Genes present in multiple copies and genes that contain regions of repetitive sequences can undergo concerted evolution, which results in homogenization of the nucleotide sequence of the genes or repetitive regions. In regions of tandem repeats, this occurs through misalignment of repeat units followed by unequal crossover, which generates two products with differing numbers of repeat units. Gene conversion is thought to lead to one of these products becoming fixed in a species. The homogenous sequence of previously studied genes that have been thought to undergo this process has made it difficult to determine the exact models involved. Here I examine concerted evolution in SM50, a sea urchin gene that encodes a protein involved in biomineralization. The repetitive region in the SM50 gene varies in length between species, and there is variability in each repeat unit as well. I examine the codon usage in SM50 in a variety of species, and discuss how purifying selection, substitutions, concerted evolution, and selection at the level of DNA sequence have played a role in the evolution of this gene. I also examine the structure and sequence of the repeat units, and purpose models that have led to the evolution of the repeat pattern seen in the different species examined. Finally, I have found variation in the number of repeat units within several species. This has allowed us to deduce the specific models of unequal crossover that led to this variation. The unique variation in the repetitive region of SM50 has enabled us to describe a model of how substitutions affect the model of misalignment and unequal crossover.

Research and development in aquaculture has supported the knowledge-based development of the sector over the last decades. In particular, species diversification is playing an important role to ensure sustainability of the industry and helping to reduce pressure on wild stocks of those aquatic species for which farming technology is still at the early stages. Due to the increasing pressures on more traditional carnivorous marine finfish species (aquafeed reliance on fishmeal and fish oil, environmental impact, market price) low trophic organisms are receiving more attention to provide sustainable alternatives and integrate production activities with the aim of reducing environmental impacts and to provide secondary high value crops. Integrated Multi-Trophic Aquaculture (IMTA) systems are therefore at the forefront of innovation in the industry. Several invertebrate species have been investigated and tested as integral part of IMTA (mussels, oysters, abalone and macroalgae) and echinoderms have also been considered as good candidates for the future development of this technology. In order to allow for a more widespread uptake of integrated aquaculture, several technical and biological challenges need to be overcome, including a reliable supply of juveniles. In recent years, this has prompted investigation on Echiniculture as a whole and on hatchery technologies in particular. This PhD investigated key constraints in edible sea urchin (Paracentrotus lividus) juvenile production with the aim to improve commercial sea urchin hatchery outputs. The research firstly focused on larval nutrition (Chapter 3 and 4) and specifically tested the hypothesis that larvae required higher dietary inputs of long chain fatty acids than those provided by Dunaliella tertiolecta, a microalgae species widely used in echinoderm larval rearing. Fatty acid composition of P. lividus eggs, investigated in Chapter 3, supported this hypothesis, which was further confirmed by the results obtained in Chapter 4 where microalgae (Cricosphaera elongata, Pleurochrisis carterae and Tetraselmis suecica) with a more balanced fatty acid profile, in particular richer in long chain fatty acids, were employed. This resulted in a significantly improved larval development and survival. Results also indicated that these alternative microalgae species could be successfully grown without modification of the microalgae production protocols in the hatchery where the experimentation had taken place. The third experimental chapter compared static and flow through systems which provides more stable water quality through constant water exchange and reduces larval handling and associated stress. Results indicated that larval survival was significantly improved by the flow-through system and the need for tank cleaning was reduced (three versus seven times per larval cycle when using flow-through and static rearing systems respectively). However, water quality, based on the parameters assessed (NH4, PO4-3, NO2 and NO3), did not show any significant differences between systems. Reduced handling could have therefore played the most important role in promoting larval survival. Both these trials resulted in a significant 5 to 20 % increased survival. A follow-up study, combining flow-through with more suitable microalgae, should be carried out and could result in even further enhanced survival. Then, chapters 6 and 7 focused on broodstock nutrition and subsequent improvement of gamete quantity and quality. These two trials aimed to explore and describe the biological effects that some important nutrients, such as proteins, lipids, fatty acids and carotenoids, have on urchins’ somatic and gonadal growth, gonad biochemical composition during gametogenesis, fecundity and maternal provisioning to developing embryos. Results from the experiment described in Chapter 6 indicated that higher protein content can improve somatic growth in P. lividus adults and that more expensive, protein-, lipid- and energy-rich diets do not significantly enhance fecundity or offspring performance. Results, moreover, highlighted the need for a specifically formulated broodstock diet and gave some insights into what its composition should be, especially in relation to carotenoids. In Chapter 7, fatty acid profiles of P. lividus gonads throughout gametogenesis were studied for the first time. It was observed that, among Long Chain Polyunsaturated Fatty Acids (LC-PUFAs), Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) are primarily accumulated during gametogenesis, whilst Arachidonic acid (ARA) appears to be independent of dietary input. In addition, it was clearly shown that ARA is the only LC-PUFA accumulated in the eggs along with Non Methylene Interrupted Fatty Acids (NMI FAs). As well as looking at the biological effects of different diets on fatty acid profiles of gonadal and larval tissues, the work also expanded on a more fundamental level to explore the metabolic pathway through which precursors could be used by sea urchins for the endogenous production of long chain fatty acids (Chapter 8). Three Expressed Sequence Tags (ESTs) for putative fatty acyl desaturases, one of which was closely related to Octopus vulgaris ∆5-like fatty acyl desaturase, were identified. The newly cloned putative desaturase of P. lividus possessed all typical features of other fatty acyl desaturases. However, because of time constraints, functional characterisation, originally planned, of the new protein could not be performed and further research effort is needed to investigate this important aspect of sea urchin physiology. Overall, the aim of this research project has been achieved as it provided a set of exploitable results and protocols to improve hatchery practices for the production of P. lividus juvenile. However, more research is required to investigate some of the underlying mechanisms behind the observed biological effects such as delay in larval development when T. suecica was used as larval feed, increased broodstock fecundity, improved larval survival in the flow-through system and higher gonadal concentration of some fatty acids (mainly DHA) than provided in the feed.

Σκοπός της συγκεκριμένης ερευνητικής εργασίας ήταν η αποκάλυψη του ρόλου και της θέσης του PlCOUP-TF στο ρυθμιστικό γονιδιακό δίκτυο της νευρογένεσης του αχινού Paracentrotus lividus, αλλά και η διερεύνηση των επιδράσεων του συγκεκριμένου μεταγραφικού παράγοντα στα υπόλοιπα γονίδια που εμπλέκονται στην δημιουργία νευρώνων. Ο PlCOUP-TF είναι ένας μητρικός μεταγραφικός παράγοντας και ορφανός πυρηνικός υποδοχέας που φαίνεται να διαδραματίζει σημαντικό ρόλο στην δημιουργία νευρώνων σε μια ποικιλία οργανισμών. Τα υπό μελέτη γονίδια της νευρογένεσης είναι επίσης μεταγραφικοι παράγοντες με εξελικτικά συντηρημένες επικράτειες. Για τον προαναφερθέντα σκοπό, πραγματοποίηθηκαν πειράματα διπλής φθορίζουσας in situ υβριδοποίησης ώστε να αποκαλυφθούν οι περιοχές συνεντοπίσμου του PlCOUP-TF και των γονιδίων της νευρογένεσης σε ώριμα στάδια της ανάπτυξης. Επίσης, διεξήχθησαν πειράματα καταστολής της μητρικής έκφρασης του PlCOUP-TF με ενέσεις με ΜΑSO σε γονιμοποιημένα ωάρια και έλεγχος της έκφρασης των νευροειδικών γονιδίων στα ενεμένα και στα αντίστοιχα control έμβρυα. Ο έλεγχος της έκφρασης ήταν τόσο ποιοτικός (in situ χρωμογόνος υβριδοποίηση) όσο και ποσοτικός (Q-PCR). Με τα πειράματα αυτά, αποκαλύφθηκε ότι πιθανότατα ο συγκεκριμένος μεταγραφικός παράγοντας λειτουργεί ενεργοποιητικά για τα εν λόγω γονίδια. Συγκεκριμένα, φαίνεται η ύπαρξη κατασταλτικής δράσης του PlCOUP-TF πάνω σε καταστολέα των γονιδίων της νευρογένεσης (double negative gate) στην περιοχή του εμπρόσθιου νευροεξωδέρματος και σε συγκεκριμένες περιοχές της βλεφαριδωτής ζώνης. / Our aim was to identify the role of the orphan nuclear receptor PlCoup-TF in sea urchin embryonic neurogenesis and especially in the determination of the anterior neuroectoderm (ANE). To this end, we cloned a set of embryonic cDNAs encoding regulatory proteins expressed specifically in the ANE and we prepared antisense RNA probes for double fluorescence in situ hybridization for the following genes: PlCoup-TF, PlHbn, Plz81 and PlFoxG. We studied the spatial pattern of expression of ANE genes in conjunction with the expression pattern of PlCoup-TF in all embryonic stages of the sea urchin Paracentrotus lividus. A neurogenic territory specified within the animal pole of the embryo, is formed as a result of the interplay of the aforementioned regulatory factors that together constitute a sub-circuit within the embryonic gene regulatory network (GRN). We wanted to determine PlCoup-TF’s place within the GRN and specifically the ANE sub-circuit. Therefore, we knockdowned PlCoup-TF expression during embryogenesis by injecting specific morpholino antisense oligonucleotides (MASO) into sea urchin eggs and determine the expression pattern of the ANE specific genes by chromogenic in situ hybridization to resulting morphant embryos. The efficiency of the knockdown was measured by QPCR, where the amount of PlCoup-TF transcripts of morphants is compared to that of control embryos. Finally, we concluded that probably PlCoup-TF activate the ANE genes, by repressing repressor(s) of these genes (double negative gate) in ANE and in specific regions of CBE.

O Coup¬TF, αποτελεί ορφανό μέλος της υπεροικογένειας των υποδοχέων των στεροειδών/θυρεοειδών ορμονών και κατέχει κυρίαρχο ρόλο στην ανάπτυξη των εμβρύων όλων των μεταζώων. Στην παρούσα Διατριβή μελετήθηκε η cis¬ ρυθμιστική περιοχή του γονιδίου του, με σκοπό την ένταξή του στο γονιδιακό ρυθμιστικό δίκτυο του εμβρύου του αχινού. Με πειράματα in situ υβριδοποίησης βρέθηκε ότι το γονίδιο PlCoup¬TF εκφράζεται στο στοματικό εξώδερμα του γαστριδίου και στη βλεφαριδωτή ζώνη στον πλουτέα, στο είδος Paracentrotus lividus. Από παλαιότερα πειράματα είχε βρεθεί ότι το τμήμα της ανοδικής περιοχής που εκτείνεται από το -232 ως το ¬532 (τμήμα a), είναι απαραίτητο και επαρκές για την έκφραση του γονιδίου αναφοράς (gfp) στη βλεφαριδωτή ζώνη του πλουτέα. Εντός της περιοχής a ανευρέθησαν τρία πιθανά ρυθμιστικά στοιχεία (¬ 453, ¬432 και ¬377) του γονιδίου PlCoup¬TF, τα οποία αναγνωρίζονται από πρωτεΐνες εμβρυικού πυρηνικού εκχυλίσματος. Στοχευμένες μεταλλάξεις των στοιχείων αυτών, οδήγησαν σε μείωση της έκφρασης του γονιδίου αναφοράς (στοιχείο ¬453) και στην εκτοπική έκφρασή του (στοιχεία ¬432 και ¬377). Περαιτέρω μελέτη των παραγόντων που αναγνωρίζουν τα στοιχεία αυτά, οδήγησε στο συμπέρασμα ότι ο μεταγραφικός παράγοντας PlElk αναγνωρίζει το στοιχείο ¬ 453 και ρυθμίζει θετικά το γονιδίο του PlCoup¬TF και ο μεταγραφικός παράγοντας PlOtx αναγνωρίζει το στοιχείο -377 και καταστέλλει την έκφραση του PlCoup¬TF στο αντιστοματικό εξώδερμα. Τα αποτελέσματα της παρούσης εργασίας οδήγησαν στην ένταξη του γονιδίου PlCoup¬TF και των δύο ρυθμιστών του στο γονιδιακό ρυθμιστικό δίκτυο που καθορίζει τη διαφοροποίηση της βλεφαριδωτής ζώνης εντός του εμβρυικού εξωδέρματος. / Coup­TF, an orphan member of the nuclear receptor super family, has a fundamental role in the development of metazoan embryos. The study of the gene's regulatory circuit in the sea urchin embryo will facilitate the placement of this transcription factor in the well­studied embryonic Gene Regulatory Network (GRN). The Paracentrotus lividus Coup­TF gene (PlCoup­TF) is expressed throughout embryonic development preferentially in the oral ectoderm of the gastrula and the ciliary band of the pluteus stage. Two overlapping λ genomic clones, containing three exons and upstream sequences of PlCoup­TF, were isolated from a genomic library. The transcription initiation site was determined and 5′ deletions and individual segments of a 1930 bp upstream region were placed ahead of a GFP reporter cassette and injected into fertilized P.lividus eggs. Module a (−532 to −232), was necessary and sufficient to confer ciliary band expression to the reporter. Comparison of P.lividus and Strongylocentrotus purpuratusupstream Coup­TF sequences, revealed considerable conservation, but none within module a. 5′ and internal deletions into module a, defined a smaller region that confers ciliary band specific expression. Putative regulatory cis­acting elements (RE1, RE2 and RE3) within module a, were specifically bound by proteins in sea urchin embryonic nuclear extracts. Site­specific mutagenesis of these elements resulted in loss of reporter activity (RE1) or ectopic expression (RE2, RE3). It is proposed that sea urchin transcription factors, which bind these three regulatory sites, are necessary for spatial and quantitative regulation of the PlCoup­TF gene at pluteus stage sea urchin embryos. Additional experiments led us to the conclusion that transcription factor PlElk binds to the ­453 regulatory element and positively regulates PlCoup­TF gene in the ciliary band. Furthermore, PlOtx binds to the ­377 regulatory element and negatively regulates PlCoup­TF gene in the aboral ectoderm. These findings lead to the hierarchical positioning of PlCoup­TF within the embryonic GRN

<p>Changes in the expression and function of genes active during metazoan development have played a critical role in the evolution of morphological differences between species and phyla, yet the origins of these changes remain poorly understood. What roles do positive and negative selection play in the evolution of development? How do evolutionary changes accumulate given the degree to which organisms are able to buffer the effects of environmental and genetic perturbations during development? The crucial insight of the Modern Evolutionary Synthesis was that divergence between species arises from variation within populations. Following this principle, I have made use of tools from quantitative and population genetics to investigate three central questions: 1) How much genetic variation is there in the networks of genes that underlie metazoan development? 2) What affect does developmental buffering have on the accumulation of selectable genetic variation? 3) To what extent does selection act to shape patterns of genetic variation among different kinds of genes and at different stages of development? I show that developmental systems can harbor extensive levels of genetic variation, and that the amount of genetic variation in individual genes at different stages of development is related to the extent to which variation in those genes is buffered by genetic interactions. I also show that while selection plays an active role in shaping genetic variation in development, the extent to which variation in a gene is visible to selection depends in predictable ways on a) the biological function of that gene and b) whether the mutations in question influence gene expression or protein function. My results as a whole demonstrate the utility of population level approaches to the study of the evolution of development, and provide key insights into the role that selection plays in generating developmental variation.</p> / Dissertation

<p>Many factors, including gene networks, developmental processes, and the environment mediate the link between the activity of genes and complex phenotypes in higher organisms. While genetic variants are the raw material for evolution, these other factors are critical for determining which variants are actually exposed to natural selection. In this dissertation, I describe three projects in which I investigate how developmental mechanisms and the environment interact to shape phenotypic variation. In each project, I use gene expression as a window into the activity of genes, and as a tool to measure variation in and among developmental mechanisms. Two projects are experimental, focusing on early development in sea urchins, and how environmental stress caused by climate change impacts the expression of genetic variation in phenotypic traits. In these projects, I explicitly incorporate information about the biochemical functions of genes and how they interact in development, and test how such mechanisms shape the impact of genetic and environmental perturbations to development. The third project is methodological, in which I propose a unified statistical framework for inferring previously unknown developmental constraints that may underlie gene expression phenotypes. Together, these projects demonstrate that an understanding of developmental mechanisms can enhance our understanding of the processes that shape variation in populations, and can help predict the biological effects of climate change.</p> / Dissertation

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-08T12:11:24Z No. of bitstreams: 1 arquivototal.pdf: 1391577 bytes, checksum: e8bbd0db33d228b40d69b547aed31f9c (MD5) / Made available in DSpace on 2017-09-08T12:11:24Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1391577 bytes, checksum: e8bbd0db33d228b40d69b547aed31f9c (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Human activities have caused climate changes and altered the salinity of the oceans. Salinity is one of the factors that limit the distribution and the survival of marine organisms. Coelomocytes are the immune system cells of the echinoderms and have been studied as biomarkers in stress situations. The aim of the present study was to investigate the effect of the salinity in the immune system cells of the tropical sea urchin Echinometra lucunter. Animals were collected in João Pessoa coast (Brazilian Northeast). Animals or coelomocytes were exposed to different salinity (25‰ to 45‰) and phagocytic parameters, production of reactive oxygen species (ROS), mitochondrial activity and ABC transporter activity analyzed. The phagocytic parameters did not change when animals or cells were exposed to low or high salinity in any time intervals monitored. However, our data showed an increase in the coelomocytes concentration when animals were exposed to 25‰. ROS levels were higher when cells were incubated at 25‰ and lower when cells were cultured at 45‰. We noted a loss of the mitochondrial inner membrane potential when coelomocytes were incubated at 45‰. The activity of ABC transporters decreased when cells were incubated at low salinity and increased when cells were incubated at high salinity. Our work shows that the immune system of the tropical sea urchins E. lucunter tolerates salinity changes from 25‰ to 45‰ and suggests two cellular parameters (ROS levels and ABC transporters activity) as potential biomarkers on the monitoring of the impact of environmental salinity changes. / As atividades humanas têm causado mudanças climáticas e alterado a salinidade dos oceanos. A salinidade é um dos fatores que limitam a distribuição e sobrevivência de organismos marinhos. Celomócitos são as células do sistema imune dos equinodermos e têm sido estudados como biomarcadores em situações de estresse. O objetivo do presente estudo foi investigar o efeito da salinidade em celomáticos do ouriço-do-mar tropical Echinometra lucunter. Os animais foram coletados na costa de João Pessoa (Nordeste do Brasil). Os animais ou os celomócitos foram expostos a diferentes salinidades (25‰ e 45‰) e parâmetros fagocíticos, produção de espécies reativas de oxigênio (ROS), atividade mitocondrial e atividade dos transportadores ABC analisados. Os parâmetros fagocíticos não alteraram quando os animais ou as células foram expostos a 25‰ ou 45‰ nos intervalos de tempo monitorados. Porém, foi observado um aumento na concentração de celomócitos quando os animais foram expostos a 25‰. Os níveis de ROS foram maiores quando as células foram incubadas a 25‰, e menores quando as células foram cultivadas a 45‰. Foi observada uma perda do potencial de membrana mitocondrial interna quando os celomócitos foram incubados a 45‰. A atividade dos transportadores ABC diminuiu quando as células foram incubadas a 25‰ e aumentou quando as células foram incubadas a 45‰. O presente trabalho demonstra que o sistema imune do ouriço-do-mar E. lucunter tolera mudanças de salinidade (25‰ até 45‰), e sugere dois parâmetros celulares (níveis de ROS e atividade de transportadores ABC) como potenciais biomarcadores no monitoramento de mudanças na salinidade ambiental.

For decades, humans have impacted marine ecosystems in a variety of ways including contamination by pollution, fishing, and physical destruction of habitats. Global change has, and will, lead to alterations in in a number of abiotic factors of our ocean in particular reduced oxygen saturation, salinity changes, elevated temperature (ocean warming or OW) and elevated carbon dioxide (ocean acidification or OA). Now and in the future, OA and OW will operate together with local anthropogenic drivers such as oil pollution. And yet, at present, very little is known about their potential combined interactive effects on physiological performance and tolerance of marine organisms. Therefore, multiple driver experiments are required if we are to understand and predict future vulnerability of species, populations and ecosystems. Early life stages of invertebrates are generally considered most vulnerable to environmental stress. However, few studies consider the combined effects OA and OW on survival and growth during early development of marine invertebrates, and to our knowledge, there is no information on the additional effects of oil pollution. Therefore, the aim of this thesis was to investigate the effects of combined exposure to OA, OW, and incorporating local drivers such as oil pollution on the development, morphology and physiology of three economically and ecologically important marine invertebrates. These are Northern shrimp Pandalus borealis, Northern krill Meganyctiphanes norvegica, and the green sea urchin Strongylocentrotus droebachiensis. All are cold-water species, assumed to have a narrower tolerance than more temperate species, and so could be particular sensitive to combined stressor affects. Both Northern krill and to a lesser extent Northern shrimp larvae survived experimental conditions, mirroring those predicted under a future global change scenario (combined OA and OW exposure). Neither was hatching success affected. Both shrimp and krill larvae exhibited accelerated developmental rates and incurred greater maintenance costs as a result of exposure to these stressors. Shrimp larvae showed accelerated developmental rates (-9 days), increased metabolic rates (+20 %), and increased feeding rates (+20 %), but reduced growth (- 9 %) when exposed to OW compared with the control. OA increased development rate but only at the control temperature. Although juvenile mortality of krill was not affected by predicted OA/OW conditions, metabolic rate increased significantly (+ 36 %), as did larval developmental rate, while number of moults, feeding rate and growth (- 67 %) decreased significantly (- 67 %, - 60 % and -8 % respectively). Accelerated development was accompanied by greater maintenance costs possibly due to experience a mismatch between energy supply and demand. Both species had an excess of food, and so growth reduction was more likely to be associated with higher metabolic demands in the future global change treatments. Food shortage in situ, due to variable food availability in the sea and/or mismatch with key prey species (algae and zooplankton) could result in more negative effects on growth and ultimately survival. Green sea urchins were also able to survive OA exposure, without detectable effects on hatching success. However, at day 44 post-fertilization, larval body length in the OA treatment was 9 % lower compared to the control. Furthermore, there was a significant tendency of urchin larvae to increase swimming activity in the OA conditions that might indicate compensatory feeding. Elevated maintenance and repair costs as a result of exposure to multi-stressors affected the energy budget of all the three species studied here resulting in reduced growth. Global drivers (OA and OW) resulted in trade-offs with more energy reallocated to swimming activity and metabolism, rather than growth. Exposure to oil reduced the acquisition of energy by reduced feeding which in turn resulted in less energy being available for growth. Both shrimp and sea urchin larvae showed reduced activity and feeding when exposed to oil. It is possible that the reduced swimming activity observed may be due to a narcotic effect of the oil. Furthermore, early stage sea urchin larvae showed increased mortality when exposed to oil while the older larvae did not, indicating a stage specific toxicity to oil for sea urchin larvae. The combination of global drivers and oil pollution acted additively on growth for both sea urchin and shrimp larvae. The impact of combined drivers on the size of shrimp larvae was equal the sum of the negative impacts observed for each driver: a 5 % reduction when exposed to OA and OW, a 9 % reduction when exposed to oil, and a cumulative 15 % reduction when exposed to all stressors. Similarly, the impact of combined drivers on the size of sea urchin larvae was equal to the sum of the negative impacts observed for each driver: a 14 % reduction when exposed to OA, a 9 % reduction when exposed to oil, and a 21 % reduction when exposed to all drivers. Therefore, the study demonstrated the additive physiological effects of OA, OW and a contaminant, and indicated that larval (sea urchin and shrimp) resilience to future changes (i.e. pollution) could be greatly reduced if larvae were already energy limited and severely stressed (reduced development) as a result of exposure to the global drivers. This study therefore shows the importance that the effective management of local drivers such as oil pollution could have against the backdrop of OA and OW, and emphasises that it is important to study impacts of toxicants, such as an oil pollution, in the context of predicted changes in the environment, as OW and OA are becoming major concerns. Finally, the fact that some local and global drivers seem to act additively should encourage local managers to act on local driver regulations, to obtain positive effects on local populations and environment and thereby rendering them more resilient to the negative impacts of future global drivers.

Earth’s population is expected to reach 9 billion by 2050. Ensuring food security for the growing world population is one of today’s society’s major challenges and responsibilities. Aquatic products have the potential to contribute significantly in the growing population’s dietary requirements. Since increasing the pressure on most natural fish stocks is now widely agreed not to be an option, the aquaculture sector needs to grow. The challenge is to increase aquaculture production without depleting natural resources or damaging the environment but also in a financially sustainable way. Integrated Multitrophic Aquaculture (IMTA) is one method of sustainable aquatic production. Integrating bioremediatory organisms that extract particulate organic matter or dissolved inorganic nutrients with monocultures of fed species has the potential of reducing the particulate and soluble waste loads from effluents, whilst producing a low-input protein source that may also increase the farm income. IMTA is a viable solution for mitigating the environmental impact of waste released from fish farms. The fish waste is exploited as a food source for lower trophic, extractive organisms giving an added value to the investment in feed. Studies up to now have shown that under experimental conditions as well as in small-scale commercial studies, various filter-feeding, deposit-feeding and grazing species can ingest fish waste particles. The aim now is to achieve IMTA optimization, where extractive organisms can ingest most of the finfish waste food and excretions. Any such design is likely to be complex incorporating a multidisciplinary approach, and therefore to date a reason why most studies have failed to prove the environmental and economic benefits of IMTA. Consequently, the aim of this study is to develop ways of selecting an ideal combination of species for a specific locality, manage the cultures in a way that ensures the maximum nutrient recycling feasible per unit of area; and ensure high growth rate of the extractive organisms while being financially beneficial. The approach taken was a combination of investigative literature reviews, computer modelling work and small-scale growth trials to determine the relative growth of extractive organisms fed fishfeed and waste, followed by the development of a systems-based model of interaction and growth efficiency for combinations of organisms within an IMTA system. This study starts by investigating, with small-scale laboratory experiments, the potential of two organic extractive species, the lugworm, Arenicola marina and the sea urchin, Psammechinus miliaris, as organic extractive components of IMTA systems. Their ability to consume and assimilate salmon faeces was evaluated as well as their remediation efficiency. This was done by comparing the carbon, nitrogen and phosphorus content of the pellet-faeces mixture to that of the sea urchin faeces and sea urchin gonad content. Their growth, gonadosomatic index (GSI) (for the sea urchins), tissue carbon, nitrogen and phosphorous content were compared between seaweed diets and a diet consisting of a mixture of salmon faeces and feed pellets. The results showed statistically significant gonad carbon content for the sea urchins fed with faeces. Similarly, statistically significant higher phosphorous content was found in the tissues of the lugworms fed with the mixture of salmon faeces and pellets than in the lugworms of the other two groups. The subsequent and main phase of this study was the development of a model for optimising IMTA performance. The modelling process included model development, run, optimization and risk assessment. The IMTA model developed consisted of Atlantic salmon Salmo salar, the sea urchin Paracentrotus lividus and the macroalgae Ulva sp. It simulates the growth as well as the uptake and release of nitrogen by these organisms under environmental conditions of a hypothetical site on the west coast of Scotland. The aim of the model was to maximize the potential of IMTA in terms of productivity and to reduce the amount of nutrients that are released in the environment, and thus to contribute towards a more sustainable and productive form of aquaculture. The IMTA model developed can be re-parameterised to simulate the growth and nutrient uptake of different species and the growth and nutrient uptake under different environmental conditions. This capacity of the model was used in order to do a comparative study of the nitrogen bioremediation potential of three different invertebrate species, cultivated as part of an IMTA. These species were the lugworm (Arenicola marina), the blue mussel (Mytilus edulis) and the purple sea urchin (Paracentrotus lividus). The results of this comparative study showed that weight for weight, M. edulis is more efficient in removing POM than P. lividus that is in turn better than A. marina with regard to the amount of nitrogen they can assimilate. But in terms of cultivation area required for the production of the same total biomass, P. lividus was better at removing POM followed by M. edulis and then by A. marina.

Globally, human populations are increasing and coastal ecosystems are becoming increasingly impacted by anthropogenic stressors. As eutrophication and exploitation of coastal resources increases, primary producer response to these drivers becomes a key indicator of ecosystem stability. Despite the importance of monitoring primary producers such as seagrasses and macroalgae, detailed studies on the response of these benthic habitat components to drivers remain relatively sparse. Utilizing a multi-faceted examination of turtle-seagrass and sea urchin-macroalgae consumer and nutrient dynamics, I elucidate the impact of these drivers in Akumal, Quintana Roo, Mexico. In Yal Ku Lagoon, macroalgae bioindicators signified high nutrient availability, which is important for further studies, but did not consistently follow published trends reflecting decreased δ15N content with distance from suspected source. In Akumal Bay, eutrophication and grazing by turtles and fishes combine to structure patches within the seagrass beds. Grazed seagrass patches had higher structural complexity and productivity than patches continually grazed by turtles and fishes. Results from this study indicate that patch abandonment may follow giving-up density theory, the first to be recorded in the marine environment. As Diadema antillarum populations recover after their massive mortality thirty years ago, the role these echinoids will have in reducing macroalgae cover and altering ecosystem state remains to be clear. Although Diadema antillarum densities within the coral reef ecosystem were comparable to other regions within the Caribbean, the echinoid population in Akumal Bay was an insufficient driver to prevent dominance of a turf-algal-sediment (TAS) state. After a four year study, declining coral cover coupled with increased algal cover suggests that the TAS-dominated state is likely to persist over time despite echinoid recovery. Studies on macroalgal diversity and nutrients within this same region of echinoids indicated diversity and nutrient content of macroalgae increased, which may further increase the persistence of the algal-dominated state. This study provides valuable insight into the variable effects of herbivores and nutrients on primary producers within a tropical coastal ecosystem. Results from this work challenge many of the currently accepted theories on primary producer response to nutrients and herbivory while providing a framework for further studies into these dynamics.