Aspects of the reproductive biology of female <i>Loligo forbesi</i> were investigated. The species is dioecious and females are generally smaller than males. The maximum size attained by females in Scottish waters is 330 mm and the largest size attained by the males is 550 mm. Variations in sex ratios do not indicate any seasonality. The demography is complex and apparently not consistent from year to year. Recruitment is possibly all year round and is associated with an extended spawning season (December to May). It is not known whether different spawning groups contribute to spawning. Maturation is associated with an increase in the size of the reproductive structures (ovary, proximal oviduct, oviducal gland, distal oviduct, nidamental glands and accessory nidamental glands) relative to body weight. This is not associated with a decrease in the wet somatic and visceral components. The glands of the female reproductive system show histological changes associated with maturation. The epithelia of these glands become secretory with maturation and their secretions are thought to provide the egg coats. An exception is the proximal oviduct which is apparently involved in the transportation of eggs and possibly has an absorptive function. Examination of eggs from the ovary of females at different stages of maturation suggests that eggs are spawned continuously over an extended period. The function of the accessory nidamental glands is unknown but they exhibit a secretory cycle associated with maturation. The accessory nidamental glands harbour symbiotic bacteria which change colour with maturation. This colour change has potential for use in experiments investigating factors controlling maturation in those species which have accessory nidamental glands. Preliminary experiments have shown that the colour of these bacteria can change when grown on a medium to which extracts of optic glands (cultured in a medium) were added. A total of twelve strains of bacteria were isolated from the accessory nidamental glands of females from different maturity stages. Based on physical and biochemical characteristics, the bacteria were tentatively identified as species of <i>Vibrio</i> and <i>Pseudomonas</i>.

Cephalopods have extremely well developed visual systems which are of particular interest due to the well known morphological similarity of the cephalopod eye to the vertebrate eye. This similarity ends at the level of the photoreceptors where vertebrates and invertebrates have been found to use different intracellular second messengers. Although the effect of extracellular ion manipulation on the light response has been examined and some very useful biochemical studies carried out, the pathway has not been investigated by the use of pharmacological intervention; a method which has proved to be useful in other preparations. This study examines various properties of the photoreceptors of the cuttlefish, Sepia officinalis, with particular interest in the second messenger signalling pathway. Both extracellular and whole cell patch clamp recording has been utilised. The second messenger signalling pathway, which mediates phototransduction in the retina of S. officinalis, was investigated by recording the electroretinogram and examining how this changed with the application of various extracellularly applied, membrane permeable pharmacological agents. Invertebrate phototransduction utilises the phosphoinositide (PI) signalling pathway therefore specific activators and inhibitors targeted at precise sites of this pathway were applied to the extracellular bathing solution. These studies indicated that cleavage of phosphatidylinositol-4,5-bisphosphate is essential for the production of a light response and that the inositol trisphophate (IP3) branch of this pathway is of greatest importance in this preparation, as opposed to the diacylglycerol branch. How this second messenger cascade transfers the incoming information into a temporally coded signal was studied by measuring maximum critical flicker fusion frequency. The effect of cell size on this property was investigated and also how cell sensitivity was affected and whether these properties appeared to fit the animal's environmental conditions or whether they were restricted by cellular properties. The animals were found to have relatively &quot;slow&quot; eyes. However the younger age group studied, with shorter photoreceptors, was found to be both faster and more sensitive. This was an unexpected finding considering temporal resolving power is often sacrificed for sensitivity. It is suggested that the observed differences between age groups was attributable to the effects of increased cell size on the cell membrane time constant and that deterioration of signalling molecules with aging may also be a contributing factor. An investigation of the cell signalling pathway at the level of individual cells was also carried out using the whole cell patch clamp technique. Using this technique, two voltage activated currents were found; an inward sodium current characterised by its voltage and tetrodotoxin sensitivity, and an outward potassium current characterised by its tetraethylammonium sensitivity. As well as finding further evidence for the involvement of the IP3 branch of the PI pathway there is also evidence of a role for cyclic guanosine monophosphate. A suitable mode of measuring light-induced fluctuations in the intracellular calcium levels was also investigated with a view to observing the impact of the pharmacological agents on intracellular calcium concentration. This investigation has enhanced the understanding of the S. officinalis visual system by greatly adding to the present knowledge of the second messenger signalling cascade and by giving an insight into how this transfers into the animal's temporal resolving power. Some preliminary information regarding the membrane currents activated by light has also been presented. This has all been possible by the development of a versatile retinal slice preparation that has been proven to be accessible to extracellular recording and whole cell patch clamp recording combined with pharmacological manipulation.

Cephalopods have one of the most sophisticated cardiovascular systems among the invertebrates; they have an enclosed high pressure blood system, characterised by a double circulation, with one main systemic heart and two gill hearts. The cephalopod cardiac tissues are basically myogenic, but there is evidence for both nervous and hormonal regulation of most parts of the cardiovascular system, although the details of the control systems remain unknown. This study investigated the physiology and innervation of the cardiac tissues of the squid Alloteuthis subulata, Loligo forbesii and L vulgaris. Histological staining techniques established that the cardiac organs in squid are innervated from the palliovisceral lobe of the brain via the paired visceral nerves. The nerves to the ventricle, branch off from each of the visceral nerves, close to a commissure that connects the two visceral nerves. The auricles of the systemic heart are innervated from a cardiac ganglion, situated at the base of the gills. Other branches, also given off at this level, innervate the lateral vena cava, afferent and efferent branchial blood vessels, branchial hearts and the muscular valve region between the branchial heart and afferent branchial vessel. Electron microscopical studies have shown the structure and number of fibres in these nerves. Pharmacological studies of isolated and perfused squid branchial hearts showed that acetylcholine had an inhibitory effect on cardiac activity, acting on both nicotinic and muscarinic receptors. An aminergic receptor system may be present in squid branchial hearts, although the transmitter substance is still unknown. The catecholamines adrenaline and noradrenaline excite the heart, although in an inconsistent manner. Whole cell patch clamp studies revealed that individual squid heart cells operated, using a combination of at least six different ionic currents; three outward potassium currents (delayed rectifier, A-current, calcium-activated current) and three inward currents (sodium current, L- and T -type calcium currents). An understanding of the functions of the various currents was obtained by recording electrically stimulated and spontaneous action potentials, using conventional intracellular recording and stimulation techniques. The ionic currents were isolated by applying known channel antagonists, and each antagonist's effect on the action potential was studied.

Over the last 50 years there has been a rapid increase in global landings of cephalopods (octopus, squid and cuttlefish). In European waters, cuttlefish are among the most important commercial cephalopod resources and within the North-East Atlantic, the English Channel supports the largest cuttlefish fishery, with the common cuttlefish, Sepia o cinalis (Linnaeus, 1758), dominating landings. S. o cinalis has a short (2 year) life cycle in the English Channel that is punctuated by seasonal migrations inshore and o shore. Using a combination of di erent métiers including beam trawling, otter trawling and coastal trapping, this shared fisheries resource is targeted at nearly every phase of the life cycle. Despite this continuing increase there remain only minimal management measures in place, with no quotas, no total allowable catches, no closed areas, no minimal landing size and no routine assessment of stocks. In order to provide sustainable fisheries management advice for S. o cinalis populations it is essential that a thorough understanding of the ecology and life history of this species, in particular the factors a ecting spawning and recruitment variability, is attained.In this thesis, I examine critical gaps in our understanding of the distribution, movements, habitat use and behaviours of spawning and sub-adult S. o cinalis. This research provides baseline data for this species within the inshore waters of the English Channel and uses a combination of novel field-based electronic tracking techniques, in situ subtidal observations of spawning patterns within natural environments and presence-only species distribution modelling. A maximum entropy (MaxEnt) modelling approach was used to predict the distribution of benthic egg clusters using presence-only data. The model showed very good performance in terms of predictive power and accuracy (test area under the receiver operating characteristics curve [AUC] = 0.909) and among the explanatory variables used to build the model, depth (gain = 1.17), chlorophyll-a concentration (used here as a proxy for turbidity; gain = 1.06) and distance from coastline (gain = 1.02) were shown to be the greatest determining factors for the distribution of S. o cinalis spawning. As part of the model output, maps (logistic and binary) of the predicted spawning distribution of S. o cinalis within the English Channel were produced.Subtidal observation were undertaken at spawning grounds on both the North and South coast of the English Channel to investigate spawning habitat and structure use. A total of 15 types of natural spawning structures were identified. The range of spawning structures used varied among sites with Zostera marina identified as the dominant spawning structure at two of the UK sites (Torbay and Poole Bay), potentially indicating a ‘preference’ for this structure within localities. Fractal dimension analysis of the seagrass beds at Torbay revealed that the spatial dynamics of seagrass beds within this site varied significantly between 2011 and 2012 (Mann- Whitney U: Z = 4.92, P < 0.0001) as a result of both anthropogenic and natural disturbance. Interannual changes in the spatial dynamics of these beds could a ect the annual pattern and intensity of spawning at a site. The use of structures with small diameters was found to occur, with cuttlefish adapting the device to their requirements by utilising multiple leaves or thalli in order to achieve a suitable diameter for egg attachment, this was evident in their use of both Chorda filum and Z. marina.This research also provided the first data on the fine-scale movements and behaviours of adult and sub-adult individuals, tracked within their natural environments, using electronic tagging methodologies. That expected patterns of short-term spawning site fidelity at a local level were observed in only two individuals, whilst larger scale movements (up to 35 km) along the coastline were observed in three individuals, indicated that a range of behaviours and movement patterns could occur among spawning adults. Similarly varied patterns of site fidelity were also observed in tagged sub-adults, tracked over an extended period (up to 73 days), using a static acoustic array. These results highlight the complex range of patterns and plasticity in behaviour that exist within natural populations.In summary, a series of di erent approaches was used within this thesis in an e ort to improve our understanding of the fine-scale movement, behaviours and habitat use of S. o cinalis (in both spawning adults and non spawning sub-adults), as well as their potential spawning distribution within the inshore waters of the English Channel. Observing the movements and behaviours of small marine animals like S. o cinalis in their natural environments has traditionally been di cult. Recent developments in technologies and techniques however, including those used within this thesis (e.g. electronic tagging), have highlighted the potential capacity of novel tools to monitor the in situ movements and behaviour of cuttlefish. By providing important insights into the ecology of this species these new tools can aid conservation and management advice for this important commercial fishery species, both within the English Channel and further afield.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O boto-cinza (Sotalia guianensis Van Beneden, 1864) é uma espécie de delfinídeo que ocorre em águas costeiras das Américas do Sul e Central, especialmente em ambientes estuarinos, baías e áreas protegidas. Estudos de ecologia alimentar podem prover informações sobre o comportamento das espécies e de suas presas. Nesse contexto, o conhecimento da dieta é o primeiro passo para entendermos como se dá as relações predador-presa do ambiente. Neste estudo são abordados aspectos do hábito alimentar do boto-cinza na Baía de Guanabara, com o objetivo caracterizar sua dieta, e realizar comparações intra e interespecíficas, utilizando para isso a análise do conteúdo estomacal de exemplares encalhados entre 1990 e 2009. Outras espécies de delfinídeos foram utilizadas para a comparação com o boto-cinza, estes apresentam hábitos costeiros e oceânicos, são elas: o golfinho-pintado-do-Atlântico (Stenella frontalis Cuvier, 1829), o golfinho-de-dentes-rugosos (Steno bredanensis Lesson, 1828), o golfinho-comum (Delphinus delphis Linnaeus, 1758) e o golfinho-nariz-de-garrafa (Tursiops truncatus Montagu, 1821), o golfinho-de-Fraser (Lagenodelphis hosei Fraser, 1956) e o golfinho-riscado (Stenella coeruleoalba Meyen, 1833). A metodologia empregada se baseia na triagem do conteúdo estomacal, e identificação dos itens alimentares recuperados (otólitos, vértebras de teleósteos e bicos de cefalópodes). O comprimento e biomassas das presas foram estimados com o uso de equações de regressão encontradas na literatura para essas espécies. Para verificar variações intraespecíficas na dieta do boto-cinza, as amostras foram separadas nas categorias: machos adultos, fêmeas adultas e juvenis, e também segundo a estação do ano em que os estômagos foram recuperados, classificados em estações quente/chuvosa (out-abr) e fria/seca (mai-set). Para a caracterização da dieta do boto-cinza e posteriores comparações intra e interespecíficas, foram utilizados os parâmetros: abundância, riqueza, ocorrência, comprimento e biomassa estimada das presas. Os teleósteos foram o grupo de presas mais freqüente e abundante na dieta do boto-cinza, seguido dos cefalópodes e por último dos crustáceos. Foi visto uma alta similaridade da dieta entre fêmeas, machos e juvenis com relação às espécies consumidas e ao perfil das presas, sendo o porte das presas diferente entre adultos e juvenis. Com relação a variações sazonais, foi visto que as espécies consumidas variaram em quantidade, porém, o tamanho e o perfil das presas se mantive o mesmo entre as épocas quente/chuvosa e fria/seca. A abundância, biomassa e riqueza de espécies foram maiores nas estações quentes/chuvosas que nas estações frias/secas, o que pode ser um reflexo da maior disponibilidade de presas nessa época. As demais espécies de delfinídeos consumiram maior variedade de presas que o boto-cinza, o que pode estar relacionado a uma maior movimentação dessas espécies ao longo da costa. Este estudo indica que o boto-cinza apresenta variações intra-específicas no seu hábito alimentar entre adultos e juvenis, e variações sazonais na composição de sua dieta. Seus hábitos alimentares diferiram das demais espécies de delfinídeos, e indicam que o boto-cinza encontra na Baía de Guanabara recursos alimentares suficientes para seu requerimento energético, não precisando sair da região da baía para sua alimentação ou realizando apenas pequenos deslocamentos. / In this work, aspects of feeding habits of the Guiana dolphin, Sotalia guianensis (Van Beneden, 1864), were studied in Guanabara Bay (N = 26), with the aim of characterizing its diet and comparing the results with those from other species of the Delphinidae family (N = 31). The stomach contents analyzed were collected from specimens stranded on the beaches of Rio de Janeiro between 1990 and 2009. The methodology is based on washing stomach contents through 1 mm mesh sieves, as well as on the identification of the food items remained, such as teleost otoliths and cephalopod beaks. The length of the food items was used to estimate the length and weight of preys using regression equations found in the literature for these species. Teleosts constituted the most abundant prey in the stomachs of Guiana dolphin (N = 325), being followed by cephalopods (N = 76) and finally by crustaceans (N = 16). Were identified 17 species of teleosts belonging to 10 families, as well as two species of cephalopods from the Loliginidae family and one species of crustacean from the Penaeidae family. The most important species in Guiana dolphin diet, according to the index of relative importance were the whitemouth croaker (Micropogonias furnieri), the weakfish (Cynoscion jamaicensis) and atlantic midshipman (Porichthys porosissimus). Among cephalopods, the squid Loligo plei was the most representative prey in the diet. Regarding the size of prey, most consumed teleosts had the length varying between 11 and 20 cm (15.4 5.5 cm), characterizing the consumption of juvenile fishes. The cephalopods mantle length ranged from 6 to 15 cm (11.8 4.2 cm). A great similarity was observed in the diet composition of females, males, and juveniles of Guiana dolphin, however, the mean length and biomass of fish differed between those consumed by adults and those preyed by juveniles (Kruskal-Wallis, p<0,05, posteriori comparison of medians). Species richness was higher in warm seasons, as well as the diversity and evenness index, which may be related to a greater availability of prey during these seasons. Proportions of the preys consumed varied, however, the composition of the diet, as well as the habits and size of prey, remained similar between seasons. While all species consumed by Guiana dolphin presented coastal habits, the other delphinids species consumed preys of larger distributions, which could be related to a greater movement of these species along the coast. This study showed that there are variations in food habits of Guiana dolphin in Guanabara Bay and indicates that this species feeds on the available resources in the region.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O boto-cinza (Sotalia guianensis Van Beneden, 1864) é uma espécie de delfinídeo que ocorre em águas costeiras das Américas do Sul e Central, especialmente em ambientes estuarinos, baías e áreas protegidas. Estudos de ecologia alimentar podem prover informações sobre o comportamento das espécies e de suas presas. Nesse contexto, o conhecimento da dieta é o primeiro passo para entendermos como se dá as relações predador-presa do ambiente. Neste estudo são abordados aspectos do hábito alimentar do boto-cinza na Baía de Guanabara, com o objetivo caracterizar sua dieta, e realizar comparações intra e interespecíficas, utilizando para isso a análise do conteúdo estomacal de exemplares encalhados entre 1990 e 2009. Outras espécies de delfinídeos foram utilizadas para a comparação com o boto-cinza, estes apresentam hábitos costeiros e oceânicos, são elas: o golfinho-pintado-do-Atlântico (Stenella frontalis Cuvier, 1829), o golfinho-de-dentes-rugosos (Steno bredanensis Lesson, 1828), o golfinho-comum (Delphinus delphis Linnaeus, 1758) e o golfinho-nariz-de-garrafa (Tursiops truncatus Montagu, 1821), o golfinho-de-Fraser (Lagenodelphis hosei Fraser, 1956) e o golfinho-riscado (Stenella coeruleoalba Meyen, 1833). A metodologia empregada se baseia na triagem do conteúdo estomacal, e identificação dos itens alimentares recuperados (otólitos, vértebras de teleósteos e bicos de cefalópodes). O comprimento e biomassas das presas foram estimados com o uso de equações de regressão encontradas na literatura para essas espécies. Para verificar variações intraespecíficas na dieta do boto-cinza, as amostras foram separadas nas categorias: machos adultos, fêmeas adultas e juvenis, e também segundo a estação do ano em que os estômagos foram recuperados, classificados em estações quente/chuvosa (out-abr) e fria/seca (mai-set). Para a caracterização da dieta do boto-cinza e posteriores comparações intra e interespecíficas, foram utilizados os parâmetros: abundância, riqueza, ocorrência, comprimento e biomassa estimada das presas. Os teleósteos foram o grupo de presas mais freqüente e abundante na dieta do boto-cinza, seguido dos cefalópodes e por último dos crustáceos. Foi visto uma alta similaridade da dieta entre fêmeas, machos e juvenis com relação às espécies consumidas e ao perfil das presas, sendo o porte das presas diferente entre adultos e juvenis. Com relação a variações sazonais, foi visto que as espécies consumidas variaram em quantidade, porém, o tamanho e o perfil das presas se mantive o mesmo entre as épocas quente/chuvosa e fria/seca. A abundância, biomassa e riqueza de espécies foram maiores nas estações quentes/chuvosas que nas estações frias/secas, o que pode ser um reflexo da maior disponibilidade de presas nessa época. As demais espécies de delfinídeos consumiram maior variedade de presas que o boto-cinza, o que pode estar relacionado a uma maior movimentação dessas espécies ao longo da costa. Este estudo indica que o boto-cinza apresenta variações intra-específicas no seu hábito alimentar entre adultos e juvenis, e variações sazonais na composição de sua dieta. Seus hábitos alimentares diferiram das demais espécies de delfinídeos, e indicam que o boto-cinza encontra na Baía de Guanabara recursos alimentares suficientes para seu requerimento energético, não precisando sair da região da baía para sua alimentação ou realizando apenas pequenos deslocamentos. / In this work, aspects of feeding habits of the Guiana dolphin, Sotalia guianensis (Van Beneden, 1864), were studied in Guanabara Bay (N = 26), with the aim of characterizing its diet and comparing the results with those from other species of the Delphinidae family (N = 31). The stomach contents analyzed were collected from specimens stranded on the beaches of Rio de Janeiro between 1990 and 2009. The methodology is based on washing stomach contents through 1 mm mesh sieves, as well as on the identification of the food items remained, such as teleost otoliths and cephalopod beaks. The length of the food items was used to estimate the length and weight of preys using regression equations found in the literature for these species. Teleosts constituted the most abundant prey in the stomachs of Guiana dolphin (N = 325), being followed by cephalopods (N = 76) and finally by crustaceans (N = 16). Were identified 17 species of teleosts belonging to 10 families, as well as two species of cephalopods from the Loliginidae family and one species of crustacean from the Penaeidae family. The most important species in Guiana dolphin diet, according to the index of relative importance were the whitemouth croaker (Micropogonias furnieri), the weakfish (Cynoscion jamaicensis) and atlantic midshipman (Porichthys porosissimus). Among cephalopods, the squid Loligo plei was the most representative prey in the diet. Regarding the size of prey, most consumed teleosts had the length varying between 11 and 20 cm (15.4 5.5 cm), characterizing the consumption of juvenile fishes. The cephalopods mantle length ranged from 6 to 15 cm (11.8 4.2 cm). A great similarity was observed in the diet composition of females, males, and juveniles of Guiana dolphin, however, the mean length and biomass of fish differed between those consumed by adults and those preyed by juveniles (Kruskal-Wallis, p<0,05, posteriori comparison of medians). Species richness was higher in warm seasons, as well as the diversity and evenness index, which may be related to a greater availability of prey during these seasons. Proportions of the preys consumed varied, however, the composition of the diet, as well as the habits and size of prey, remained similar between seasons. While all species consumed by Guiana dolphin presented coastal habits, the other delphinids species consumed preys of larger distributions, which could be related to a greater movement of these species along the coast. This study showed that there are variations in food habits of Guiana dolphin in Guanabara Bay and indicates that this species feeds on the available resources in the region.

A idade e crescimento da lula Doryteuthis plei da região do litoral norte do Estado de São Paulo é descrita com base em 424 exemplares entre 1,58 a 267 mm de comprimento de manto (ML). Os estatólitos foram utilizados como ferramenta de análise de idade, bem como foi realizado um estudo ontogenético de sua morfometria e morfologia. Com o objetivo de analisar e aprimorar a curva e a taxa de crescimento de D. plei, o presente estudo deu ênfase aos indivíduos em estágios iniciais do ciclo de vida. O formato dos estatólitos foi descrito e demonstrou semelhança à de outros loliginídeos, porém com características particulares, suficientes para a identificação da espécie. Houve relação entre ML e o comprimento dos estatólitos (SL), obtido pelo modelo de ajuste logarítmico. A idade dos exemplares amostrados foi estimada e com isto foi possível obter a curva de crescimento (relação idade e ML) da população em estudo, a qual foi ajustada pelo modelo logístico. O mesmo modelo de ajuste houve para os machos, porém para as fêmeas e para os juvenis, o melhor modelo foi o Gompertz. A longevidade da espécie no local de estudo foi estimada em aproximadamente oito meses. A taxa de crescimento e suas variações conforme as estações do ano foram estimadas. Foram evidenciados valores sendo em mm/dia quando em estádio imaturos em relação aos adultos e decrécimo acelerado após atingir 60 mm ML. Foram observadas diferenças da taxa de crescimento entre os sexos quando os exemplares atingem 150 mm de ML. Espécimes nascidos no inverno apresentaram maiores taxas de crescimento em relação aos nascidos em outras estações do ano. Através do retro-cálculo da idade, foi observado que durante todo o ano ocorrem nascimentos de D. plei, em maior frequência no primeiro semestre. É possível estimar a idade de D. plei tendo apenas as medidas de SL ou comprimento dos domos, sendo que para cada relação, o ajuste obtido foi o logarítmico. Os parâmetros de crescimento apresentados no presente estudo oferecem recursos para estudos de avaliação do estoque pesqueiro e biologia populacional. / Age and growth of the squid Doryteuthis plei from the northern shelf off São Paulo are described based on 424 specimens from 1.58 to 267 mm mantle length (ML). Statoliths were used as microstructures for aging, and an ontogenetic study of their morphological and morphometric was performed. The present study aimed to analyze and improve the growth curve and growth rate estimate of individuals of D. plei, emphasizing those in early stages of life cycle. The shape of statoliths was described and demonstrated similarity other loliginids, with particular characteristics sufficient to identify the species. There was a relationship between ML and statoliths length (SL) by the logarithmic model. The age of sampled specimens was estimated and it was possible to obtain the growth curve (relationship between age and ML) of the population in study, was better fitted by the logistic model. The same adjustment was found for males, but for females and juveniles, the Gompertz model was best suitable. The longevity of the species from place of study was estimated at approximately eight months. The growth rate and its variations were estimated according to the seasons, which evidenced higher values in mm/day in immature stage when compared to adults. In addition, individuals after reaching 60 mm ML accelerated a decrease in growth rate. Differences in growth rate between sexes were found after specimens reach 150 mm ML. Specimens born in winter had higher growth rates when compared to those born in other seasons. Through retro-calculation of age, it was observed that births of D. plei occur throughout the year, but most frequently in the first half. The study enables the age estimate of D. plei having only measures of SL or length of the statolith\'s domes. Growth parameters obtained provide information useful for stock assessment and population biology.

Les sacs rénaux des céphalopodes sont communément le siège d’infections parasitaires spectaculaires provoquées principalement par des ciliés apostomes du genre Chromidina et des mésozoaires, les dicyémides. Les formes végétatives sont vermiformes et peuvent atteindre plusieurs mm de long. Elles sont fixées par leur partie antérieure aux cellules rénales. La diversité de ces parasites énigmatiques, basée sur des morpho-espèces, reste très mal connue pour les chromidinés et aucune donnée moléculaire n’est disponible pour ces ciliés. Les Dicyémides ont été en revanche plus étudiés mais les données morphologiques et moléculaires sont parcellaires et leurs relations phylogénétiques restent débattues. Au cours de cette thèse, nous nous sommes proposés d’étudier ces deux groupes afin d’évaluer leur biodiversité chez les céphalopodes méditerranéens et d’Atlantique, et de fournir plus d’éléments pour la compréhension de leur systématique et de leur phylogénie. Ainsi, à partir d’un large échantillonnage de céphalopodes d’Atlantique (Concarneau, France) et méditerranéens (La Goulette, Tunisie) prélevés sur 3 années consécutives, nous avons entrepris de caractériser morphologiquement et génétiquement ces parasites en réalisant une analyse moléculaire du marqueur ADNr 18S afin d’aborder la notion d’espèce, de préciser leur spécificité d’hôte, de corréler la diversité morphologique et génétique, et de mesurer la variabilité génétique au sein d’une même morpho-espèce.Dans nos échantillonnages, la prévalence de l’infection par des chromidinés s’est révélée faible, certainement biaisée par les milieux échantillonnés, essentiellement benthiques, alors que les chromidinés infectent préférentiellement les céphalopodes pélagiques. Nous pouvons cependant prédire que leur biodiversité est sous-estimée, car deux nouvelles espèces ont pu néanmoins être décrites. Nous avons pu obtenir pour la première fois des séquences de l’ADNr 18S de ces parasites. Les analyses phylogénétiques robustes montrent que les chromidinés se positionnent parmi les ciliés apostomes au sein de la classe des Oligohymenophorea.La prévalence des dicyémides était beaucoup plus importante (88%). Un problème important dans la systématique des dicyémides est que bon nombre de morpho-espèces nommées sont basées sur des descriptions morphologiques incomplètes qui entrainent des ambiguïtés dans leur détermination. Ceci nous a amenés à redécrire de façon détaillée sur tous les stades de développement l’espèce Dicyemennea eledones rencontrée dans nos échantillonnages. Les analyses phylogénétiques des séquences du marqueur ADNr 18S obtenues au cours de cette étude et intégrant les séquences de dicyémides déjà connues, nous ont permis de définir 10 clades robustes. Les morpho-espèces ne se distribuent pas de façon non ambigüe au sein de ces clades puisque certaines morpho-espèces se retrouvent dans plusieurs clades et que certains clades peuvent regrouper plusieurs morpho-espèces. Ceci pose la question de la pertinence de ce marqueur ADNr 18S pour l’identification des espèces, voire même des genres, et pose également la question de la pertinence des marqueurs morphologiques utilisés à ce jour pour la systématique des dicyémides. / The renal sacs of cephalopods are an uncommon habitat for two phylogenetically distant parasites, dicyemid mesozoa and ciliate chromidinids. These are wormlike organisms, which can reach a few millimetres in length and are attached to the renal epithelia by their anterior part. Information was mainly based on morphological criteria and little is known about the biodiversity and taxonomy of these enigmatic parasites. Concerning the chromidinids, only 4 morpho-species have been described and there are no molecular data available. Dicyemids have been a little more studied, but morphological criteria are still incomplete for some species and not all life stages have been studied. The molecular data are sparse and the phylogenetic position of these organisms is uncertain.During this thesis, we have undertaken to study these two groups with regard to their biodiversity in cephalods from the Mediterranean Sea and the Atlantic Ocean. The aims are to obtain more elements for the comprehension of their systematics and phylogeny. Starting from a large sample of cephalopods from the Atlantic Ocean (Concarneau, France) and the Mediterranean Sea (La Goulette, Tunisia), fished during 3 consecutive years, we characterized the parasites from a morphological and molecular point of view. We realised a molecular analysis of the 18S rDNA marker in order to look at the notion of species, to precise their host specificity, to correlate the morphological and genetic diversity and to measure the genetic variability within one morpho-species.In our samples, the prevalence of the infection by chromidinids was revealed to be weak, probably biased by the position of the collection in the water column. The sampled hosts were predominantly benthic, while chromidinids mainly infect pelagic cephalopods. We predict that the diversity is underestimated, even if we were able to describe two new species. We obtained for the first time molecular data from the 18S rDNA marker. The robust phylogenetic analyses show that chromidinids are positioned within the apostome ciliates, belonging to the Oligohymenophorea.In our samples, the prevalence of the infection by dicyemids was shown to be more important (88%). A major problem in the dicyemid systematics is that a large number of named morpho-species are based on incomplete morphological descriptions, entailing ambiguities in their identification. We therefore redescribed all stages of the development of the species Dicyemennea eledones of our samples in detail. Phylogenetic analyses of the 18S rDNA marker sequences of our samples, combined with the sequences available in databases, allowed us to define 10 robust clades. The morpho-species are not distributed in a non ambiguous manner within these clades, as some can be found in different clades and some clades can regroup more than one morpho-species. This leads to the question of the relevance of the 18S rDNA marker for the identification of species and even genera. It also challenges the relevance of morphological criteria currently used for the dicyemid systematics.

Controlled Exposure Experiments revealed lesions in the statocysts of four cephalopod species of the Mediterranean Sea (Sepia officinalis, Loligo vulgaris, Illex coindetii and Octopus vulgaris), when exposed to relatively low intensity low frequency sounds. The analysis was performed through: scanning (SEM) and transmission (TEM) electron microscopy techniques of the whole inner structure of the cephalopod statocysts, especially on macula and crista; SEM of the epidermal lines of cephalopod hatchlings; and proteomic studies (2DE/MALDI –MS) of the statocyst’s endolymph. All exposed adult individuals presented the same lesions and the same incremental effects over time, consistent with a massive acoustic trauma observed in land species that were exposed to much higher intensities of sound. Immediately after exposure, the damage was observed in the macula statica princeps (msp) and in the crista sensory epithelium. Kinocilia on hair cells were either missing or were bent or flaccid. A number of hair cells showed protruding apical poles and ruptured lateral plasma membranes, most probably resulting from the extrusion of cytoplasmic material. Hair cells were also partially ejected from the sensory epithelium, and spherical holes corresponding to missing hair cells were visible in the epithelium. The cytoplasmic content of the damaged hair cells showed obvious changes, including the presence of numerous vacuoles and electron dense inclusions not seen in the control animals. The appearance of these lesions became gradually more pronounced in individuals after 12, 24, 48, 72, and 96 hours. Special attention was given to validate these findings with control animals that were caught, maintained and sequentially sacrificed following the same protocol as the exposed individuals. The statocyst ultrastructure was therefore revisited and a comparative analysis was carefully conducted to assess the lesions triggered by the exposure to noise This study also presents preliminary results of the sound effects on epidermal lines of cephalopod hatchlings. The lesions, consistent with an acoustic trauma, were identic in the three species that were exposed, but their evolution over time, in opposition with what was observed in the statocysts, were different, suggesting that the animal size and metabolic response might play a role in a possible recovery process. The analysis of noise effects in the statocyst endolymph by proteomic techniques was only conducted on Sepia officinalis. The presence of differential staining of gels from control and subjected to sound exposure individuals demonstrate that the injuries could be related to a possible physiological imbalance that would affect the protein levels of the endolymph. The lesions and findings described here are new to cephalopod pathology. Given that lowfrequency noise levels in the ocean are increasing (e.g. due to shipping, offshore industry, and naval maneuvers), that the role of cephalopods in marine ecosystems is only now beginning to be understood, and that reliable bioacoustic data on invertebrates are scarce, the present study and future investigations will bring an important contribution to the sustainable use of the marine environment. / Després de sotmetre'ls a experiments d'exposició controlada a sons de baixa intensitat i baixa freqüència es van observar lesions en els estatocists de quatre espècies de cefalòpodes de la mar Mediterrània (Sepia officinalis, Loligo vulgaris, Illex coindetii i Octopus vulgaris). L'anàlisi es va realitzar per mitjà de de microscòpia electronica d'escombratge (SEM) i de transmissió (TEM) de tota l'estructura interna de l'estatocist dels cefalòpodes, especialment en la màcula i en la crista, per SEM de les línees epidèrmiques de les larves dels cefalòpodes i per tècniques de proteòmica (2DE/MALDI-MS), de l'endolimfa de l'estatocist. Tots els estatocists d'individus adults de cefalòpodes exposats presentaven les mateixes lesions i aquests efectes eren més greus a mesura que passava el temps després de l'exposició als sons. Tots els animals exposats al soroll van mostrar lesions consistens amb trauma acústic massiu observat en altres espècies terrestres que havien estat exposades a intensitats molt més altes de so. Immediatament després de l'exposició, es van observar danys a la macula statica princeps (msp) i en l'epiteli sensorial de la crista. Els quinocilis de les cèl·lules ciliades desapareixien o es doblegaven i es tornaven flàccids. Un nombre important de cèl·lules ciliades mostraven els pols apicals sobresortint de l'epiteli sensorial, així com el trencament de les membranes plasmàtiques laterals, molt probablement com a resultat de l'extrusió de material citoplasmàtic. Les cèl·lules ciliades també van ser parcialment expulsades de l'epiteli sensorial deixant visibles forats esfèrics en el mateix. El contingut citoplasmàtic de les cèl·lules ciliades danyades va mostrar canvis obvis, com ara la presència de nombrosos vacúols i inclusions electrodenses que no es veien en els animals control. L'aparició d'aquestes lesions es va tornar gradualment més pronunciada en els individus analitzats després de 12, 24, 48, 72 i 96 hores. Es van validar curosament aquests resultats per mitjà de la comparació amb els animals control que van ser capturats, mantinguts i sacrificats de forma seqüencial seguint el mateix protocol que els individus exposats. La ultraestructura de l'estatocist va ser revisada i es va dur a terme un curós anàlisi comparatiu per tal d'avaluar les lesions provocades per l'exposició al soroll. Aquest estudi també presenta els resultats preliminars dels efectes del so en les línies epidèrmiques de cefalòpodes recent nascuts. Les lesions, consistens amb trauma acústic, eren idèntiques en les tres espècies que van ser exposades, però la seva evolució en el temps, en oposició amb el que es va observar en els estatocists, era diferent, cosa que suggereix que la grandària dels animals i la resposta metabòlica podria tenir influència en un possible procés de recuperació. L'avaluació dels efectes en l'endolimfa de l'estatocist per tècniques de proteòmica es va dur a terme només en Sepia officinalis. La presència de taques diferencials en els gels dels individus control i els sotmesos a exposició a so demostren que les lesions podrien estar relacionades amb un possible desequilibri fisiològic que tindria repercusions en els nivells proteics de l'endolimfa. Les lesions descrites aquí són noves pel que fa a la patologia dels cefalòpodes. Atès que els nivells de soroll de baixa freqüència a l'oceà estan augmentant (per exemple, a causa del transport, la indústria petrolera i les maniobres navals), que el paper dels cefalòpodes en els ecosistemes marins només ha començat a ser entès recentment, i que les dades bioacústiques fiables sobre els invertebrats són escasses, el present estudi i les investigacions futures aportaran una important contribució a l'ús sostenible del medi marí.

A idade e crescimento da lula Doryteuthis plei da região do litoral norte do Estado de São Paulo é descrita com base em 424 exemplares entre 1,58 a 267 mm de comprimento de manto (ML). Os estatólitos foram utilizados como ferramenta de análise de idade, bem como foi realizado um estudo ontogenético de sua morfometria e morfologia. Com o objetivo de analisar e aprimorar a curva e a taxa de crescimento de D. plei, o presente estudo deu ênfase aos indivíduos em estágios iniciais do ciclo de vida. O formato dos estatólitos foi descrito e demonstrou semelhança à de outros loliginídeos, porém com características particulares, suficientes para a identificação da espécie. Houve relação entre ML e o comprimento dos estatólitos (SL), obtido pelo modelo de ajuste logarítmico. A idade dos exemplares amostrados foi estimada e com isto foi possível obter a curva de crescimento (relação idade e ML) da população em estudo, a qual foi ajustada pelo modelo logístico. O mesmo modelo de ajuste houve para os machos, porém para as fêmeas e para os juvenis, o melhor modelo foi o Gompertz. A longevidade da espécie no local de estudo foi estimada em aproximadamente oito meses. A taxa de crescimento e suas variações conforme as estações do ano foram estimadas. Foram evidenciados valores sendo em mm/dia quando em estádio imaturos em relação aos adultos e decrécimo acelerado após atingir 60 mm ML. Foram observadas diferenças da taxa de crescimento entre os sexos quando os exemplares atingem 150 mm de ML. Espécimes nascidos no inverno apresentaram maiores taxas de crescimento em relação aos nascidos em outras estações do ano. Através do retro-cálculo da idade, foi observado que durante todo o ano ocorrem nascimentos de D. plei, em maior frequência no primeiro semestre. É possível estimar a idade de D. plei tendo apenas as medidas de SL ou comprimento dos domos, sendo que para cada relação, o ajuste obtido foi o logarítmico. Os parâmetros de crescimento apresentados no presente estudo oferecem recursos para estudos de avaliação do estoque pesqueiro e biologia populacional. / Age and growth of the squid Doryteuthis plei from the northern shelf off São Paulo are described based on 424 specimens from 1.58 to 267 mm mantle length (ML). Statoliths were used as microstructures for aging, and an ontogenetic study of their morphological and morphometric was performed. The present study aimed to analyze and improve the growth curve and growth rate estimate of individuals of D. plei, emphasizing those in early stages of life cycle. The shape of statoliths was described and demonstrated similarity other loliginids, with particular characteristics sufficient to identify the species. There was a relationship between ML and statoliths length (SL) by the logarithmic model. The age of sampled specimens was estimated and it was possible to obtain the growth curve (relationship between age and ML) of the population in study, was better fitted by the logistic model. The same adjustment was found for males, but for females and juveniles, the Gompertz model was best suitable. The longevity of the species from place of study was estimated at approximately eight months. The growth rate and its variations were estimated according to the seasons, which evidenced higher values in mm/day in immature stage when compared to adults. In addition, individuals after reaching 60 mm ML accelerated a decrease in growth rate. Differences in growth rate between sexes were found after specimens reach 150 mm ML. Specimens born in winter had higher growth rates when compared to those born in other seasons. Through retro-calculation of age, it was observed that births of D. plei occur throughout the year, but most frequently in the first half. The study enables the age estimate of D. plei having only measures of SL or length of the statolith\'s domes. Growth parameters obtained provide information useful for stock assessment and population biology.