This PhD Thesis deals with the study of marine natural products as candidates in either drug discovery and development. In particular, the investigation focused on benthic organisms, i.e. those fixed or in a close relation with the sea bottom. These organisms, many of which are sessile or slow-moving invertebrates, thus unable to escape quickly from their predators, and devoid of physical protection (shell or hard texture), have developed alternative protective strategies as well as the production of chemical weapons. Compounds involved in the chemical defence, besides the ecological function, can display interesting pharmacological activities. Organisms of different phyla (Algae, Molluscs and Cnidarians) and from diverse geographical areas (Mediterranean Sea and China) were investigated. The benthic diatom Cocconeis scutellum is able to affect the sex reversal in the protandric shrimp Hippolyte inermis, inducing apoptosis (programmed cell death) in the crustacean's androgenic gland. Thus, we wondered whether these diatoms could act also in other crustaceans and on human cancer cells, in order to evaluate their applications in aquaculture and medicine. In fact, some commercially important crustaceans show a bimodal pattern of growth, in which males grow faster and are bigger than females. For this reason, the production of all-male populations is more advantageous. The administration of proapoptotic compounds in an early larval phase could be a method to manipulate the sex in these crustaceans. Nevertheless, our study demonstrated that diatom compounds did not induce apoptosis in the androgenic gland of other species, suggesting a targeted and specific action of C. scutellum metabolites. On the other hand, the biological assays on human cancer cell lines showed that solid tumours were more responsive to C. scutellum than haematological tumours and normal cells, suggesting a specific toxic action of the diatom compounds. In addition, we demonstrated that the most active fraction to induce apoptosis in cancer cells was that containing the highest amount of eicosapentaenoic acid (EPA), described in the literature for its anticancer and proapoptotic properties. This fraction detained the cell cycle at G2/M phase and the results in terms of apoptosis and reduced cell viability produced by this fraction were comparable with those induced by EPA. Western blotting experiments showed that diatoms triggered apoptosis by the extrinsic pathway, i.e. by means of activation of caspase 8, and confirmed the involvement of effector caspase 3. In addition, C. scutellum was studied for the first time from the chemical point of view. By GC-MC we identified more than 100 compounds, mainly fatty acids, sterols and glycerides, and excluded the presence of short chain aldehydes.In collaboration with the University of Athens, the chemical study on the anaspidean mollusc Aplysia fasciata was carried out. From its digestive and hermaphroditic glands 16 metabolites were isolated by a combination of chromatographic techniques, and they were identified by spectroscopic analysis. Six of them (6-hydroxy-1-brasilene, epibrasilenol acetate, (3Z,9Z)-7-chloro-6-hydroxy-12-oxopentadeca-3,9-dien-1-yne, 6-epi-beta-snyderol, 16-acetoxy-15-bromo-7-hydroxy-9(11)-parguerene), belonging to different classes of sesquiterpene, acetogenin and diterpene compounds, are new and related with metabolites isolated from different species of the red alga Laurencia, the animal's diet. In the Istituto di Chimica Biomolecolare (Italy), the chemical and biological investigation on the Chinese soft coral Sinularia sp. was performed. We isolated four new casbene compounds, i.e. bicyclic diterpenes characterized by a cyclopropane fused with a 14-membered macrocycle, which are usually found in Euphorbiaceae plants and rarely in marine organisms. Contrastingly to most natural cis ring-fused casbenes, in Sinularia co-existed cis and trans compounds and this suggested a non-stereospecific biosynthetic mechanism. The 4 casbenes were active against Gram+ bacteria and just casbene 3 also against Gram- bacteria, only hydroxylated casbenes displayed anti-feeding properties, and they did not exhibit antiproliferative activity. / "Metabolitos secundarios de organismos bentónicos: aspectos ecológicos y químicos" Depositada por Michela NappoTEXTO:El objetivo de esta Tesis es el estudio de los productos naturales marinos y su posible relevancia en el desarrollo de nuevos medicamentos. La investigación se enfocó sobre los organismos bentónicos, i.e. los que viven en contacto con el fondo marino y que, a pesar de la falta de protección física y la escasa movilidad de la mayoría de las especies, han desarrollado mecanismos de defensa alternativos, como la producción de armas químicas. Estos compuestos, dotados de propiedades ecológicas, a menudo exhiben también importantes actividades farmacológicas. Se consideraron organismos pertenecientes a diferentes phyla (algas, moluscos y cnidarios) y áreas geográficas. Las diatomeas Cocconeis scutellum afectan la reversión sexual del decápodo proterándrico Hippolyte inermis, induciendo apoptosis (muerte celular programada) en la glándula androgénica del crustáceo. En esta Tesis evaluamos también la capacidad de dichas diatomeas en afectar el cambio de sexo de otros crustáceos, en particular los de interés comercial, y de actuar sobre las células tumorales humanas, a fin de encontrar posibles aplicaciones en acuicultura y medicina. También se valoró el patrón metabólico de las diatomeas mediante cromatografía de gases/espectrometría de masas, identificando más de 100 compuestos (ácidos grasos, esteroles y glicéridos), no constatándose la presencia de aldéhidos de cadena corta.En la Universidad de Atenas, se realizó el estudio químico del molusco anaspídeo Aplysia fasciata. Mediante diferentes técnicas cromatográficas y espectroscópicas, se aislaron 16 metabolitos, de los cuales seis eran nuevos (6-hidroxi-1-brasileno, epibrasilenol acetato, (3Z,9Z)-7-cloro-6-hidroxi-12-oxopentadeca-3,9-dien-1-ino, 6-epi-beta-snyderol, 16-acetoxi-15-bromo-7-hidroxi-9(11)-parguereno), relacionados con metabolitos de algas rojas del género Laurencia, que constituyen la dieta del animal.En el Instituto de Química Biomolecular (Pozzuoli, Italia) se realizó tanto el estudio químico como los experimentos biológicos con el coral blando Sinularia sp. procedente de China. De esta especie se aislaron 4 nuevos compuestos casbénicos (diterpenos bicíclicos formados por un anillo ciclopropánico fusionado a un macrociclo de 14 átomos) cuya estructura es frecuente en plantas de la familia Euphorbiaceae y bastante más rara en los organismos marinos. Los casbenos hidroxilados eran repelentes alimenticios; todos son activos frente a las bacterias Gram+ y sólo el casbeno 3 es también activo frente a las bacterias Gram- .

Coral reefs constitute some of the most prolific and diverse ecosystems on our planet, but also among the most threatened. This thesis investigates the effects of environmental stressors on corals’ metabolism and symbiont diversity. Paper I shows that the coral Turbinaria mesenterina withstood a single stressor while a combination of two stressors (decreased salinity and increased seawater temperature) lead to decreased metabolism. Increased seawater temperature in combination with two stressors (enhanced nutrients and decreased salinity) lead to rapid mortality of all specimens. Paper II shows that chronic stress in combination with increased seawater temperature affects coral species differently. Porites lutea did not show any difference in response to temperature increase, regardless of environmental disturbance history, while Galaxea fascicularis’ metabolism was negatively affected in chronically disturbed corals but not in corals from less disturbed areas. The main explanation for the difference in response between the two species is different compositions of endosymbionts as found in paper III. P. lutea only harboured the symbiont C15, regardless of environment, whilst D1a dominated the nearshore G. fascicularis and C1 dominated offshore corals. In paper IV there was a clear inshore-offshore pattern of D1a along the whole coast of Vietnam, where D1a dominated inshore. In contrast, the five symbionts belonging to group C displayed a strong latitudinal gradient, with diversity increasing from north to south. The coral host showed higher diversity offshore than inshore. The thesis emphasizes the importance of improving water quality (paper I and II) and protecting marginal areas since tolerant coral hosts and symbionts can be found there (paper III and IV), as well as safeguarding areas with high symbiont diversity (paper IV) to increase the ability of corals to withstand future environmental changes. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

This thesis is composed of two articles. The first is an analysis of long-term survival among A. cervicornis outplants in the Florida Keys, from 2007 to the present. The second is a review of literature that informs coral restoration and guides both restoration practitioners and coral researchers towards greater effectiveness in outplant survival and understanding biological processes involved in restoration. In the first article, despite promising initial evidence of outplant survival and health, the long-term results, using Weibull survival analysis, are discouraging with almost all out planted corals over an 8 year long study exhibiting steep declines in percent live tissue and survival between three and five years. Not only is this 3-5 year collapse apparent in all the outplanted cohorts, but the evidence is highly significant that the length of outplant survival is decreasing with each passing year (diminished resilience). These findings suggest that some shared, likely environmental factor, is increasingly impacting all outplants. Further, no cohorts appear to adapt to the environmental conditions in which these declines are occurring (diminished adaptive capacity), a trend that would be evident if their declines slowed or reversed and Weibull beta-parameterization would show. The second article, reviews several areas of recent study which offer avenues for future research: these include, ecological history and biogeography, developmental pathways of colonial form and function, polarity and symmetry, genetics, wound healing, fecundity, reproduction, sexual maturity and community interactions. The thesis concludes with questions for further research and understanding in the field of coral restoration.

126 f. / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-08-26T15:03:19Z No. of bitstreams: 1 Tese doutorado Gilmara - versao final.pdf: 18224828 bytes, checksum: 1f3f2af3ca28cc20aa72bad42c59c0f2 (MD5) / Approved for entry into archive by Ana Hilda Fonseca(anahilda@ufba.br) on 2013-08-26T15:05:14Z (GMT) No. of bitstreams: 1 Tese doutorado Gilmara - versao final.pdf: 18224828 bytes, checksum: 1f3f2af3ca28cc20aa72bad42c59c0f2 (MD5) / Made available in DSpace on 2013-08-26T15:05:14Z (GMT). No. of bitstreams: 1 Tese doutorado Gilmara - versao final.pdf: 18224828 bytes, checksum: 1f3f2af3ca28cc20aa72bad42c59c0f2 (MD5) Previous issue date: 2013 / CNPq / Contaminantes traços ocorrem naturalmente nos diversos compartimentos do ambiente marinho. Entretanto, as atividades antrópicas proporcionam um aporte elevado de elementos traços e maiores para o ambiente, o qual pode impactar negativamente nos compartimentos abióticos, bem como na biota, comprometendo os serviços ambientais e a ecologia dos diversos ambientes costeiros. Neste contexto, este estudo teve dois objetivos. O primeiro visou otimizar métodos para a determinação de elementos traços e maiores em tecidos de poliqueta e esqueletos de coral. O segundo propôs identificar candidatos potenciais (poliqueta - Chaetopterus variopedatus e caranguejo simbionte Polyonyx gibbesi; moluscos bivalves - Crassostrea rhizophorae, Anomalocardia brasiliana, Mytella guyanensis, Lucina pectinata e Braquidontes exustus) a organismos biomonitores de contaminação por elementos traço e maiores para a Baía de Todos os Santos (BTS). Amostras de bivalves foram coletadas na BTS e Baía de Camamu em 2010 e 2011. As amostras de poliquetas foram coletadas na BTS, entre 2010 e 2012. Amostras de sedimento foram coletadas para análise química e granulométrica. Na BTS, tubo de poliquetas e caranguejo simbionte (P. gibbesi) foram coletados juntamente com poliqueta. Amostras para a análise química foram secas, moídas e submetidas à digestão ou extração com solução ácida para solubilização dos analitos. Em laboratório foram feitos testes de otimização para a digestão ácida das amostras de esqueleto de coral e tecido de poliqueta. Para poliqueta, o método otimizado para digestão por microondas com cavidade, com o uso de mistura ácida de 8,1 mol L-1, forneceu bons resultados e foi aplicado às amostras coletadas na BTS. O método otimizado para digestão de esqueleto de coral, usando solução ácida de 6 mol L-1, forneceu bons resultados para um maior número de elementos traço e pode ser aplicado em pesquisa futura com amostras de esqueletos de corais, para monitorar a contaminação de recifes por Cr, Cd, Co, Cu, Mn e Sn. As concentrações de elementos traço das amostras foram determinadas por espectrometria de massa (ICP-MS) e/ou emissão (ICP OES) com plasma indutivamente acoplado. Os resultados mostraram que cada organismo estudado respondeu de modo diferenciado ao nível de contaminação do ambiente em que vive, incorporando elementos traço e/ou maiores nos tecidos. A incorporação foi influenciada por vários fatores, tais como o tipo de contaminante e a biodisponibilidade no ambiente, nível de contaminação, habitat, hábito alimentar e a fisiologia. Esses fatores foram avaliados em conjunto e considerados na escolha dos melhores candidatos a biomonitores de uma área. Como os níveis de contaminação variam ao longo da BTS, devido às inúmeras fontes de contaminantes, é importante utilizar mais de um grupo de organismo para representar a contaminação de toda a área amostrada. Dentre as espécies estudadas, a ostra C. rhizophorae armazenou concentrações bem mais altas de Cu, Cd e Zn, devido à própria fisiologia. Porém, considerando a distribuição geográfica dos organismos estudados na BTS, as espécies de bivalves M. guyanensis e A. brasiliana, e o poliqueta C. variopedatus, por apresentarem concentrações similares para um maior número de elementos (Al, Ba, Co, Cr, Mn, e Zn), são as mais indicadas a biomonitoras destes contaminantes para a BTS, podendo ser utilizadas no monitoramento de outras regiões costeiras tropicais. / Salvador

Submitted by Johnsson Rodrigo (r.johnsson@gmail.com) on 2013-09-02T12:08:06Z No. of bitstreams: 1 ROBERTA.pdf: 21017159 bytes, checksum: 41e6a847bf3ae9866b8f2160a17c81bf (MD5) / Approved for entry into archive by Alda Lima da Silva(sivalda@ufba.br) on 2013-09-26T18:00:23Z (GMT) No. of bitstreams: 1 ROBERTA.pdf: 21017159 bytes, checksum: 41e6a847bf3ae9866b8f2160a17c81bf (MD5) / Made available in DSpace on 2013-09-26T18:00:23Z (GMT). No. of bitstreams: 1 ROBERTA.pdf: 21017159 bytes, checksum: 41e6a847bf3ae9866b8f2160a17c81bf (MD5) / CNPq, FAPESB / O gênero Mussismilia (M. hispida, M. harttii e M. braziliensis) é endêmico do litoral brasileiro e inclui as principais espécies de corais construtores. Estudos com a carcinofauna associada à Mussismilia são escassos e restritos a M. hispida no setor sudeste. Dentre os organismos registrados em associação com M. hispida, destacam-se os caranguejos-de-galha e copépodes sifonostomatóides. Para todo Atlântico, são registrados apenas cinco gêneros de Cryptochiridae (Decapoda, Brachyura) (Detocarcinus, Cecidocarcinus, Opecarcinus, Troglocarcinus e Kroppcarcinus – o último, recentemente descrito). Para o Brasil, considerando os dados da literatura, três espécies são registradas para a costa brasileira: Opecarcinus hypostegus, Troglocarcinus corallicola e Kroppcarcinus siderastreicola. Uma nova espécie de Troglocarcinus, gênero até então monoespecífico, foi encontrada em associação às espécies de Mussismilia no litoral da Bahia. Troglocarcinus n. sp. difere de sua congênere por (1) possuir numerosos espinhos bem desenvolvidos na carapaça (delimitando as regiões protogástrica e mesogástrica), pelo (2) segmento basal da antênula (que não se estende até o pedúnculo ocular), pela (3) presença de cerdas no esternito do primeiro pereópodo e, finalmente, pelo (4) terceiro pleópodo da fêmea unirreme ou birreme. Para a inclusão da nova espécie, foi necessária a elaboração de uma emenda na diagnose do gênero. No Atlântico Sul, a família Entomolepididae (Copepoda, Siphonostomatoida) está representada apenas por Spongiopsyllus. Uma nova espécie de Spongiopsyllus foi encontrada em amostras de Mussismilia hispida provenientes da Praia da Boa Viagem, na Baía de Todos-os-Santos. Spongiopsyllus n. sp. se distingue de S. adventicius pela redução no número de segmentos da antênula, ornamentação do endópodo da antena e pela cerdação do maxilípede e da P1. Spongiopsyllus n. sp. é o segundo registro do gênero para o Atlântico Sul e a primeira ocorrência da família associada com Scleractinia. Adicionalmente, foi elaborada uma chave de identificação para todas as espécies de Entomolepididae. O inventário da biodiversidade marinha na Província Brasileira ainda é incipiente. Deste modo, os resultados obtidos no presente estudo contribuem ao conhecimento da carcinofauna, com ênfase às formas associadas de hábito críptico, revelando que novas espécies e ocorrências geográficas permanecem amplamente desconhecidas pela comunidade acadêmica e científica. / Salvador

Corals face serious worldwide population declines due to global climate change in combination with direct anthropogenic impacts. Global climate change is difficult to manage locally, but policy makers can regulate the magnitude of local stressors affecting reefs. The objective of this experiment is to investigate if reducing sedimentation will enable reef corals to better endure global climate change. It has been shown that some coral species can handle climate change stress when provided with additional energy resources. Here I tested if the capacity of corals to cope with climate change can be improved when their ability to feed and photosynthesize was not compromised by increased sedimentation. Sedimentation can impede coral feeding and their ability to photosynthesize due to direct polyp blocking and increased turbidity, which reduces light availability. To evaluate the potential of enhancing coral ability to tolerate climate change by reducing a local stressor, I examined the survival and growth of brooding coral Porites astreoides juveniles when exposed to ambient and elevated water temperatures under differing sedimentation rates. I also assessed if sediment composition has significant impacts on these results. I used sediment from a reef and sediment from a boat basin within a port to mimic natural and anthropogenic sediment types and processes (e.g. dredging). Experiments were conducted to quantitatively assess the synergistic effects of sediment concentration and composition, along with increased temperature on the survival and growth of juvenile P. astreoides. The most detrimental effects were observed with anthropogenic sediment, when both sediment concentration and water temperatures were high. However, increased natural reef sediment was found to be beneficial to juvenile corals. More interestingly, I found that corals capacity to deal with higher temperatures is improved when anthropogenic sedimentation is maintained at minimal levels and turbidity resulting from sedimentation was low. Therefore, this information will aid managers in making decisions that regulate dredging and construction activities to minimize sedimentation, which will contribute to increase coral survival under climate change.

Cold-water corals, similar to tropical corals, contain a diverse and complex microbial landscape. Comprised of vital microscopic organisms (i.e. bacteria, viruses, archaea), the coral microbiome is a driving factor in the proliferation and survival of the coral host. Bacteria provide essential biological functions within coral holobionts, facilitating increased nutrient utilization and production of antimicrobial compounds. To date, few cold-water octocoral species have been analyzed to explore the diversity and abundance of their microbial associates. For this study, 23 samples of the family Anthothelidae were collected from Norfolk (n = 12) and Baltimore Canyons (n = 11) from the western Atlantic in August 2012 and May 2013. Genetic testing found that these samples comprised two Anthothela species (Anthothela grandiflora and Anthothela sp.) and a new genus. DNA was extracted and sequenced with primers targeting the V4-V5 variable region of the 16S rRNA gene using 454 pyrosequencing with GS FLX Titanium chemistry. Results demonstrated that the host genus was the primary driver of bacterial composition. The new coral genus, dominated by Alteromonadales and Pirellulales, had much higher species richness and a distinct bacterial community compared to Anthothela samples. Anthothela species had very similar bacterial communities, dominated by Oceanospirillales and Spirochaetes. Core bacterial diversity present across 90% of the Anthothela samples revealed genus-level conservation. This core included unclassified Oceanospirillales, Kiloniellales, Campylobacterales, and Spirochaeta; the functional abilities of which contribute to a nearly complete nitrogen cycle. Dominant bacterial members of the new coral genus also had functional capabilities in nitrogen cycling. Overall, many of the bacterial associates identified in this study have the potential to contribute to the acquisition and cycling of nutrients within the coral holobiont.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22513号 / 農博第2417号 / 新制||農||1078(附属図書館) / 学位論文||R2||N5293(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 渡邊 隆司, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Over the last four decades, coral reefs have suffered a ~50% decline of across the tropics. Consequently, most research efforts have focused on the impacts of anthropogenic pressures on corals, including ocean warming, ocean acidification, and overfishing. However, recent discoveries indicate that coral reefs are becoming increasingly vulnerable to acute deoxygenation events, which can drive severe and widespread coral bleaching, and in some cases, mortality of corals and other reef organisms. On unimpacted coral reefs, dissolved oxygen (DO) availability can vary between 50% and 200% air saturation, depending on the location, proximity to the open-ocean, and time of the day. During the daytime, Symbiodiniaceae spp. produce more O$_2$ than the coral host can consume, releasing excess O$_2$ to the surrounding tissues. However, at nighttime Symbiodiniaceae spp. cease O$_2$ production. Hence, corals may suffer to O$_2$ deprivation at nighttime when the photosynthesis ceases, and holobiont respiration consumes oxygen. To assess the O$_2$ thresholds and aftereffects of two Red Sea coral species: ${{P. lobata}}$ and ${{G. fascicularis}}$ corals were exposed to reduced DO concentrations. Coral fragments from both species were exposed to one control treatment (6.8 mg O$_2$ l$^{−1}$) and three reduced DO concentrations treatments (5.25 mg O$_2$ l$^{−1}$, 3.5 mg O$_2$ l$^{−1}$, and 1.25 mg O$_2$ l$^{−1}$). Experiments were held at a stable temperature (32°C ± 0.25) and stable pH levels (pH 8.2 ± 0.08). Corals in these experiments displayed different thresholds to low O$_2$ concentrations. ${{P. lobata}}$ coral fragments didn’t exhibit any bleaching symptoms throughout complete experiment. However, ${{G. fascicularis}}$ fragments showed signs of bleaching after the third night of exposure to the low O$_2$ treatment (1.25 mg O$_2$ l$^{−1}$). Physiological variables such as maximum and effective photochemical efficiency, Chl ${{a}}$, cell density, and dark respiration experienced the lowest values under the low O$_2$ treatment for both species. These results highlight the need for further experimental assessments of deoxygenation thresholds for corals across the globe. These assessments are of great importance to create better conservation strategies for the preservation of coral reefs.

In this study reef corals from two Caribbean and adjacent regions in different periods were investigated. Sclerochronological records of corals from well preserved Miocene to Pleistocene reefs were used to document potential changes in seasonal and interannual climate associated with CAS uplift and closure. In southern Florida the Plio/Pleistocene Caloosahatchee Formation is cropping out. During the deposition of this stacked shallow marine sediments at the Plio/Pleistocene Florida carbonate platform, a rich coral fauna existed. Corals from the Caloosahatchee Formation were investigated herein, regarding to reveal high resolution (bimonthly) climatic archives in their skeletons. The second region investigated herein is the Cibao Valley in the Dominican Republic, where sediments of the Neogene Yaque Group are outcropping. Corals from the Late Miocene Cercado Formation and the Mio/Pliocene Gurabo Formation (both upper part of the Yaque Group) are discussed here. Both formations are mixed carbonate-siliciclastic deposits of the prograding inner-shelf and contain a well preserved faunal record. Sclerochronological methods are used to identify seasonal and interannual climatic variability. Especially radiography, stable isotope analysis and laser ablation measurements (LA-ICP-MS) were used and discussed with a special focus on the pristine preservation of the analysed coralline material.