O presente projeto analisou o perfil dos ácidos graxos, embriogênese e ontogenia do sistema digestório das larvas da garoupa verdadeira Epinephelus marginatus durante os primeiros dias de desenvolvimento. Além disso, descrevemos como os embriões se dividem, eclodem e desenvolvem suas principais características, e como se desenvolve seu sistema digestório ao longo dos primeiros dias de desenvolvimento, e como utilizam os AG durante os primeiros 8 dias de desenvolvimento. Observamos que as larvas da garoupa apresentam desenvolvimento de suas estruturas digestórias relativamente lento, no entanto, estas são capazes de capturar, ingerir e digerir presas já a partir do 4º DAE. Os ovos da garoupa verdadeira são compostos por elevados percentuais de PUFA nos fosfolipídios e para o processo de eclosão utilizam preferencialmente os SFA dos fosfolipídios. Além disso, os PUFA da série n3 sobrepõe-se aos da série n6, principalmente o DHA, que apresentaram valores elevados em comparação com outras espécies marinhas tanto nos fosfolipídios quanto nos triglicérides nos três primeiros dias de desenvolvimento. As larvas apresentam uma elevada necessidade dos HUFAs DHA/EPA, e durante essa fase é importante a utilização de alimento vivo de tamanho reduzido (copépodes ou “SS strain” Brachionus rotundiformes) enriquecidos com valores da relação entre DHA/EPA acima de 2,0. Com isso, todo o conhecimento gerado durante esses dias de desenvolvimento já permitem a aplicação deste conhecimento no processo de embriogênese e larvicultura da garoupa verdadeira contribuindo para alavancar sua domesticação e produção em cativeiro e elaborar futuramente um programa de repovoamento desta espécie contribuindo para sua conservação. / This project analyzed the fatty acids profile, embryogenesis and ontogeny of the digestive system of dusky grouper Epinephelus marginatus larvae during the first days of development. Furthermore, we described how the embryos are divided, hatch and develop its main features, and how develops its digestive system during the first days of development, and how they use the FA during the first 8 days of development. We observed that the grouper larvae present relative slow development of their digestive structures, however, the larvae are able to capture, ingest and digest preys already from 4º DAE. The dusky grouper eggs are composed by high percentages of PUFA in phospholipids and for the hatching process it uses preferably the SFA of phospholipids. Additionally, the n3 series PUFAs overlaps the n6 series, especially DHA, which exhibited high values compared to other marine species both on phospholipids as in the triglycerides during the first three days of development. The larvae exhibit a high requirement of HUFAs DHA/EPA, and during this phase is important to use live food of small size (copepods or “SS strain” Brachionus rotundiformes) enriched with the ratio of DHA/EPA levels above 2.0. Thus, all the knowledge generated during these days of development allow us the application of this knowledge in the embryogenesis and hatchery process of the dusky grouper contributing to leverage its domestication and production in captivity and eventually draw up a restocking program of this species contributing to their conservation.

The dusky grouper Epinephelus marginatus (Lowe 1834), is a protogynous sequential hermaphrodite and is considered to be one of the most important fish species in the Mediterranean Sea. It is a K-strategist, being slow growing and late maturing, and this, coupled with its reproductive biology and relatively sedentary behaviour, has made it extremely sensitive to overexploitation, leading it to be classified by the IUCN as an endangered fish species. Wild dusky grouper have suffered from disease outbreaks in the past decade, leading to mass mortalities across the Mediterranean Sea, including Libyan coastal waters. These mortalities have mostly been attributed to Nodavirus infections. In Europe and Brazil, efforts are in place to culture this fish for commercial grow-out and stock enhancement programmes. In Libya, the dusky grouper is consumed regularly and is considered a prime-eating fish. Its importance for the Libyan internal market, as well as its potential for export, makes it an ideal candidate for future Libyan aquaculture activities. Given the scarce literature regarding the dusky grouper in Libya, this study aimed first to assess dusky grouper fisheries, spawning seasons and to identify the main threats that the fishing sector poses for wild stocks. Second this study aimed to determine the health status of wild dusky grouper offered at a local fish market in the capital Tripoli, in order to identify pathogens, pathologies or other health issues that might pose a hazard to cultured populations but also to remaining wild dusky grouper stocks. To achieve these aims, twelve field surveys spanning the period of 2013-2015 were conducted. From these surveys, it was established that the dusky grouper is captured throughout the year, including the spawning season. Fish sizes offered for sale ranged between 20-92 cm total length (TL), with the fish being sold from local fishing grounds around Tripoli, but also from as far as Benghazi, 1300 km to the east of Tripoli. The dusky grouper is principally caught in artisanal fisheries and by spearfishing, with approximately 300 spear-fishermen serving one particular fish market in Tripoli that was a focus in this study, and with dusky grouper being one of their main targets. Over the period of the survey, 267 landed dusky grouper were inspected for visible lesions prior to sampling. A total of 50 dusky grouper with sizes ranging from 27- 66 cm TL including the gonads from a further five fish measuring 66-92 cm TL that were sampled separately and examined to assess the stage of sexual maturity and to look for the presence of parasitic infections mainly affecting the gills, skin and gonads. The spawning season was found to extend from May to early September, with females ranging between 39-68 cm TL, males measuring 57-92 cm TL, and transient fish measuring 58-68 cm TL. From otolith readings of 8 fish, the youngest fish was a 3 year old juvenile of 28 cm TL and the oldest was an 8-9 year old 56 cm TL female. Whilst the highest prevalence of parasitic infection was found to be monogenean infection of the gills, with 100% prevalence, followed by gnathiid isopods infecting the oral cavity with 92% prevalence, it was the nematode Philometra sp. infecting post-spawning ovaries at 52% prevalence, that gave the highest apparent pathological impact. Necrosis potentially attributed to Philometra sp. in one particular ovary, was at a level likely to have caused complete parasitic castration, while others showed varying levels of probable functional reduction. The pathologies described need further investigation, especially in relation to possible synergies between Philometra sp. and bacteria in causing the necrosis. From the 267 inspected dusky grouper, 55 fish ranging in size from 42-92 cm TL were observed to be affected by external skin lesions of unknown aetiology. Twenty-six of these fish were sampled, having lesions at various stages of severity, and 5 further unaffected fish were used for histological assessment of the skin as negative controls. Histopathologically, the lesions comprised a multifocal, unilateral or bilateral dermatitis, involving the epidermis, superficial dermis and scale pockets, and sometimes, in severe cases, the hypodermis. Severe lesions had marked epidermal spongiosis progressing to ulceration. Healing was observed in some fish. Bacteria and fungi could be isolated from severe lesions, although they were not seen histopathologically in early-stage lesions. By contrast, metazoan parasite eggs were observed in the dermis and epidermis of some fish with mild and moderate dermatitis. Unidentified gravid digenean trematodes, carrying similar eggs, were also seen within the blood vessels of the deep and superficial dermis. The newly described condition was termed dusky grouper dermatitis (DGD). DGD’s geographical distribution along the Libyan coastline was investigated using a novel application of the social media network Facebook. Using Facebook, it was possible to document skin lesions of dusky grouper in Libyan waters from images attached to the entries of spear-fishermen. Thirty two Facebook accounts and 8 Facebook groups posting from 23 Libyan coastal cities provided a retrospective observational dataset comprising a total of 382 images of dusky grouper caught by spearfishing from December 2011-December 2015. Skin lesions were observable on 57 / 362 fish, for which images were of sufficient quality for analysis, giving a minimal prevalence for lesions of 15.75%. Only dusky grouper exceeding an estimated 40 cm total length exhibited lesions. The ability to collect useful data about the occurrence and geographical distribution of pathological conditions affecting wild fish using social media networks, demonstrates their potential utility as a tool to support epidemiological studies and monitor the health of populations of aquatic animals. The gravid digenean trematode described from mild lesions of five fish was identified using reconstruction through histological sectioning as belonging to the Family Aporocotylidae Odhner, 1912. This is the first description of a blood fluke from the dusky grouper, as well as from dermal blood vessels. The parasite was relatively long; the longest section of the parasite that could be measured was 1500 µm and 20-80 µm in width, while the total length of the parasite was estimated at 1500-2000 µm. Minute tegumental spines, possibly covering only a few parts of the parasite, were seen from some cross-sections. The parasite had one post-testicular ovary, which might overlap the testis, a pre-ovarian ascending uterus, and a post-ovarian descending uterus. It also possessed an oesophagus surrounded by oesophageal glandular cells and a pre-ovarian and pre-testicular extension of the vitelline cells, mostly at the level of the ascending uterus. The parasite was observed to be intra-vascular, the uterine lumen varies in size to accommodate between 1-7 eggs. The uterine eggs were embryonated and observed to span several stages of maturation. Eggs were also found in the dermal blood vessels, in the dermis, and in the epidermis, with the latter appearing to provide a potential route of egress of eggs into the environment. The extra-uterine eggs were 23.5 to 37.52 µm long and contained a ciliated miracidium. The eggs seemed to elicit a mixed inflammatory reaction, with degranulation of eosinophilic granular cells attached to the external surface of some of the eggs within the blood vessels but also the dermis. From observations made in the current study, this parasite appears to be a new species, most closely allied to none of the currently described Aporocotylidae genera. / In summary, the present study has demonstrated that the dusky grouper is extensively fished in Libya without discrimination to sizes and season, by both artisanal and spearfishing, with the latter as one of the main fishing methods, posing treats to the spawning potential and conservation of dusky grouper in Libya. The philometrid infecting the ovaries has a potential to reduce fecundity or to result in parasitic castration of wild broodstock. Gill-infecting monogeneans might represent a hazard for all stages of dusky grouper production. Dusky grouper dermatitis is a skin lesion, although there are no indications that infections may result in mortalities. Under culture conditions, however, this might change due to increase bacterial loads, which might lead to secondary bacterial infection. The presence of skin lesions would undoubtedly reduce the market value of whole fish. These findings are important for existing wild stocks, and for future plans regarding the aquaculture of dusky grouper. Future studies need to focus on the pathology of DGD, describing the disease process and aetiology using laboratory techniques such as TEM and virology as well as using morphology and molecular-based tools to describe the blood fluke and to determine their potential role in the initiation the disease. The novel approach to disease surveillance using social media Facebook posts could be further expanded by attracting citizen scientists, for future research assessing disease in wild fish, for sightings of mortality events and/or the appearance of disease outbreaks, or, for mapping marine mammal stranding’s and/or turtle nesting activity.

O presente projeto analisou o perfil dos ácidos graxos, embriogênese e ontogenia do sistema digestório das larvas da garoupa verdadeira Epinephelus marginatus durante os primeiros dias de desenvolvimento. Além disso, descrevemos como os embriões se dividem, eclodem e desenvolvem suas principais características, e como se desenvolve seu sistema digestório ao longo dos primeiros dias de desenvolvimento, e como utilizam os AG durante os primeiros 8 dias de desenvolvimento. Observamos que as larvas da garoupa apresentam desenvolvimento de suas estruturas digestórias relativamente lento, no entanto, estas são capazes de capturar, ingerir e digerir presas já a partir do 4º DAE. Os ovos da garoupa verdadeira são compostos por elevados percentuais de PUFA nos fosfolipídios e para o processo de eclosão utilizam preferencialmente os SFA dos fosfolipídios. Além disso, os PUFA da série n3 sobrepõe-se aos da série n6, principalmente o DHA, que apresentaram valores elevados em comparação com outras espécies marinhas tanto nos fosfolipídios quanto nos triglicérides nos três primeiros dias de desenvolvimento. As larvas apresentam uma elevada necessidade dos HUFAs DHA/EPA, e durante essa fase é importante a utilização de alimento vivo de tamanho reduzido (copépodes ou “SS strain” Brachionus rotundiformes) enriquecidos com valores da relação entre DHA/EPA acima de 2,0. Com isso, todo o conhecimento gerado durante esses dias de desenvolvimento já permitem a aplicação deste conhecimento no processo de embriogênese e larvicultura da garoupa verdadeira contribuindo para alavancar sua domesticação e produção em cativeiro e elaborar futuramente um programa de repovoamento desta espécie contribuindo para sua conservação. / This project analyzed the fatty acids profile, embryogenesis and ontogeny of the digestive system of dusky grouper Epinephelus marginatus larvae during the first days of development. Furthermore, we described how the embryos are divided, hatch and develop its main features, and how develops its digestive system during the first days of development, and how they use the FA during the first 8 days of development. We observed that the grouper larvae present relative slow development of their digestive structures, however, the larvae are able to capture, ingest and digest preys already from 4º DAE. The dusky grouper eggs are composed by high percentages of PUFA in phospholipids and for the hatching process it uses preferably the SFA of phospholipids. Additionally, the n3 series PUFAs overlaps the n6 series, especially DHA, which exhibited high values compared to other marine species both on phospholipids as in the triglycerides during the first three days of development. The larvae exhibit a high requirement of HUFAs DHA/EPA, and during this phase is important to use live food of small size (copepods or “SS strain” Brachionus rotundiformes) enriched with the ratio of DHA/EPA levels above 2.0. Thus, all the knowledge generated during these days of development allow us the application of this knowledge in the embryogenesis and hatchery process of the dusky grouper contributing to leverage its domestication and production in captivity and eventually draw up a restocking program of this species contributing to their conservation.