<p>Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development. </p><p> The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.</p><p> This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control. </p><p>In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.</p><p> In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.</p><p> The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.</p> / Dissertation

p. 1-82 / Submitted by Santiago Fabio (fabio.ssantiago@hotmail.com) on 2013-04-10T20:04:49Z No. of bitstreams: 1 Dissert seg01.pdf: 499245 bytes, checksum: 0655b17411b2a8d56e0ae1742341678e (MD5) / Rejected by Alda Lima da Silva(sivalda@ufba.br), reason: Corrigir Instituto de Biologia on 2013-04-15T20:24:41Z (GMT) / Submitted by Santiago Fabio (fabio.ssantiago@hotmail.com) on 2013-04-23T20:51:38Z No. of bitstreams: 1 Dissert seg01.pdf: 499245 bytes, checksum: 0655b17411b2a8d56e0ae1742341678e (MD5) / Approved for entry into archive by Alda Lima da Silva(sivalda@ufba.br) on 2013-05-03T20:58:38Z (GMT) No. of bitstreams: 1 Dissert seg01.pdf: 499245 bytes, checksum: 0655b17411b2a8d56e0ae1742341678e (MD5) / Made available in DSpace on 2013-05-03T20:58:38Z (GMT). No. of bitstreams: 1 Dissert seg01.pdf: 499245 bytes, checksum: 0655b17411b2a8d56e0ae1742341678e (MD5) Previous issue date: 2005 / Inúmeras substâncias químicas que compõem os produtos manufaturados e estão presentes nos efluentes de origem industrial, são corriqueiramente dispostas no meio ambiente através do carreamento para corpos d´água naturais. Os testes de toxicidade aquática são empregados com o intuito de avaliar o efeito destes agentes químicos nos organismos, fornecendo assim uma abordagem ecológica complementar às analises químicas normalmente empregadas. Para serem validados os testes ecotoxicológicos precisam ser realizados seguindo protocolos específicos e fazerem uso de substâncias de referência, que são químicos de efeito conhecido sobre os organismos de maneira a atestarem a confiabilidade dos resultados obtidos. O objetivo deste trabalho foi avaliar a utilização das substâncias orgânicas Fenol e 4-Clorofenol, como alternativas para referência em testes ecotoxicológicos com embriões do ouriço-do-mar Echinometra lucunter. Para isto, quinze testes crônicos de curta duração foram conduzidos para cada substância investigada, mais o Dodecil Sulfato de Sódio (DSS), que foi utilizado como controle positivo. Os valores de CE50-36h encontrados variaram de 125,88 a 143,84 mg/L para o Fenol e de 16,90 a 26,31 mg/L para o 4-Clorofenol. A concentração de efeito alternativo foi determinada com base na DMS crítica. O nível equivalente à concentração estimada de efeito crônico foi CE10. A partir de tal concentração é possível observar efeitos deletérios significativos na população do organismo-teste. O valor médio de CE10-36h encontrado para o Fenol foi de 36,63 mg/L e para o 4- Clorofenol foi de 4,58 mg/L. Os coeficientes de variação dos resultados obtidos indicaram que os valores encontrados para o Fenol no nível de efeito de 50% apresentam maior precisão analítica (4,04%), enquanto que no nível de efeito alternativo, o 4-Clorofenol apresentou maior precisão dentre as substâncias utilizadas (27,98%). A plotagem em carta-controle, dos resultados obtidos com os testes utilizando o DSS, que foram realizados em paralelo à outras substâncias, atestaram a saúde dos organismos utilizados e a obtenção de resultados confiáveis nos experimentos conduzidos. Embriões de E. lucunter são mais sensíveis ao 4- Clorofenol do que ao Fenol, uma vez que sob as mesmas concentrações, o valor de CE50-36h obtido para o Fenol foi mais alto que o obtido para o 4-Clorofenol. Aliadas às características peculiares a cada substância, o 4-Clorofenol apresentou-se como mais indicado para a utilização como referência em testes ecotoxicológicos com embriões de E. lucunter. / Salvador

O aquecimento global é uma realidade e seus efeitos são bastante estudados atualmente. No entanto, pouca atenção tem-se dado para as alterações que ocorrem com invertebrados marinhos em decorrência dessa alteração climática. Sabe-se que uma das alterações que ocorrem em consequência do aumento da temperatura da água do mar, é o aumento do aparecimento de doenças no ambiente marinho. Para tanto, é importante que se avalie sua resposta imune inata frente a esse fator estressor. Os ouriços-do-mar foram escolhidos como modelo por serem considerados bons biondicadores ambientais, além de serem filogeneticamente próximos aos cordados, partilhando com estes, amplo repertório de genes e receptores envolvidos no sistema imune inato. O presente trabalho avaliou a resposta imune inata de ouriços-do-mar antárticos (S. neumayeri) e tropicais (L. variegatus e E. lucunter) frente ao estresse térmico por diferentes temperaturas e períodos de exposição. No que se refere aos ouriços-do-mar tropicais, foram estudadas duas espécies, uma que habita regiões menos submetidas a variações de marés (L. variegatus) e outra constantemente exposta a variações de maré (E. lucunter). Constatou-se uma diferença entre a resposta ao estresse térmico nas três espécies estudadas. A espécie antártica demonstrou alterações mais significativas de aumento na porcentagem de esferulócitos vermelhos (EV) e da capacidade fagocítica no período agudo de exposição (24h) quando submetidos a temperaturas intermediárias. Por outro lado, a espécie tropical E. lucunter apresentou alteração somente na porcentagem de EV no período de exposição crônico (7 e 14 dias) a temperaturas altas, enquanto que L. variegatus apresentou alteração neste tipo celular em todas as temperaturas experimentais e períodos avaliados, além de uma importante redução da capacidade fagocítica nos mesmos períodos que foi diretamente proporcional ao aumento da temperatura. Buscando compreender tais diferenças entre animais que habitam uma mesma região, verificou-se que a espécie L. variegatus apresentou alteração na adesão e espraiamento celular, no citoesqueleto de actina dos amebócitos fagocíticos (AF), na migração celular sem adição de substância quimiotática e na resposta quimiotática frente a leveduras S. cerevisiae, além de diminuição do estresse oxidativo em elevadas temperaturas. Constatou-se também que os AF desta espécie apresentaram alterações morfométricas importantes, como diminuição na circunferência celular e alteração na área de espraiamento. Por outro lado, a espécie E. lucunter não apresentou alteração em nenhum parâmetro analisado. Conclui-se, assim, que o estresse térmico atua de maneiras diferentes em espécies polares e tropicais de ouriços-do-mar. No entanto, os mecanismos moleculares pelos quais a espécie E. lucunter apresenta maior termotolerância em comparação à espécie L. variegatus permanecem imcompreendidos e merecem ser estudados. / Global warming is a reality and its effects are widely studied today. However, little attention has been given to the changes that occur to marine invertebrates due to this climate change. It is known that one of the changes that occur as a result of the seawater temperature rise is the increased occurrence of diseases in the marine environment. Therefore, it is important to evaluate the innate immune response against this stressor. The sea urchins were chosen as a model for being considered good environmental bioindicators, and are phylogenetically close to chordates, sharing with them large repertoire of genes and receptors involved in the innate immune system. This study evaluated the innate immune response of Antarctic (S. neumayeri) and tropical (L. variegatus and E. lucunter) sea urchins against thermal stress by different temperatures and exposure periods. With regard to tropical sea urchins, two species, one that inhabits sites less subject to tidal variations (L. variegatus) and the other constantly exposed to tidal oscilations (E. lucunter) were studied. A difference between the response to thermal stress in the three species was observed. The Antarctic species showed more significant changes regarding increase of the percentage of red spherule cells and phagocytic capacity in acute exposure period (24 h) when subjected to mild temperatures. On the other hand, tropical species E. lucunter presented only change in the percentage of red spherule cells during chronic exposure (7 and 14 days) at extreme temperatures, while L. variegatus showed a change in this cell type in all experimental temperatures and exposure periods evaluated, as well as a significant reduction in phagocytic capacity in the same periods that was directly proportional to the temperature increase. Trying to understand the differences between animals that inhabit the same region , it was found that the species L. variegatus showed changes in cell adhesion and spreading, actin cytoskeleton of phagocytic amoebocytes, cell migration without the addition of chemotactic substance and chemotactic response against yeasts S. cerevisiae, besides a reduction of oxidative stress at elevated temperatures. It was also found that the phagocytic amoebocyte of this species showed significant morphological changes, such as reduction in cell circunference and change in the area of spreading. On the other hand, the species E. lucunter presented no change in any parameter analyzed. Thus, it is concluded that the thermal stress acts differently in polar and tropical species of sea urchins. However, the molecular mechanisms by which the species E. lucunter shows higher thermotolerance compared to the species L. variegatus remain obscure and deserve to be studied.

A acidificação oceânica, resultante do aumento da concentração de CO2 atmosférico, vem alterando a química dos oceanos resultando na diminuição de seu pH. Diversos estudos avaliaram as consequências dessa diminuição no pH oceânico nas taxas de calcificação, reprodução e desenvolvimento em diversos modelos marinhos, entretanto estudos relacionados a outros processos fisiológicos, como a imunidade, e estudos com indivíduos adultos são escassos. Ouriços-do-mar são espécies aderidas ao substrato, importantes para a ciclagem de nutrientes no ambiente marinho, sendo também animais utilizados como bioindicadores para monitoramento ambiental; assim o estudo da resposta imune inata desses animais frente à acidificação dos oceanos é de extrema importância para prever possíveis alterações fisiológicas desses animais e sua capacidade de adaptação. O presente trabalho teve como objetivo avaliar as alterações provocadas pela acidificação oceânica na resposta imune e no balanço ácido base de duas espécies de ouriço-do-mar tropicais: Lytechinus variegatus e Echinometra lucunter durante as estações de verão e inverno; para isso foram analisados os índices fagocíticos, a capacidade de adesão e espraiamento celular dos amebócitos fagociticos além do balanço acido base do liquido celomático após o período de 24 horas e cinco dias de exposição aos pHs 7,6 e 7,3. Foi também avaliada a capacidade de recuperação dessas espécies com o objetivo de verificar se os parâmetros alterados pela exposição conseguiam ser reestabelecidos. Os resultados mostraram que a redução no pH da água do mar alterou a proporção celular, reduziu a capacidade de fagocitose e espraiamento dos amebócitos fagocíticos assim como também afetou o balanço ácido-base do líquido celomático. Foram encontradas diferenças também entre as estações do ano sendo estas encontradas apenas na espécie Lytechinus variegatus. O teste de recuperação mostrou que os parâmetros alterados pela exposição tendem a retornar aos valores controles, mostrando que em curto prazo essas alterações podem não ser irreversíveis, entretanto, mais estudos são necessários principalmente avaliando períodos de exposição prolongados. Juntos nossos resultados mostram que a acidificação oceânica prejudica parâmetros imunes extremamente importantes para a eliminação de patógenos e consequentemente a sobrevivência desses animais em um futuro oceano acidificado. / Ocean acidification due to increased atmospheric CO2 concentration is altering ocean chemistry resulting in the decrease of its pH. Several studies evaluated the effects of this decrease in ocean pH on calcification rates, reproduction and development in different marine models, however studies related to other physiological processes such as immunity and studies with adult animals are scarce. Sea urchins are species adhered to the substrate, important for nutrient cycling in the marine environment, also being used as bioindicators for environmental monitoring. Thus the study of the innate immune response of these animals due to the acidification of the oceans is extremely important to predict possible physiological changes of these animals and their ability to adapt to this condition. This study aimed to evaluate the changes caused by ocean acidification in the immune response and in the acid-base balance of two sea urchin tropical species: Lytechinus variegatus and Echinometra lucunter during seasons of summer and winter; for this, indexes of phagocytosis, cell adhesion and spreading ability were analyzed in addition to the acid-base balance of the coelomic fluid after 24 hours and five days of exposure to pH 7.6 and 7.3The recover ability of these species were also evaluated in order to verify if the parameters altered by exposition could be reestablished. The results shows that a reduction in the seawater pH changed the cell proportion, reduced the ability of phagocytosis and phagocytic amoebocyte spreading as well as affected the acid-base balance of the coelomic fluid. Differences were also found between seasons, but only in the specie Lytechinus variegatus. The recovery test showed that the parameters altered by exposure tend to return to control values, showing that in the short term these changes may not be irreversible, however, further studies are necessary mainly those related to prolonged periods of exposure. Together our results show that ocean acidification impairs immune parameters extremely important for the elimination of pathogens and consequently the survival of these animals in a future acidified ocean.

Submitted by Viviane Lima da Cunha (viviane@biblioteca.ufpb.br) on 2016-03-30T12:47:08Z No. of bitstreams: 1 arquivototal.pdf: 5765501 bytes, checksum: d810d02b334ee8a11679326d3a4f57aa (MD5) / Made available in DSpace on 2016-03-30T12:47:09Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 5765501 bytes, checksum: d810d02b334ee8a11679326d3a4f57aa (MD5) Previous issue date: 2015-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Ultraviolet radiation (UV) environmental levels have increased in last years and high values are expected until half of this century. Gametes and embryos of marine organisms, whose embryonic development occurs externally, are particularly sensitive to UVA and UVB radiations. The first line of cellular defense against chemical or physical stress is the activity of ABC transporters. The aim of present work was investigate the involvement of ABC transporters on UVA or UVB effects on gametes or embryos of the sea urchin Echinometra lucunter, and investigate the photoactivity and the ABC transporter blocker activity of coumarin and 3-hydroxycoumarin in gametes and embryos of E. lucunter. Gametes or embryos were exposed to UVA (3.6 – 14.4 kJ m-2) or UVB (0.112 – 14.4 kJ m-2), and the embryonic development was monitored by optical microscopy at different developmental stages in the presence or absence of the ABC-transporter blockers reversin205 (ABCB1 blocker) ou MK571 (ABCC1 blocker). E. lucunter eggs, spermatozoa and embryos were resistant to UVA exposure. Resistance to the harmful effects of UVB was strongly associated to ABC transporter activity (embryos > eggs > spermatozoa). ABCB1 or ABCC1 blockage promoted injurious effects on spermatozoa exposed to UVA. ABCC1 transporter blockage increased UVB-dependent damage in eggs while ABCB1 transporter inhibition increased harmful effects of UVB in embryonic cells. ABC transporters blockers also induced abnormalities in larval development derived from spermatozoa or eggs irradiated to UVB. However, ABC transporter activity was not affected by UVB exposure. Additionally, we demonstrate the photoprotective activity of coumarin and 3-hidroxycoumarin against the deleterious effects of UVB on E. lucunter gametes and embryonic cells, and the ABC transporter blocker activity of the 3-hydroxycoumarin. In conclusion, the present study is the first report of the protective role of ABC transporters against UVA and UVB injurious effects in sea urchins gametes and embryonic cells. Furthermore, our results inserts new alternative for the treatment of various diseases related to UVB excessive exposure and also provides alternatives for clinical reversal of multidrug resistance phenotype. / Os níveis ambientais de radiação ultravioleta (UV) têm aumentado significativamente nas últimas décadas e a tendência é que os valores elevados permaneçam até meados deste século. Gametas e embriões de organismos marinhos, cujo desenvolvimento embrionário ocorre no ambiente externo, são particularmente sensíveis às radiações UVA e UVB. A primeira linha de defesa desses organismos contra estresses químicos e físicos é a atividade de efluxo de bombas conhecidas como transportadores ABC. O objetivo do presente trabalho foi investigar o efeito fotoprotetor de transportadores ABC em gametas e embriões do ouriço-do-mar Echinometra lucunter expostos à UVA ou UVB, bem como investigar a fotoatividade e a atividade bloqueadora de proteínas ABC da cumarina e de seus derivados em gametas e embriões de E. lucunter. Os gametas ou embriões foram expostos à UVA (3,6 a 14,4 kJ m-2) ou à UVB (0,112 a 14,4 kJ m-2), na presença ou ausência dos bloqueadores de transportadores ABC: reversina205 (bloqueador de ABCB1); e MK571 (bloqueador de ABCC1). O desenvolvimento embrionário foi monitorado em microscopia óptica comum. Os gametas e zigotos de E. lucunter foram resistentes à UVA. A resistência das células supracitadas à UVB foi fortemente associada à intensidade da atividade dos transportadores ABC (zigotos > óvulos > espermatozoides). O bloqueio dos transportadores ABCB1 e ABCC1 promoveu inibição no desenvolvimento de embriões derivados de espermatozoides expostos à UVA. O bloqueio do transportador ABCC1 aumentou, em 35,9 + 5,8%, os danos aos óvulos expostos à UVB enquanto que o bloqueio de ABCB1 intensificou, em 36,1 + 2,7%, os danos aos zigotos, induzidos pela UVB. Os bloqueadores de transportadores ABC também induziram malformações ao desenvolvimento larval derivado de espermatozoides ou óvulos expostos à UVB. Entretanto, a atividade dos transportadores ABC não foi afetada pela exposição à UVB. Adicionalmente, demonstramos a atividade fotoprotetora da cumarina e seus derivados contra os efeitos deletérios da UVB em gametas e células embrionárias de E.lucunter, e a atividade bloqueadora de transportadores ABC da 3-hidroxicumarina. Em conclusão, o presente estudo é o primeiro relato do papel protetor de transportadores ABC contra os efeitos deletérios da UVA ou UVB em gametas e células embrionárias de ouriços-do-mar. Além disso, esse trabalho também relata, de forma inédita, a atividade fotoprotetora da cumarina e da 3-hidroxicumarina contra a UVB em gametas e embriões irradiados. Estudos posteriores serão necessários para a inserção terapêutica desses compostos para o tratamento de diversas enfermidades relacionadas com a exposição excessiva à UVB.

A acidificação oceânica, resultante do aumento da concentração de CO2 atmosférico, vem alterando a química dos oceanos resultando na diminuição de seu pH. Diversos estudos avaliaram as consequências dessa diminuição no pH oceânico nas taxas de calcificação, reprodução e desenvolvimento em diversos modelos marinhos, entretanto estudos relacionados a outros processos fisiológicos, como a imunidade, e estudos com indivíduos adultos são escassos. Ouriços-do-mar são espécies aderidas ao substrato, importantes para a ciclagem de nutrientes no ambiente marinho, sendo também animais utilizados como bioindicadores para monitoramento ambiental; assim o estudo da resposta imune inata desses animais frente à acidificação dos oceanos é de extrema importância para prever possíveis alterações fisiológicas desses animais e sua capacidade de adaptação. O presente trabalho teve como objetivo avaliar as alterações provocadas pela acidificação oceânica na resposta imune e no balanço ácido base de duas espécies de ouriço-do-mar tropicais: Lytechinus variegatus e Echinometra lucunter durante as estações de verão e inverno; para isso foram analisados os índices fagocíticos, a capacidade de adesão e espraiamento celular dos amebócitos fagociticos além do balanço acido base do liquido celomático após o período de 24 horas e cinco dias de exposição aos pHs 7,6 e 7,3. Foi também avaliada a capacidade de recuperação dessas espécies com o objetivo de verificar se os parâmetros alterados pela exposição conseguiam ser reestabelecidos. Os resultados mostraram que a redução no pH da água do mar alterou a proporção celular, reduziu a capacidade de fagocitose e espraiamento dos amebócitos fagocíticos assim como também afetou o balanço ácido-base do líquido celomático. Foram encontradas diferenças também entre as estações do ano sendo estas encontradas apenas na espécie Lytechinus variegatus. O teste de recuperação mostrou que os parâmetros alterados pela exposição tendem a retornar aos valores controles, mostrando que em curto prazo essas alterações podem não ser irreversíveis, entretanto, mais estudos são necessários principalmente avaliando períodos de exposição prolongados. Juntos nossos resultados mostram que a acidificação oceânica prejudica parâmetros imunes extremamente importantes para a eliminação de patógenos e consequentemente a sobrevivência desses animais em um futuro oceano acidificado. / Ocean acidification due to increased atmospheric CO2 concentration is altering ocean chemistry resulting in the decrease of its pH. Several studies evaluated the effects of this decrease in ocean pH on calcification rates, reproduction and development in different marine models, however studies related to other physiological processes such as immunity and studies with adult animals are scarce. Sea urchins are species adhered to the substrate, important for nutrient cycling in the marine environment, also being used as bioindicators for environmental monitoring. Thus the study of the innate immune response of these animals due to the acidification of the oceans is extremely important to predict possible physiological changes of these animals and their ability to adapt to this condition. This study aimed to evaluate the changes caused by ocean acidification in the immune response and in the acid-base balance of two sea urchin tropical species: Lytechinus variegatus and Echinometra lucunter during seasons of summer and winter; for this, indexes of phagocytosis, cell adhesion and spreading ability were analyzed in addition to the acid-base balance of the coelomic fluid after 24 hours and five days of exposure to pH 7.6 and 7.3The recover ability of these species were also evaluated in order to verify if the parameters altered by exposition could be reestablished. The results shows that a reduction in the seawater pH changed the cell proportion, reduced the ability of phagocytosis and phagocytic amoebocyte spreading as well as affected the acid-base balance of the coelomic fluid. Differences were also found between seasons, but only in the specie Lytechinus variegatus. The recovery test showed that the parameters altered by exposure tend to return to control values, showing that in the short term these changes may not be irreversible, however, further studies are necessary mainly those related to prolonged periods of exposure. Together our results show that ocean acidification impairs immune parameters extremely important for the elimination of pathogens and consequently the survival of these animals in a future acidified ocean.

Planktonic larvae of marine organisms are increasingly being exposed and required to respond to a changing physical environment. Adult sea urchins occupy both intertidal and subtidal waters and broadcast spawn gametes into the water column to contend with variable physical conditions. To answer how populations of invertebrates residing at different depths adequately prepare their offspring to cope with different levels of ultraviolet radiation (UVR), we collected adult purple sea urchins, Strongylocentrotus purpuratus, from four sites (two intertidal and two subtidal (~15 m deep)) on the central coast of CA to compare UV tolerance in offspring. Our measurements of UVA (321–400 nm) and UVB (280–320 nm) irradiance at all collection sites showed UVA and UVB were low or absent in subtidal sites compared to intertidal sites. Our study found that offspring from intertidal populations experience a less severe developmental delay when exposed to environmentally relevant levels of UVR (using artificial lighting) than offspring from subtidal populations. The mean percent cleavage delay for UV-treated embryos relative to the controls was 16% (± 2.3 SE) for intertidal sites and 21.1% (± 2.7 SE) for subtidal sites. This suggests that environmental UV cues or additional environmental cues experienced by intertidal mothers may prepare offspring to resist effects of UV exposure during early development. To further understand differences in biochemistry of the eggs released from mothers of different populations (i.e. differences in maternal investment), we assessed differences in protein abundance among batches of eggs from intertidal and subtidal populations. We identify a range of candidate proteins involved in various cellular processes such as cell cycle regulation, signaling and transport, oxidative stress and metabolism that may help developing embryos cope with UVR stress. These candidate proteins may also help us understand adaptations important for survival of developing marine organisms in changing ocean conditions.

Die Grundlage der Entwicklung multizellulärer Lebewesen bildet die Herausbildung stabiler Zelllinien aus einer einzelnen Zygote. Die Expression spezifischer Kombinationen von Transkriptionsfaktoren ist von zentraler Bedeutung für diesen Prozess. Des Weiteren sind epigenetische Veränderungen des Genoms von Bedeutung, über deren Verhältnis zu Änderungen der Zell-Identitäten weniger bekannt ist. In dieser Arbeit nutze ich Daten aus scATAC-seq und scRNA-seq (mit metabolischer Markierung; scSLAM-seq) um die regulierenden Elemente im Seeigel zu charakterisieren – vom 4-Zell-Stadium, über die Aktivierung des zygotischen Genoms, bis hin zu den Strukturen der frühen Pluteus-Larve. Die Schicksale der einzelnen Zellen des Seeigels sind gut erforscht und ab der vierten Zellteilung etabliert (auch wenn einzelne Veränderungen bis zum 128-Zell Stadium induziert werden können), wonach die Keimblätter fest verankert sind. Plastizität innerhalb der Keimblätter ist mindestens bis zum Ende der Gastrulation möglich. Mithilfe dieser Daten, sowie den bekannten Ergebnissen der Erforschung der Zellschicksale vergangener Jahrzehnte, ist eine Diversifizierung von Zelltypen ersichtlich, die beim 128-Zell-Stadium mit der großen Welle der zygotischen Genomaktivierung (ZGA), einer Veränderung des Chromatinzustandes und dem Verlust der Entwicklungsplastizität einhergeht. Ein kleiner Teil von Genen, im Allgemeinen zelltypspezifische Transkriptionsfaktoren, wird schon vor der großen Welle der ZGA exprimiert, während sich gut offene Chromatinstrukturen auf wenige, distinkte regulatorische Sequenzen beschränken. Von diesem Zeitpunkt an, wird die Genexpression zelltypspezifisch, auch wenn viele Chromatinelemente weiterhin zelltypübergreifend offen sind. Insgesamt sind die Chromatinelemente recht kompakt und Elemente innerhalb der Gene häufig. Danach porträtiere ich noch die regulatorischen Veränderungen die mit der neuralen Entwicklung, sowie der Diversität von skelettbildenden Zellen im Seeigel einhergehen. Zuletzt identifiziere ich ~ 100 Gene, die in die Kalzifizierung des Skelettes des Seeigels involviert sind. / The emergence of multiple, stable cell lineages from a single-cell zygote is the essence of multicellular development. Combinatorial transcription factor expression is central to this process, as well as epigenetic changes whose relationship to changes in cell-identity are far less well understood. In this thesis, I use data from scATAC-seq and scRNA-seq (with metabolic labeling; scSLAM-seq) to characterize the regulatory landscapes of sea urchin development spanning from the 4-cell embryo through maternal zygotic transition (MZT), gastrulation, and the early pluteus larvae with its ecologically relevant structures (~72h). The early fate-map in sea urchins is well understood, providing an ideal model for this analysis; the basic fate-map is established by the fourth cleavage (though inducible lineage changes are possible up to the 128-cell stage) after which germ-layer identity is locked, though there remains considerable plasticity within lineages at least through gastrulation. Using these data, along-side classic research into cell fate maps in the early embryo, I find that cell-type diversification and a loss of plasticity at 128-cell stage corresponds to a major wave of zygotic genome activation (ZGA) and a clear resolution of the chromatin landscape in the sea urchin. However, a subset of genes, often cell-type-specific transcription factors, shows evidence of pre-MZT zygotic expression with early chromatin accessibility limited to few sites with distinct regulatory sequences. From this time, gene expression profiles become highly cell-type-specific, though many regulatory elements remain ubiquitously accessible, suggesting differential transcription factor occupancy at broadly accessible sites. Overall, the regulatory landscape is fairly compact with accessible intragenic elements being frequent. Subsequently, I profile the regulatory changes underlying neurodevelopment and the diversity of skeletogenic cells in the sea urchin. Finally, I also identify ~ 100 genes that are associated with calcification of the sea urchin skeleton.

<p>In light of the paucity of information on the toxicity of lead (Pb), zinc (Zn), copper (Cu) and nickel (Ni) in the marine environment, the aim of this thesis was to generate data on the mechanisms of toxicity of Pb, Zn, Cu and Ni in the early life stages of a very sensitive marine organism, the purple sea urchin (<em>Strongylocentrotus purpuratus</em>). Previous studies in other systems indicated that these metals can impact ionoregulation, especially calcium homeostasis, so an initial study focused on ionoregulatory changes during the first 96h of development under control conditions in embryonic and larval stages of <em>S. purpuratus</em>. Control tests showed that patterns of Ca, K, Na and Mg had an interesting pattern of accumulation over 96 h development and that the most pronounced changes were observed during the gastrulation stage. A variety of biological endpoints were subsequently utilized to examine potential mechanisms of toxicity. Toxicity tests were performed to determine median toxicity threshold values (i.e. EC50: median effective concentration and LA50: median lethal accumulation) for Zn and Pb. Growth, unidirectional Ca uptake rates, whole body ion concentrations (Na, K, Ca, Mg), and Ca ATPase activity were also monitored every 12 h over the first 84 or 96 h of early development to investigate the mechanisms of toxicity during acute and chronic exposures to lethal and sublethal concentrations of Pb, Zn, Cu and Ni in 100% sea water.</p> <p>Sea urchin embryos were very sensitive to Zn with an EC50 of 2.3 µmol/L (95% C.I. = 1.97–2.71 μmol/L) and LA50 of 4.8 (2.16-11.33) μmol/kg. Embryos displayed even higher sensitivity to Pb with an EC50 of 0.36 (0.25-0.49) µmol/L and LA50 of 1.92 (1.67-2.78) μmol/kg. The toxic effects of these metals were increased when tested in combination with extracts of dissolved organic carbon (DOC) from marine and freshwater sources.</p> <p>From studying larvae chronically exposed to Pb (60 µg/L), Zn (139 µg/L), Cu (6 µg/L) and Ni (47 µg/L) it was apparent that these metals rendered their toxic effects, at least in part, through disruption of Ca homeostasis. Unidirectional Ca uptake rates as well as Ca ATPase activity were significantly inhibited at various time points over development, in larvae in these metal exposures. This resulted in significantly lower levels of Ca accumulated in the larvae. Interestingly, larvae showed some capacity for recovery as Ca uptake rates and internal Ca levels returned to control values periodically over development. Also metal initially accumulated in the larvae often returned to control levels at 72 h of development with the exception of Pb, which was the only non essential metal out of the four tested. Metal effects on the whole body levels of three other ions (Na, K, Mg) as well as larval weight were modest. Surprisingly, acute exposure to much higher levels of these same metals at various stages during development had negligible inhibitory effects on unidirectional Ca uptake rate, suggesting a mechanism other than direct competition for the Ca uptake sites. We propose studying the toxicity of contaminants periodically over development as an effective way to detect sub-lethal effects, which may not be displayed at the traditional endpoint of 72 h.</p> / Master of Science (MS)

This study focused on the management of the red sea urchin Loxechinus albus fishery in Chile. The main objective was to design, construct, implement and assess a computer-based simulation model to analyse the biological effects, socio-economic consequences and spatial dynamics resulting from coastal management plans applied to this resource under the system of AMEBR. This was accomplished by using systems dynamics (SD) and geographical information systems (GIS) modelling, in a process of model development, run, optimisation, sensitivity analysis and risk management, and a series of field-based activities carried out at the cove of Quintay. The GIS model developed for allocating sea urchins restocking sites offered a flexible, cost-effective, user-friendly and descriptive technique for support decision-making on management of this species and other benthic resources. Final site selection for restocking was based on the identification, quantification and selection of higher suitability¦availability combinations (site categories). This map showed 16 different suitability¦availability combinations or site categories, ranging from 4¦100 to 8¦100 (suitability points¦availability %). These had an average of 6.44¦69.37 (covering an area of 82.5 Ha overall equivalent to 81.21% of the study area. This site classification demonstrated high heterogeneity between options, and revealed the full variety of alternatives for decision-making. More importantly, the generally high suitability indexes as well as available area emphasised the prospects for restocking sea urchins in this study area. Over and above of the quantitative outcomes obtained from running the GISRM (suitable and available restocking sites) and the BSESM (alternative strategic management plans), the case study-based analysis made it possible to disclose the wider issues related to the red sea urchin coastal management. These results demonstrated the biological inefficiency of traditional size/seasonal restriction-based approach (macro-scenario 1) for sustainable management of the target species. More importantly, final outcomes strongly suggested that a combination of adaptive restocking-based enhancement activities and flexible exploitation constituted a highly attractive approach (macro-scenario 3) for stock management of this fishery in terms of harvestable stock and related incomes. However from the economic analysis, stocking was also found to be economically unfeasible, being a rather cost intensive exercise negatively affected by high natural mortality rates. A single-variable optimisation analysis demonstrated that a higher survival rate is needed to generate sufficient profits to cover major restocking costs and a positive payment, or a cost reduction is essential to make up for the loss. On top to these practical constraints, based on the distinctive modest economic situation prevailing for most Chilean coves and hence their limited capacity to pay for stocking material, unless adequate and constant funding is available to support artisanal associations, they are very unlikely to develop mass release programmes. Given the economic (i.e.: high operating costs) and technical (i.e.: low survival rates) limitations conditioning stocking-based management cost-effectiveness and applicability, wide implementation of mass releases as a major approach for management of the red sea urchin fishery is very unlikely to take place in Chile. This study presents a methodology and offers a tool to design, evaluate and optimise coastal management plans for the red sea urchin in a dynamic, interactive, systematic, integrated and flexible way. The optional strategic management plans proposed on this study may not be applied equally to any AMEBR, as they are the outputs arising from a single cove-specific analysis. Still, the complete methodological framework and analysis procedures developed may be applied to run the BSESM and optimise management of a red sea urchin fishery at any other AMEBR case of study.