para A. anguilla, tendo em conta a deteção de dano citogenético e no ADN, respetivamente. Além disso, em ambos os casos a genotoxicidade mostrou ser de natureza temporária, uma vez que os peixes demonstraram a capacidade de reverter completamente as lesões previamente induzidas. A exposição ao inseticida Decis® levou ao aumento da frequência de ANE, como resultado das suas propriedades clastogénica e/ou aneugénica. Este parâmetro assumiu níveis semelhantes aos medidos nos indivíduos do grupo controlo 7 dias após a cessação da exposição ao contaminante. A recuperação do dano citogenético parece resultar de uma remoção preferencial das células com morfologia nuclear anómala, tendo em conta que não se observaram alterações na dinâmica eritropoética. O herbicida Roundup® confirmou a sua capacidade de induzir dano no ADN de células hepáticas e, apesar de não se refletir num aumento de quebras resultantes dos sítios sensíveis à FPG e à EndoIII, o envolvimento dos processos oxidativos foi confirmado. O dano no ADN de enguias expostas a este agroquímico deixou de ser percetível 1 dia após a cessação da exposição, demonstrando ser um tipo de dano de fácil reparação. As enzimas de reparação de ADN revelaram ser vulneráveis a pressões inibitórias associadas a elevados níveis de Roundup® (constituintes e/ou metabolitos) e/ou espécies reativas de oxigénio (ERO) que provavelmente ocorrem no fígado durante o período de exposição. Contudo, no período de pós-exposição, observou-se um aumento da reparação do dano oxidativo no ADN, tornando-se deste modo, uma importante via para a recuperação completa do dano genético. No que diz respeito às metodologias adotadas na concretização da presente tese (teste das ANE e técnica do cometa), ambas revelaram ser adequadas enquanto ferramentas de deteção dos tipos de dano descritos, confirmando a relevância da sua aplicação na avaliação do risco genotóxico de contaminantes em peixes. Tendo em conta a especificidade dos tipos de dano genético associados a cada uma das técnicas, a sua utilização complementar deverá ser considerada como uma mais-valia em diferentes momentos, considerando períodos de exposição/pós-exposição. Globalmente, os resultados obtidos através deste trabalho indicaram que as comunidades aquáticas expostas a pesticidas se encontram sob risco, no que concerne à sua integridade genómica. Neste sentido, os dados obtidos apontam ainda para a necessidade de incrementar o número de estudos de avaliação do risco ambiental para as ictiopopulações. Deve ainda ser investigado o impacte de exposições curtas de pesticidas, tendo em conta a capacidade dos peixes recuperarem rapidamente dos efeitos causados a curto prazo. Neste sentido, é importante estabelecer medidas rigorosas que apontem para a diminuição da utilização de pesticidas e de comportamentos negligentes, assim como conduzir à escolha de compostos menos tóxicos.

The European eel (Anguilla anguilla, Linnaeus 1758) is a catadromous fish which has a great cultural, scientific, and commercial value. The protection of this species is particularly difficult because the biological eel’s life cycle remains in many aspects still unknown. The European eel (A. anguilla) population has declined alarmingly over the past 30 years, this condition has led to questions about the long-term welfare status of this species. The objective of this thesis is to perform a histological analysis on gonad samples of European eels from 4 different lagoons of the North Adriatic at different stages of silvering and to evaluate the maturation of the gonads. For this study were captured a total of 859 wild eels from 4 different lagoons using the “lavoriero”. Subsequently the biometric parameters were collected. A total of 79 female eels were randomly selected, dissected and the gonads were removed for histological analyzes. Sections of 4 μm were cut and labeled with hematoxylin and eosin (H&E). Histological observations of germ cells, at the level of light microscopy, allowed the characterization of six steps of oocyte maturation. Valle di Comacchio is the one with the highest levels of oocyte maturation while Valle Ca Pasta has the lowest. What needs to be stressed is that eels with silver index III have an oocyte maturation nearly equal to eels at stage IV and V of silvering. Considering the results, we can affirm that eels from North Adriatic lagoons are eels with high levels of oocyte maturation accompanied by high indices of GSI values at silver stages III. The following experimental work showed that the levels of oocyte maturation are higher even at lower silver index levels. It can be hypothesized that in the case of North Adriatic eels the transition from yellow to silver eel occurs faster in relation to the high trophic availability in North Adriatic lagoons.

[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

Data över förekomst av två storleksklasser (0-150 mm och >150 mm) av Europeisk ål (Anguilla anguilla) i vattendrag på svenska västkusten hämtades från SERS (Svenskt ElfiskeRegiSter vid SLU). Dessa analyserades genom logistisk regression för att avgöra vilka olika makrohabitat-, landskaps- och hindervariabler som bäst kunde användas för att förutspå förekomst av ål. Resultatet visade att det för makrohabitatvariablerna var vattendragets bredd, vattenhastighet och vegetationsmängd som bäst förutspådde förekomst av båda storleksklasserna. För landskapsvariablerna var det avstånd till mynningen, fosforkoncentration och vattentemperatur som bäst förutspådde förekomsten av båda storleksklasserna. Därtill var även sjöprocent en viktig variabel för att förklara förekomsten av den större klassen ål. För variabler som beskriver vandringshinder var det dammar, ålyngelledare och naturlika fiskvägar som bäst förutspådde ålförekomst av båda storleksklasserna. Därtill var även avstånd till uppströms hinder viktigt för att förklara förekomsten av större ål (>150 mm). Mängden lämpliga tillväxthabitat för ålen kan därför troligtvis ökas genom åtgärder riktade mot lokalens vattendragsbredd, vattenhastighet och vegetationsmängd.

The European eel, Anguilla anguilla, is designated a threatened species and parasites are considered to be one of the contributory factors associated with the species decline. To address the lack of knowledge concerning parasite infections in the UK European eel population, a total of 140 specimens were obtained from the Environment Agency at 14 river sites across England and Wales. Each specimen was dissected and infection status determined for the different classes of helminth using a combination of morphological and molecular approaches, including a PCR-based restriction digestion approach to discriminate between the gill monogeneans Pseudodactylogyrus anguillae and P. bini. Overall, 101 eels (72.1 %) were infected with a total of 1504 helminths and these included gill monogeneans (prevalence = 35.7%), gastrointestinal nematodes (prevalence = 33.6%), the swim bladder nematode Anguillicoloides crassus (prevalence = 25.0%), acanthocephalans (prevalence = 30.0%) and cestodes (prevalence = 9.3%). Seven identified helminth species; the pathogenic pseudodactylids P. anguillae and P. bini, the pathogenic swim bladder nematode A. crassus, the gastrointestinal nematodes Spinitectus inermis and Paraquimperia tenerrima, and the tapeworms Proteocephalus macrocephalus and Bothriocephalus claviceps, are known eel specialists. A further 4 non-specialist eel helminths; the acanthocephalans Acanthocephalus clavula, A. lucii and the category 2 parasite Pomphorhynchus laevis, plus the gastrointestinal nematode Raphidascaris acus, were also identified. Helminth infection appeared to have a significant association with the larger eels and this was particularly evident for infections involving acanthocephalans. This presumably reflects the age and hence prolonged exposure to infection risk, as well as a more diverse diet of the larger, compared to the smaller, eels. Interestingly, the condition factor of the eels infected with helminths was also significantly greater than that of the uninfected eels. Helminth co-infections were commonly observed (54.5% of the infected eels) and the majority of these co-infections (75%) involved one, or both, of the pathogenic A. crassus and pseudodactylids. With regard to catchment sites, interesting differences were noted in the primary helminth infection data. Eel specimens analysed from the rivers in South East England contained the most numerous and diverse range of helminths. Indeed, helminth prevalence was 100% and co-infections, including up to 5 taxa, were common; pseudodactylids and acanthocephalans were present in all the observed co-infections. Eels sampled from rivers in North West England also contained a rich diversity of helminths and although pseudodactytlids were common, the gastrointestinal nematodes were the dominant taxa observed in co-infections. A. crassus and the pseudodactylids were the dominant helminths in co-infected eels sampled from South Wales and interestingly, even though this region had the second highest helminth prevalence, tapeworm infections were not observed. Only 50% of eels sampled from North Wales were infected with helminths; specifically, acanthocephalans and A. crassus were not observed in eels from this region and gastrointestinal nematodes were present in all the co-infections. Taken together, the data in this thesis confirms that European eels in the UK are commonly infected with a plethora of helminths. Some of the eel specimens exhibited known pathologies associated with their infections. However, the precise impacts that these helminths have upon eel health, and importantly the migratory capacity of the host, remain unknown and are worthy of further investigation.

1) Closed recirculating intensive aquaculture potentially offers major advantages over existing technologies including reduction in normal production time, reduced water input and output and beneficial environmental effects. 2) The major aim of this study was to produce a basic scientific understanding of the factors that affect intensive recirculating culture of the European eel Anguilla anguilla in order to increase efficiency and economic viability of eel aquaculture in the E.U. 3) Unlike some intensively farmed fish such as salmonids little is known of the stress factors affecting optimal growth rates in intensive eel culture. The primary effects of stress are mediated by corticosteroids and catecholamines which may have profound effects on growth, appetite and ion and water balance. 4) Growth rates of the eel Anguilla anguilla were investigated in closed water recirculating systems utilising fresh water or saline water (12 ppt)at 23°C. Eels were initially graded into two similar populations consisting of three categories, small (12g), medium (24g) and large (48g) based on initial growth rates. 5) During a 300 day period the medium and large group's growth rates were significantly greater in 12 ppt saline water than in fresh water, although for the small fish group there was no such difference. Stocking densities were maintained at commercial levels of approximately 30-100 kg/m3. 6) Plasma cortisol concentrations increased throughout the growth period in both fresh and saline water, although there were no significant differences between the two groups during the experiment. Metabolic clearance rates of cortisol were however consistently higher in saline water fish. 7) Both groups showed an increase in plasma glucose concentration throughout the experiment. However there were no significant differences between fresh water and saline water fish for plasma concentrations of glucose, free fatty acids or lactate. 8) Eels held at stocking densities of 130 kg/m3 continued to grow in the saline water whereas the control fish in fresh water ceased growing. The results suggest that maintaining water salinity at 12 ppt in closed recirculating aquaculture systems produces increased growth rates and possibly increased efficiency of food conversion. 9) In response to acute grading stress, plasma osmolality and glucose concentrations were elevated in both fresh and salt water groups 20 minutes after grading but returned to pre-grading values within 90 minutes. Plasma cortisol concentrations were elevated after 20 and 40 minutes in saline water but returned to control values after 90 minutes. In fresh water fish, plasma cortisol concentrations were elevated after 20 minutes and remained elevated throughout the experiment. 10) Acute netting stress (tank transfer) resulted in a transient increase in plasma osmolality within 20 minutes after net transfer. Plasma cortisol concentrations were significantly elevated after 20 minutes in saline water but returned to control values after 60'minutes. In fresh water fish, plasma cortisol concentrations were elevated throughout the 90 minute period monitored after net transfer. 11) In both cases of acute stress (netting and grading), plasma catecholamines were elevated within a five minute period after the stressor was applied. This study has developed techniques to assess both long-term and short-term stress in eels and has optimised the environmental conditions leading to improved growth rates. Improvements in the performance of recirculating aquaculture for on-growing eels have been demonstrated and suggestions for future possible improvements as a way forward in commercial aquaculture have been suggested. These factors will, hopefully, lead to increased economic efficiency and increased profits in eel aquaculture within the E.U.

En milieu naturel, évaluer l’effet des contaminants sur les organismes aquatiques s’avère difficile dû aux nombreux facteurs (température, oxygénation, prédation, parasitisme, …) agissant sur les organismes. Le but de ces travaux de thèse était alors de tester la possibilité de détecter et hiérarchiser les effets de divers facteurs naturels et anthropiques sur des individus prélevés in situ. Le modèle d’étude utilisé était l’anguille Européenne (Anguilla anguilla) et Américaine (Anguilla rostrata). Pour appréhender les effets in situ des polluants, nous avons tenté d’identifier des gènes pour lesquels le niveau de transcription était spécifiquement corrélé à un polluant ou un facteur naturel mesuré sur le terrain au moyen de deux outils transcriptomiques : le séquençage haut débit et la puce à ADN. Les profils transcriptomiques obtenus à partir des foies d’anguilles directement prélevées sur le terrain ont été comparés dans un deuxième temps, à ceux obtenus à partir d’individus exposés au laboratoire à divers facteurs naturels et anthropiques de façon séparée. La comparaison de ces profils a montré une différence notable des réponses des individus exposés en laboratoire et ceux prélevés in situ, soulevant un effet non négligeable du stress induit par la captivité en mésocosme. La réalisation d’une étude similaire en utilisant la nageoire caudale comme méthode non invasive s’est révélée pertinente dans la capacité de cet organe à discriminer les différentes conditions expérimentales et de terrain. L’utilisation de la puce sur des gonades d’anguilles européennes maturées artificiellement a indiqué que la pollution pourrait affecter les capacités de reproduction des futurs géniteurs et contribuer alors au déclin massif de cette espèce observé depuis les dernières décennies
In the natural environment, assessing the impact of contaminants on aquatic organisms remains difficult due to many factors (temperature, oxygenation, predation, parasitism, ...) acting on organisms. The purpose of this thesis was then to test the ability to detect and prioritize the effects of various natural and anthropogenic factors on individuals sampled in the natural environment. The models used were the European (Anguilla anguilla) and American (Anguilla rostrata) eels. To understand the effects of pollutants in situ, weat tempted to identify genes for which the transcription level was correlated to a specific pollutant or a natural factor measured in the field with two transcriptomic tools: high through put sequencing and DNA microarray. Transcriptomic profiles obtained from livers of eels taken directly from the field were compared in a second phase to those obtained from individuals exposed in the laboratory to various natural and anthropogenic factors separately. Comparison of these profiles showed a significant difference in responses of individuals exposed in the laboratory and those collected in the field, showing a significant effect induced by captivity mesocosm stress. Conducting a similar study using the tail fin as a non-invasive method was relevant for the ability of this tissue to discriminate different experimental and field conditions. Finally, the use of the microarray on the gonads of artificially matured European eels indicated that the pollution could affect the reproductive capabilities of futures pawners and contribute to massive decline of this species observed in last decades

En el presente trabajo se han estudiado los posibles efectos que concentracionessubletales de un herbicida de uso común en los arrozales del Parque Natural de laAlbufera, el propanil, pueden tener sobre una especie típica de este ecosistema, laanguila europea, Anguilla anguilla. La anguila se encuentra en fase de crecimiento yengorde durante su permanencia en el Lago, de modo que resulta relevante determinar sila contaminación por estos plaguicidas altera su equilibrio fisiológico, lo que afectaría asu normal crecimiento y desarrollo y que en último término repercutiría negativamenteen funciones vitales como la reproducción y la supervivencia.En primer lugar se procedió a la evaluación de la toxicidad aguda del propanil sobrela citada especie determinando para ello su efecto sobre la mortalidad de los individuos.Posteriormente se realizó un seguimiento de las alteraciones fisiológicas inducidas enesta especie bajo la influencia de dos concentraciones subletales del mismo herbicida,durante una fase de exposición de 72 h de duración, seguida de una fase de recuperaciónen agua exenta de herbicida (144 h) mediante un sistema de flujo continuo decirculación de medio (Flow-through test system). Paralelamente se efectuó unexperimento Control efectuado bajo las mismas condiciones experimentales, aexcepción de la adición del herbicida al medio.Así, se observó una clara respuesta de estrés en la anguila, caracterizada porhiperglucemia, hiperlactemia, hiperlipemia e hipoproteinemia, que se acompañó de unareducción de los niveles de glucógeno, proteínas totales y lípidos en diferentes órganosy tejidos, en los que un aumento de lactato fue también cuantificable como resultado dela situación de hipoxia provocada. Los animales necesitaron movilizar sus reservasenergéticas, como puso de manifiesto el descenso del contenido calórico en órganos dereserva como el hígado y el músculo. El periodo de recuperación estipulado fueinsuficiente para la restauración de los parámetros estudiados una vez eliminado elherbicida del medio, independientemente de la concentración de propanil utilizada.El hígado resultó un órgano muy "extenuado" durante la exposición al propanil,como se deduce del descenso observado de los niveles plasmáticos de albúmina yproteínas totales, junto con el aumento de las actividades enzimáticasAminotransferasas, Fosfatasa alcalina y Lactato deshidrogenasa hepáticas, y el descensode la actividad gamma-Glutamil transferasa hepática, apoyado por el aumento del índicehepatosomático, y del contenido hídrico de este órgano. Este deterioro hepático fue másacusado en los animales expuestos a la concentración de herbicida más alta.Se cuantificó la actividad enzimática Fosfatasa alcalina, que mostró una gransensibilidad a la presencia de propanil en el medio. Su actividad en general aumentó entodos los órganos y tejidos analizados, indicativo de lesiones en los tejidos y necrosiscelular. Su grado de alteración fue mayor con la concentración más elevada de herbiciday su posterior recuperación fue función del tejido u órgano, de la concentración deherbicida y del tiempo de exposición.La función gamma-Glutamil transferasa, indicativa de la activación de rutas dedetoxificación de xenobióticos y de estrés oxidativo, mostró una disminución de suactividad en el hígado paralela a su aumento en el músculo esquelético y el corazón,durante la exposición al tóxico. Tras la transferencia a agua libre de propanil, laactividad se "normalizó" en el corazón; mientras que continuó alterada en el músculo yen el hígado.El periodo de recuperación de seis días fue suficiente para observar la tendencia alrestablecimiento de la mayoría de las actividades enzimáticas medidas en las anguilasexpuestas a la concentración menor de propanil. Sin embargo, en los animalesexpuestos a la concentración más elevada, el periodo de recuperación estipulado (eldoble que el periodo de exposición) no fue suficiente.
Propanil is one of the major herbicides employed on rice farming around the worldbeing widely used in the Albufera Natural Park (Valencia, Spain). Populations ofEuropean eel, Anguilla anguilla, commonly inhabit this protected ecosystem. It isessential to determine whether this herbicide affects the individuals of this species.Alterations in the physiology, biochemistry or even behaviour of fish are beinginvestigated as potential diagnostic tools for assessing environmental effects of thecontaminants. The lethal toxicity (LC50) of propanil in the European eel wasdetermined resulting in 31.55 ppm at 96 hours exposure. In order to investigatesublethal effects of propanil on fish physiology, two sublethal concentrations (1/10 and1/50 LC50-96h) were selected. Eels from Albufera Lake were exposed to 0.63 and 3.16mg/L of propanil for 72 hours in a continuous flow-through system and allowed torecover in propanil-free water for 144 additional hours. Parallel a third group of eelswas kept in clean water as Control experiment. The parameters studied were not alteredduring the control experiment. Propanil induced a stress response in the eels. A increasein glucose, lactate and lipids (cholesterol and triglycerides) joint to a decrease in totalproteins and albumin levels in the plasma were observed. Glycogen, total proteins andlipids decreased in several organs and tissues, accompanied by a reduction in hepaticand muscular caloric content, indicative of the elevated cost of propanil exposure to theeel in terms of energy reserves. A general increase in lactate levels in all tissues sampledwas observed, indicative of anaerobiosis condition. Propanil also induced disturbancesin the activity of key enzymes as Alanine aminotransferase, Aspartate aminotransferase,Alkaline phosphatase, Lactate dehydrogenase and gamma-Glutamil Transferase. Watercontent increased in several tissues studied as well as Liver Somatic Index (LSI). Mostof the metabolic disorders persisted after a recovery period in pesticide-free waterduring 144 hours. These results revealed that propanil affects the intermediarymetabolism of A. anguilla, and most of the parameters assayed can be used as goodbiomarkers of herbicide contamination.

L’anguilla europea, Anguilla anguilla, è una specie gravemente minacciata, sia da impatti diretti legati al sovrasfruttamento della specie a tutti gli stadi del ciclo vitale, che indiretti come l’urbanizzazione costiera e la perdita di habitat lagunari. Negli ultimi 45 anni è stata documentata una riduzione del tasso di reclutamento di anguilla europea del 90%. Lo scopo di questo studio è stato approfondire le modalità riproduttive di A. anguilla per via indiretta, attraverso un’analisi di paternità. Il Centro di ricerca universitario di Cesenatico (Laboratori di Acquacoltura ed Igiene delle Produzioni Ittiche – Università di Bologna) ha avviato le prime sperimentazioni su A. anguilla, al fine di mettere a punto un protocollo di riproduzione artificiale. Nell’estate 2015 i ricercatori hanno ottenuto sette riproduzioni spontanee in ambiente controllato, da queste sono state campionate casualmente e genotipizzate circa 40 larve per ogni mandata riproduttiva e i relativi riproduttori per condurre l’analisi di paternità. In ogni riproduzione è stata utilizzata sempre e soltanto una femmina e tre o quattro maschi; le analisi genetiche, condotte utilizzando 9 loci microsatelliti, si sono focalizzate sull’individuazione dei padri e l’assegnamento di paternità è avvenuto con un livello di confidenza medio dell’89%. Dalle analisi effettuate è emerso che: 1) i maschi di questa specie, precedentemente sottoposti a stimolazioni ormonali per indurne la riproduzione e la fertilità, sono in grado di partecipare con successo a più di una riproduzione; 2) più esemplari riescono a fecondare gli ovociti di una sola femmina e sembrano stabilirsi modelli gerarchici di dominanza in quanto si è osservato generalmente che un maschio prevale sugli altri, generando da solo più del 50% della prole. Questo studio pilota rappresenta, quindi, un punto di partenza per approfondimenti futuri sulle modalità riproduttive dell’anguilla europea.

In the last 40 years, the population of European eel (Anguilla anguilla L.) has declined dramatically and is now considered to be outside safe biological limits. In 2007 the European Union implemented an “Eel Recovery Plan” regulation, in order to assist in the recovery of the species back to the previous sustainable levels. The major factors regulating eel populations are still unknown and until there is an understanding of the factors causing the low recruitment of eels, the success of any management plans and conservation measures may be limited. One factor considered important in regulating the eel population is pollution. The major aim of these studies was to investigate the impact of environmental contaminants on eel throughout their life cycle. Laboratory and field studies were carried out to assess the impact of environmental levels of pesticides (atrazine, fenitrothion, pendimethalin, chlortoluron, flusilazole, copper oxychloride, metaldehyde and chlorpyrifos), metals (copper, lead, zinc and chromium) and flame retardants (tributyl phosphate and hexabromocyclododecane) on the transition of juvenile and adult eels between freshwater and the marine environment and growth and feeding during freshwater residency. Exposure to tributyl phosphate (TBP) in freshwater had some effect on physiological (plasma levels of glucose, sodium and chloride and kidney Na⁺/K⁺ ATPase) parameters associated with the silvering process in the eel but not on the morphology or the migratory behaviour during the transition from freshwater to the marine environment. Exposure to a mixture of pesticides (pendimethalin, chlortoluron, flusilazole, copper oxychloride, metaldehyde and chlorpyrifos) did modify the migratory behaviour of eels during the early stages of the freshwater migration but did not have any effects on the physiology of saltwater adaptation. Exposure to atrazine did not impair the olfactory system of eels and they were able to detect compounds released by prey items. Exposure of juvenile (glass eels) to 0.5 μg l^-1 of tributyl phosphate did not have an impact on their freshwater adaptation as they were able to survive the movement between salt and freshwater. Glass eels exposed to a range of low concentration of metals (copper, lead, zinc and chromium) all demonstrated significant damage to their DNA. The long term impact of DNA damage is not known or whether this would reduce survival in the eels. The results of the study indicate that exposure to contaminants as the eels migrate between the freshwater and marine environments has probably only a minor role in regulating the eel population.

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 no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34625
TESIS
Premiado

Im Jahr 2006 wurden insgesamt 767 Aale des Bodensee-Obersees auf den Befall mit dem Schwimmblasenwurm A. crassus und dessen Folgen untersucht. Zusätzlich wurden die Befallsdaten von weiteren 2.326 Aalen aus den Jahren 1988 bis 2005 und 2007 bis 2009 ausgewertet. Weiterhin wurden in den Jahren 2006 bis 2008 eine Anzahl von 383 Kaulbarschen und zahlreiche Copepoden auf den Befall mit dem Larvenstadium von A. crassus untersucht. Auf der Ebene der echten Zwischenwirte ließ sich A. crassus im Bodensee-Obersee im Freiwasserplankton nicht nachweisen. Jedoch konnten Copepoden der Gattung C. abyssorum experimentell mit A. crassus infiziert werden. Der Kaulbarsch ist für A. crassus der wichtigste Stapelwirt im Bodensee-Obersee. Die untersuchten Kaulbarsche waren zwischen 18,8% und 52,1% mit A. crassus infiziert, wobei die Infektion einen deutlichen Frühjahrespeak aufwies. Im Jahr 2006 lag die Prävalenz befallener Aale im Bodensee bei 55,6%, die durchschnittliche Befallsintensität lag bei 4,4 adulter Parasiten je Aal. Durch die Datenauswertung von insgesamt 3.425 Aalen konnte der Verlauf der Anguilli­coloidose im Zeitraum von 1989 bis 2009 rekonstruiert werden. Der Höhepunkt der Infektion lag demnach im Jahr 1993 mit einer durchschnittlichen Prävalenz des Erregers von 58,3% und einer durchschnittlichen Befallsintensität von 16,6. Seit 1996 werden die Schadwirkungen von A. crassus auf die Schwimmblase aufgezeichnet und wurden nun in dieser Arbeit ausgewertet. In diesem Zeitraum nahm die Anzahl der Aale mit schweren Schwimmblasenschäden deutlich zu. Im Jahr 2006 besaßen 89,9% der Aale eine Schwimmblase mit geringer bis sehr starker Schädigung. Bei den abwanderungswilligen Blankaalen besaßen insgesamt 10% eine Schwimmblase mit sehr starken Schäden. Es bestand eine positive Korrelation zwischen der Größe der Aale und der Ausprägung der Schwimmblasenschäden. Weiterhin wurde ein negativer Einfluss durch den Befall mit Larven von A. crassus auf das Milzgewicht beobachtet. Der Befall mit abgestorbenen Adulten war mit Anämie korreliert. Durch den kumulativen Effekt der Schwimmblasenschäden, die durch die Infektion mit A. crassus hervorgerufen werden, werden vor allen Dingen größere Aale im Bodensee-Obersee beeinträchtigt. Eine parasiteninduzierte Mortalität von A. crassus liegt jedoch nicht vor.

Depuis plusieurs dizaines d’années, la population des anguilles européennes a souffert d’un dramatique déclin et est classée parmi les espèces hors de leurs limites biologiques depuis 1998. Différents phénomènes, tels que la pêche, les obstacles aux migrations, ou le réchauffement climatique, en sont à l’origine. Cependant, les perturbations environnementales, telles que les contaminations métalliques et organiques ou les épisodes d’hypoxie, participent probablement à la vulnérabilité de cette espèce. Or, l’estuaire de la Gironde est soumis à des contaminations poly-métalliques historiques, ainsi qu’à des contaminations organiques de différentes origines, et à des épisodes hypoxiques réguliers. Les travaux menés au cours de cette thèse, se sont donc composés d’études de terrain visant à identifier les contaminants majeurs chez les anguilles jaunes de l’estuaire de la Gironde, ainsi que leur voie de bio-accumulation et leurs impacts au niveau physiologique, biochimique et moléculaire. Ces études ont nécessité la mise en place d’expériences préliminaires ayant pour objectifs de vérifier les impacts de certaines procédures, telles que l’anesthésie et la mise en cage des anguilles jaunes. D’autre part, les impacts des deux principaux contaminants métalliques et organiques des anguilles européennes de l’estuaire de la Gironde, ont été testés, de façon individuelle et combinée, ainsi que ceux de l'hypoxie sur des anguilles pré-contaminées ou non, lors d’une étude expérimentale.Ainsi, ces travaux ont mis en évidence que les anguilles européennes installées dans la zone avale de l’estuaire, étaient susceptibles d’être soumises à des contaminations poly-métalliques plus importantes, essentiellement d’origine trophique, responsables d’une croissance pondérale moins élevée et de perturbations transcriptionnelles hépatiques et cérébrales. De plus, bien que les contaminations métalliques de ces anguilles ne mettent pas ne danger la santé humaine, les contaminations en PCB sont, en revanche, supérieures aux normes de consommation. D’autre part, ces travaux ont également mis en évidence des dérèglements mitochondriaux ainsi qu’un stress oxydant, au niveau branchial et cérébral chez des anguilles contaminées au Cd, et au niveau cérébral, branchial, hépatique et rénal chez les anguilles contaminées aux PCB. Enfin, la concomitance de ces deux contaminants et/ou d’un épisode d’hypoxie, réduit et/ou retarde les capacités de réponses transcriptionnelles de ces anguilles. Il semble donc que les différentes perturbations chimiques subies par les anguilles européennes au stade jaune au sein de l’estuaire de la Gironde participent de façon non négligeable à la vulnérabilité de cette espèce
For several decades, the European eel has been suffering from a dramatic decline and has been classified among species beyond their biological limit since 1998. Different phenomena, as fishing, migration barriers, or global warming, are to blame. Environmental perturbations, as metallic and organic contaminations, or hypoxic episodes, probably take part to the vulnerability of this species. The Gironde estuary has been submitted to historic poly-metallic contaminations, to organic contaminations from different origins, and to regular hypoxic episodes. This thesis work is composed of field studies aimed to identify the main contaminants in yellow eel living in the Gironde estuary, their major bioaccumulation way and impacts on physiological, biochemical and molecular parameters. These studies needed preliminary experiments aimed to verify whether some field and handling procedures are consistent with ecotoxicological analyses. Moreover, impacts of the two main contaminants in European eels from the Gironde estuary have been assessed individually or combined, with those of hypoxia on pre-contaminated eels.Thus, these studies have demonstrated that European eels installed in the downstream area of the estuary, were likely to be subject to largest poly-metallic contaminations, mostly by food web, and responsible for a less weight gain and high liver and brain transcriptional disturbances. Furthermore, although the metal contamination of eels does not endanger human health, PCB contaminations are higher than consumption standards. On the other hand, these studies have also revealed oxidative stress and mitochondrial disorders in gills and brain of Cd-contaminated eels, and in brain, gills, liver and kidneys of PCB-contaminated eels. The combination of these two contaminants and/or an episode of hypoxia, reduces and/or delays the transcriptional responses ability of these eels. It seems that the different chemical disturbances, suffered by the yellow eels in the estuary of the Gironde, participate significantly to this species vulnerability

L'anguille a un cycle de vie complexe avec une reproduction océanique et une croissance dans les eaux continentales. La phase de croissance se termine par une "métamorphose secondaire " ou argenture, dernier stade accessible dans les conditions naturelles. La maturation sexuelle naturelle n'a encore jamais été observée et le développement des gonades reste bloqué du fait d'un déficit en hormones gonadotropes, LH et FSH. Comprendre les mécanismes de régulation de LH et FSH chez l'anguille présente un intérêt fondamental pour l'évolution du contrôle de la puberté et un enjeu appliqué pour la maîtrise de la reproduction chez cette espèce en danger. Après avoir développé les outils moléculaires pour l'étude de l'expression des sous-unités de LH et FSH, nous montrons qu'au cours de l'argenture seul l'axe gonadotrope est activé, avec une augmentation de l'expression de FSHβ puis de LHβ, sans activation des axes thyréotrope et somatotrope, ce qui apparente l'argenture à une puberté plutôt qu'à une métamorphose. Nous observons aussi une régulation différentielle de l'expression de LHβ et FSHβ au cours de la maturation sexuelle expérimentale avec une amplification sélective de l'expression de LHβ. Grâce à l'utilisation de culture primaire de cellules hypophysaires d'anguille, nous montrons que les stéroïdes sexuels (estradiol et androgènes), les peptides gonadiques (activine/follistatine) et les facteurs métaboliques (IGF-1, cortisol) exercent des effets directs sur l'expression de LHβ ou FSHβ et peuvent être impliqués dans leur régulation différentielle.

Šio darbo metu naudojant 5 mikrosatelitinius DNR žymenis buvo bandoma išsiaiškinti, ar į Lietuvą natūraliai atplaukiantys upiniai unguriai skiriasi nuo į Lietuvą introdukuotų upinių ungurių genetiškai. Vietines Anguilla anguilla populiacijas atstovavo Baltijos jūros ir Kuršių marių imtys, o introdukuotas – Dringio ir Siesarčio ežerų imtys. Tikrinama buvo, tiek izoliacijos dėl atstumo (IBD), tiek laikinės izoliacijos (IBT), įtaka A. anguilla genetinei diferenciacijai. Nors genetinė diferenciacija tarp didžiosios dalies lyginamų imčių buvo maža (FST = 0,0042) ir nepatikima (p > 0,05), tačiau remdamiesi gautais tyrimo rezultatais negalime patvirtinti panmiksinės hipotezės A. anguilla rūšyje, kadangi mažos (FST = 0,0143) ir patikimos (p = 0,0018) genetinės diferenciacijos nustatymas tarp Siesarčio ežero introdukuotų upinių ungurių leidžia teigti, kad genetinė diferenciacija Anguilla anguilla rūšyje egzistuoja.
In order to find out whether native and introduced Anguilla anguilla populations differ one from another genetically I have used 5 microsatellite markers and compared 2 native between 2 introduced European eel samples. Native A. anguilla samples were taken from Baltic sea and Curonian lagoon, while introduced samples were taken from Dringis and Siesartis lakes. During this work I tried to find out not only IBD, but also IBT impact to A. anguilla genetic differentiation. Experiment results do not reject panmixia hypothesis in European eel, because there are small (FST = 0,0042) and not significant (p > 0,05) genetic differentiation among all samples used in this experiment. However, there is no enough data to say that A. anguilla genetic differentiation does not exist at all, because there is small (FST = 0,0143) and significant (p = 0,0018) genetic differentiation among lake Siesartis samples.

The European eel Anguilla anguilla (Linnaeus, 1758) is a critically endangered species whose survival in the wild is largely threatened by human activity. In addition, this vertebrate's unique ecology and biology make it even more vulnerable, and the IUCN listed A. anguilla as severely endangered on the Red List of Threatened Species. In this scenario, captive breeding can represent a significative help for the species' future and many efforts have been dedicated to the artificial reproduction. Ten years ago, dedicated research programs on A. anguilla artificial reproduction started at the DIMEVET (Bologna University). In 2020, based on parentage assignment, some lights were thrown on the reproductive performance in semi-natural conditions. The present study aims to verify if the artificial mixing of male and female gametes would lead to different results in males’ fertilization rates, improving the final rate of genetic variability. We analysed six successful reproduction events. 22 adults and 258 larvae were genotyped by 10 specie-specific microsatellite loci and paternity was allocated to 250 larvae, with a rate of success in parentage assignment of 94%. Even if a locus had to be discarded from the data set for technical reasons, this high rate in allocation success accounts for the good resolution power of the remaining nine loci (mean PIC 0.812). In each reproduction we observed a single male who contributed with 40-70% of the offspring generated, two subordinate males who contributed with 10-30% of the F1 and an “ineffective” one which contributed with 8% or less to F1. This result is homologous to the previous results in semi-natural conditions. The presence of a similar pattern of fertilization rate by different males in both studies, suggest that the behavioural component is unlikely to be the cause of the offspring generation disparity, and other explanations should be considered, like sperm quality and factors that would impact on it.

L’objectif de ce travail de thèse est de contribuer à l’étude des effets de l’environnement sur le recrutement de civelles d’Anguille européenne. Nous avons adopté une approche de modélisation, et cela pour deux raisons. Tout d’abord, l’observation de la phase larvaire durant la migration trans-atlantique est très difficile à mettre en œuvre. Ensuite la démarche de modélisation permet d’explorer la complexité de l’écologie de l’Anguille. Des hypothèses physiques et biologiques sont formulées pour comprendre le processus par lesquels l’environnement agit sur le recrutement, en fort déclin depuis 25 ans. Deux approches de modélisation sont menées : dans une première partie, une modélisation déterministe basée sur des modèles hydrodynamiques permet d’étudier la dérive passive de larves relâchées dans la zone de production, la mer des Sargasses. Une des originalités de ce travail est de rendre ces simulations réalistes en implémentant des hypothèses de comportement et de mortalité. D’une part, nos simulations montrent qu’une traversée trans-océanique en 6 mois, suggérée par l’analyse de la microstructure des otolithes, impliquerait une nage active rapide et donc trop coûteuse en énergie dans un milieu oligotrophe comme l’océan ouvert. La durée moyenne de migration est estimée à 22 mois, en conformité avec des approches préalables d’analyse de cohortes. D’autre part nos résultats suggèrent un faible impact de l’intensité du transport océanique sur le succès du recrutement. Ils démontrent aussi l’influence des dates et lieux de départ des larves sur leur histoire de vie au cours de la migration ainsi que sur leur aire d’arrivée en conformité avec les résultats préalables de génétique et de microchimie des otolithes
The PhD thesis amis to contribute to the study of environmental impacts on European glass eel recruitement. We chose a modelling approach for two reasons. First, the observation of the larval stage over the trans-atlantic migration is dIFcult to carry out. The, the modelling approach enables us to explore the complexity of eel ecology. Different physical an biological hypotheses are tested to understand processes wereby environment may impact on recruitment that is declining for 25 years. Two modelling approaches are developed. In a first Part, a deterministic modelling using hydrodynamic models enables us to study the passive drift of larvae released in the spawning area, the Sargasso Sea. One of the originalities of this work is to implement realistic hypotheses of behaviour and mortality in simulations. One of the originalities of this woik is to implement realistic hypotheses of behaviour and mortality in similations. On one hand, our simulations show that a 6-month migration duration, as suggested by the analysis of otolith microstructure, would imply a fast active swimming. This energy expenditure would be too high in a oligotrophic environment such as open ocean. The mean migration duration is estimated to 22 months in accordance with previous approaches of cohort analyses. On the other hand our results suggest a weak impact of oceanic transport intensity on recruitment success. Simulations also provide evidence of the influence of starting locations and dates on arrival areas and larva live histories in accordance with previous results of genetic and otolith microchemistry. In a second part, a statistical modelling focuses on the links between environmental conditions experienced by larvae (oceanic transport and food availabiblity) and the recruitment assessed by glass eel quantities entering European Estuaries. Recruitment is not impacted by transport conditions described by large-scale oceanic indices. Whe show that glass ell recruitment variations are related to primary production int the Sargasso Sea. The recent increase in sea temperature in the spawing area led to a decrease in food availability and thus, in glass eel recruitment

[EN] During the last 25 years, eel populations have declined considerably, this species is considered outside safe biological limits. The life cycle of the eel is quite complex including transoceanic migration whose conditions are still so unknown that even the natural spawning process has never been observed. Eels cease feeding during migration and that is why their energy reserves such as lipids play a crucial role and thus their in deep study is essential to obtain gametes (eggs and sperm) quality as the first step for their reproduction in captivity. In the last years, many efforts have been directed at the study of the influence of fatty acids on the reproductive performance in females, but not in male eels. The present manuscript describes the results obtained in two experiments. The first experiment (which led to the three first articles included in the memory) focused on the effect of fatty acids along maturation, their relationship with steroid hormones and their effect on sperm quality parameters. The second experiment involved the comparison of different diets designed, considering the previously acquired insights into the most influential fatty acids on male eel reproduction, and the assessment of their influence on the reproductive performance of males. The first experiment was used to assess the changes in the fat content, as well as fatty acids, in different tissues of male eels hormonally induced to sexual maturation under different thermal regimes (two variables and one constant). Males finished spermatogenesis earlier with a constant temperature of 20 ºC, suggesting that eel spermatogenesis is tightly regulated by temperature. The fat content did not change significantly in the muscle, but was increased in the liver and decreased in testes during testicular development. With regard to fatty acids, during sperm maturation, the liver was highlighted as the main site of synthesis. Finally, EPA, ARA and DHA remained constant in testes, while the level of the rest of fatty acids decreased significantly. Using samples from the same experiment, the correlation between the main steroid hormones and fatty acids at different stages of testicular development were investigated. Similarly as occurs in mammals, EPA and DHA were highlighted as possible modulators of androgen synthesis. The set of the results obtained suggests new perspectives concerning the functions and interactions between fatty acids and steroids in fish spermatogenesis. Also, with samples from the first experiment, a study was carried out seeking to establish the possible correlations between different fatty acids and several sperm quality parameters. Correlations between several highly unsaturated fatty acids with parameters such as volume, the percentage of motile sperm and sperm velocity were found. Finally, with the acquired knowledge so far, a second experiment was carried out designing feeds with different fatty acid percentages in order to evaluate the influence of the diets on sperm quality. The results showed that high levels of DHA and EPA in the diets induce higher volumes and high sperm motility. This study allowed obtaining results applicable to the design of optimum broodstock diets for this species.
[ES] Durante los últimos 25 años las poblaciones de anguila han disminuido considerablemente, de modo que la especie se considera fuera de los límites biológicos de seguridad. El ciclo vital de la anguila es bastante complejo incluyendo una migración transoceánica cuyas condiciones son todavía tan desconocidas que incluso el proceso natural de desove no se ha observado nunca. Las anguilas dejan de alimentarse durante la migración y es por ello que sus reservas energéticas en forma de lípidos tienen un papel crucial y su estudio en profundidad es importante para obtener gametos (huevos y esperma) de calidad como primer paso para su reproducción en cautividad. En los últimos años se han dirigido muchos esfuerzos al estudio de la influencia de los ácidos grasos en el rendimiento reproductivo de las hembras, pero no en el de los machos de anguila. El presente documento describe los resultados obtenidos en dos experimentos. El primer experimento (que dio lugar a los tres primeros artículos incluidos en la memoria) se centró en el efecto de los ácidos grasos durante la maduración, su relación con las hormonas esteroideas y su efecto sobre los parámetros de calidad espermática. El segundo experimento consistió en la comparación de diferentes dietas, diseñadas considerando los conocimientos adquiridos previamente sobre los ácidos grasos más influyentes en la reproducción de machos de anguila, y en la evaluación de su influencia en el rendimiento reproductivo de los machos. El primer experimento sirvió para evaluar los cambios en el contenido graso, así como en los ácidos grasos, en diferentes tejidos de machos de anguila hormonalmente inducidos a la maduración sexual bajo diferentes regímenes térmicos (dos variables y uno constante). Los machos concluyeron la espermatogénesis más temprano a una temperatura contante de 20 ºC, sugiriendo que la espermatogénesis de anguila está estrechamente regulada por la temperatura. El contenido en grasa no varió significativamente en el músculo, se incrementó en el hígado y bajó en el testículo a lo largo del desarrollo testicular. Con respecto a los ácidos grasos, durante la maduración del esperma, el hígado destacó como el principal sitio de síntesis. Finalmente, EPA, ARA y DHA permanecieron constantes en el testículo, mientras que el nivel del resto de ácidos grasos bajó significativamente. Usando muestras del mismo experimento, se investigaron las correlaciones entre las principales hormonas esteroideas y los ácidos grasos en los diferentes estadios del desarrollo testicular. De forma similar a lo que ocurre en mamíferos, EPA y DHA destacaron como posibles moduladores de la síntesis de andrógenos. El conjunto de los resultados obtenidos sugiere nuevas perspectivas sobre las funciones y las interacciones entre los ácidos grasos y los esteroides en la espermatogénesis de peces. También con muestras del primer experimento, se llevó a cabo un estudio que buscó las posibles correlaciones de los diferentes ácidos grasos con varios parámetros de calidad espermática. Se encontraron correlaciones entre varios ácidos grasos altamente insaturados con parámetros como el volumen, el porcentaje de espermatozoides móviles y la velocidad del esperma. Finalmente, con los conocimientos adquiridos hasta el momento, se realizó un segundo experimento para el que se diseñaron piensos con diferentes porcentajes de ácidos grasos con el fin de evaluar la influencia de las dietas sobre la calidad del esperma. Los resultados mostraron que niveles altos de DHA y EPA en las dietas inducen mayores volúmenes y una alta motilidad del esperma. Este estudio permitió obtener resultados aplicables al diseño de dietas óptimas para reproductores de esta especie.
[CAT] Durant els últims 25 anys les poblacions d'anguila han disminuït considerablement, de manera que l'espècie es considera fora dels límits biològics de seguretat. El cicle vital de l'anguila és bastant complex incloint una migració transoceànica les condicions de la qual són encara tan desconegudes que fins i tot el procés natural de fresa mai ha estat observat. Les anguiles deixen d'alimentar-se durant la migració i és per això que les seves reserves energètiques en forma de lípids tenen un paper crucial i el seu estudi en profunditat és important per a obtenir gamets (ous i esperma) de qualitat com a primer pas per a la reproducció en captivitat. En els últims anys s'han dirigit molts esforços a l'estudi de la influència dels àcids grassos en el rendiment reproductiu de les femelles, però no en el dels mascles d'anguila. El present document descriu els resultats obtinguts en dos experiments. El primer (que dona lloc als tres primers articles inclosos en la memòria) es va centrar en l'efecte dels àcids grassos durant la maduració, la seva relació amb les hormones esteroides i el seu efecte sobre els paràmetres de qualitat espermàtica. El segon experiment va consistir en la comparació de diferents dietes, dissenyades considerant els coneixements adquirits prèviament sobre els àcids grasos més influents en la reproducció dels mascles d'anguila, i en l'avaluació de la seva influència en el rendiment reproductiu dels mascles. El primer experiment va servir per avaluar els canvis en el contingut de gras, així com en els àcids grassos, en els diferents teixits de mascles d'anguila hormonalment induïts a la maduració sexual baix diferents règims tèrmics (dos variables i un constant). Els mascles van concloure la espermatogènesis més prompte a una temperatura de 20 ºC, suggerint que l'espermatogènesis d'anguila està estretament regulada per la temperatura. El contingut en greix, no va variar significativament en el múscul, es va incrementar en el fetge i va baixar en els testicles al llarg del desenvolupament testicular. Pel que fa als àcids grassos, durant la maduració de l'esperma, el fetge va destacar com al principal lloc de síntesi. Finalment, EPA, ARA y DHA van romandre constants en el testicle, mentre que el nivell de la resta d'àcids grassos va baixar significativament. Emprant mostres del mateix experiment, es van investigar les correlacions entre les principals hormones esteroides i els àcids grassos en els diferents estadis de desenvolupament testicular. De manera semblant al que ocorre en mamífers, EPA i DHA van destacar com a possibles moduladors de la síntesi d'andrògens. El conjunt dels resultats obtinguts suggerixen noves prespectives sobre les funcions i les interaccions entre els àcids grassos i els esteroides en l'espermatogènesi dels peixos. També amb mostres del primer experiment, es va dur a terme un estudi que va buscar les possibles correlacions dels diferents àcids grassos amb diversos paràmetres de qualitat espermàtica. Es van trobar correlacions entre diversos àcids grassos altament insaturats amb paràmetres com el volum, el percentatge d'espermatozoides mòbils i la velocitat de l'esperma. Finalment, amb els coneixements adquirits fins al moment, es va realitzar un experiment per al qual es van dissenyar pinsos mb diferents percentatges d'àcids grassos per tal d'avaluar la influència de les dietes sobre la qualitat de l'esperma. Els resultats van mostrar que nivells alts de DHA i EPA en les dietes indueixen a majors volums i una alta motilitat de l'esperma. Aquest estudi va permetre obtenir resultats aplicables al disseny de dietes òptimes per a reproductors d'aquesta espècie.
Baeza Ariño, R. (2015). Roles of lipids and fatty acids through the spermatogenesis of European eel (Anguilla anguilla) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/49988
TESIS

L’anguille a un cycle de vie complexe avec une reproduction océanique et une croissance dans les eaux continentales. La phase de croissance se termine par une «métamorphose secondaire » ou argenture, dernier stade accessible dans les conditions naturelles. La maturation sexuelle naturelle n’a encore jamais été observée et le développement des gonades reste bloqué du fait d’un déficit en hormones gonadotropes, LH et FSH. Comprendre les mécanismes de régulation de LH et FSH chez l’anguille présente un intérêt fondamental pour l’évolution du contrôle de la puberté et un enjeu appliqué pour la maîtrise de la reproduction chez cette espèce en danger. Après avoir développé les outils moléculaires pour l’étude de l’expression des sous-unités de LH et FSH, nous montrons qu’au cours de l’argenture seul l’axe gonadotrope est activé, avec une augmentation de l’expression de FSHβ puis de LHβ, sans activation des axes thyréotrope et somatotrope, ce qui apparente l’argenture à une puberté plutôt qu’à une métamorphose. Nous observons aussi une régulation différentielle de l’expression de LHβ et FSHβ au cours de la maturation sexuelle expérimentale avec une amplification sélective de l’expression de LHβ. Grâce à l’utilisation de culture primaire de cellules hypophysaires d’anguille, nous montrons que les stéroïdes sexuels (estradiol et androgènes), les peptides gonadiques (activine/follistatine) et les facteurs métaboliques (IGF-1, cortisol) exercent des effets directs sur l’expression de LHβ ou FSHβ et peuvent être impliqués dans leur régulation différentielle
The eel has a complex life cycle with an oceanic reproduction phase and a growth phase in continental waters. The growth phase ends with a « secondary metamorphosis » or silvering. This is the last stage in the eel’s life cycle which is accessible in natural conditions. Natural sexual maturation has never before been observed and the development of the gonads stays blocked because of a deficit in the gonadotropins LH and FSH. Understanding the regulation mechanisms of eel LH and FSH is of fundamental interest for the development of eel pubertal control and a challenge which must be overcome in order to succeed in regulating the reproduction of this endangered species. After the development of molecular tools we have studied the LH and FSH subunits; this shows that only the gonadotropic axis is active during silvering. An increase in FSHß then in LHß was seen without any activation of the thyrotropic or somatotropic axes, meaning that silvering is a pubertal event rather than a metamorphosis. There was also observed a differential regulation of expression of LHß and FSHß during experimental sexual maturation with a selective amplification of LHß. Thanks to the use of primary cell cultures of eel pituitary glands we can show that sex steroids (estradiol and androgens); gonadal peptides (activin/follistatin) and metabolic factors (IGF-I and cortisol) exert direct effects on the expression of LHß and FSHß and could be involved in the differential regulation of gonadotropins