Rozprawy doktorskie na temat „Sea squirts”

This dissertation examines two issues related to the ecological and evolutionary consequences of sexual and asexual reproduction in colonial marine invertebrates. The first two chapters explore the extent to which the planktonic larval phase limits the distribution and abundance of a colonial ascidian, Oiplosoma similis. The third chapter examines some of the fitness consequences of alterations in the pattern of asexual reproduction by colony fragmention in similis. All research was carried out on the fringing coral reef surrounding Coconut Island which is located in Kaneohe Bay, Oahu, Hawaii.
Typescript. Thesis (Ph. D.)--University of Hawaii at Manoa, 1989. Includes bibliographical references.

The ascidian species Ciona savignyi and C. intestinalis are invasive species but show interspecific differences in their population response to hypo-saline stress associated with heavy winter-run off events that are predicted to become more frequent due to climate change. Despite an almost world-wide distribution, C. intestinalis seems to be more susceptible to hypo-saline stress than the geographically more limited C. savignyi. Given that the genomes of both species are fully sequenced, we were able to compare their proteomic response to both acute and chronic salinity to characterize the mechanisms that are responsible for setting tolerance limits to hyposaline conditions in these two congeneric species. For the acute hypo-saline stress experiment, we exposed each species to decreasing salinities, 100%, 85% and 70% full-strength seawater, for 6 hours followed by a 4-hour recovery at 100%. In the chronic salinity stress experiment, each species was kept at 100% or 85% with individuals removed for analysis during a 16-day time course. Organisms were dissected to remove the tunic, and 2D SDS-PAGE was performed to separate proteins and characterize changes in protein abundances. In the acute experiment, we determined 5% and 19% of the proteins to be significantly changing abundance in C. savignyi and C. intestinalis, respectively, due to the treatment effect. For both species in the chronic experiment, we determined over 40% of the proteins to be significantly changing abundance given the treatment, time, or interaction effect. Analysis of these proteins with MALDI TOF-TOF mass spectrometry has identified numerous proteins implicated in cellular stress responses, including energy metabolism (glycolysis, ATP & NADH production), cytoskeletal restructuring, and protein turnover, providing insights into the intense cellular restructuring that occurs following hypo-saline exposure.

This thesis describes the chemical investigations of several ascidian species collected from the Great Barrier Reef, Queensland, Australia. The thesis is divided into two separate components, Part A focuses on the isolation and structure elucidation of 11 previously undescribed ascidian metabolites. All structures were assigned using a combination of spectroscopic and/or chemical methods. Part B relates to the isolation and chemical conversion of a natural product to a combinatorial template. The natural product template was subsequently used in the generation of a solution-phase combinatorial chemistry library. A further two combinatorial libraries were generated from a synthesised model compound that was related to the natural product template. Part A. Investigation of Aplidium longithorax collected from the Swains Reefs resulted in the isolation of two new para-substituted cyclofarnesylated quinone derived compounds, longithorones J (30) and K (31). The former compound had its absolute stereochemistry determined by the advanced Mosher method. From an Aplidium longithorax collected from Heron Island, two new cyclofarnesylated hydroquinone compounds, longithorols C (46) and D (47) and a novel macrocyclic chromenol, longithorol E (48) were isolated. Longithorol C (46) had its absolute stereochemistry determined by the advanced Mosher method. Chemical investigation of the deep-purple colonial ascidian, Didemnum chartaceum collected from Swains Reefs led to the isolation of five new lamellarin alkaloids, which included the 20-sulfated derivatives of lamellarins B (94), C (95) and L (96), the 8-sulfated derivative of lamellarin G (97) and the non-sulfated compound, lamellarin Z (98). The known lamellarins A (63), B (80), C (64), E (65), G (67), and L (71) plus the triacetate derivatives of lamellarin D (82) and N (83) were also isolated. An aberration in the integration of signals in the 1H NMR spectra of the 20-sulfated derivatives (94-96) led to NMR relaxation studies. T1 values were calculated for all protons in the sulfated lamellarins (94-97) and their corresponding non-sulfated derivatives (80, 64, 71, 67). The protons ortho to the sulfate group in compounds (94-97) had T1 values up to five times larger than the corresponding protons in their non-sulfated derivatives (80, 64, 71, 67). A specimen of Eudistoma anaematum collected from Heron Island was shown to contain a new b-carboline alkaloid, eudistomin V (130), in addition to the two known metabolites, eudistomin H (105) and I (106). Part B. The known natural products, 1,3-diphenethylurea (29), 1,3-dimethylxanthine (30), 1,3-dimethylisoguanine (31) and the salts of tambjamine C (16), E (18) and F (19) were isolated from the ascidian, Sigillina signifera collected in Blue Lagoon, Lizard Island. Base hydrolysis on mixtures of the salts of tambjamine C (16), E (18) and F (19) resulted in the production of 4-methoxy-2,2-bipyrrole-5-carbaldehyde (26). This natural product template (26) was used in the generation of an enamine combinatorial chemistry library (98, 103-111) using solution-phase parallel synthesis. The biaryl compound, 4-(2-thienyl)-1H-pyrrole-2-carbaldehyde (59) was successfully synthesised using Suzuki-Miyaura coupling conditions and subsequently used as a template in the generation of an amine (67, 77, 80-87) and imine (78, 92-95) combinatorial library using solution-phase parallel synthesis.

The ascidian (subphylum Tunicata: class Ascidiacea) fauna along the southern African coast constitutes an important component of the sessile benthic reef fauna. Little is known of the species composition, biodiversity and distribution of ascidians on southern African intertidal and subtidal reefs. Past research on the ascidian fauna of South Africa was sporadic and limited, with only about one hundred and seventy species recorded during the last one hundred and twenty years. This is the first taxonomic study undertaken by a South African resident scientist. The study focused on six genera in four families (Euherdmaniidae, Polyclinidae, Pseudodistomidae and Didemnidae) belonging to the class Ascidiacea and suborder Aplousobranchia. Five species new to science, two Polyclinum, two Pseudodistoma and one Polysyncraton species are described. New additional information on the distribution of five species previously known to science, including one recorded for the first time along the South African coast, is presented. A literature review and comparison of the taxonomic important characteristics is made of all species known globally for the six genera. These six genera comprise twelve Euherdmania, fifteen Aplidiopsis, forty Polyclinum thirty-one Pseudodistoma, six Atriolum and sixty-eight Polysyncraton species.

The sea squirts Ciona intestinalis and C. savignyi have disparate distribution patterns, which may result from differences in their thermal tolerance limits. Because C. intestinalis, an almost cosmopolitan species, has a more widespread distribution, it is thought that it is better adapted to endure a wide range of temperatures. In order to compare the heat stress response between these two congeners, we studied global changes in protein expression, using a proteomics approach. To characterize the response to extreme heat stress, animals of both species were exposed to temperatures of 22°C, 25°C, and 28°C for 6 h, and then were left to recover at a control temperature (13°C) for 16 h. An additional experiment was conducted to assess the effect of mild-to-moderate heat stress including a 6 h exposure to temperatures of 18°C, 20°C, and 23°C, and a 16 h recovery at a control temperature (16°C). A quantitative analysis, using 2D gel electrophoresis and gel-image analysis, showed that in the high heat stress (HHS) experiment, 15% and 18% of the all protein spots detected demonstrated changes in expression in C. intestinalis and C. savignyi, respectively. In the low heat stress (LHS) experiment, 4% of the total number of proteins detected changed significantly in both C. intestinalis and C. savignyi. Using matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight mass spectrometry, we were able to identify proteins with a 65-100% success rate, depending on species. Our results indicate that C. intestinalis maintains higher baseline levels of molecular chaperones and launches a quicker response to thermal stress than C. savignyi, suggesting it may be the more thermally tolerant of the two. In addition, actins, tubulins, and ATP-synthase F1 β-subunits were the most susceptible to proteolytic degradation, which may indicate that they have relatively higher thermal sensitivities.

Estuaries worldwide are under threat from urbanisation and development and will need effective management for their successful conservation. Coastal lakes and lagoons have been identified as one of the estuary types most susceptible to human impacts largely because of their isolated nature and slow flushing times. Management of estuaries will be most effective when based on a sound scientific understanding of the patterns of distribution, biology and ecology over the full range of biodiversity of these systems, however, such an understanding is lacking for many systems and many taxa. Studies of sponges and ascidians in coastal lakes and lagoons are rare compared to other taxa, particularly in the southern hemisphere. This study represents the first detailed scientific investigation of the ecology of sponges and ascidian in coastal lakes and lagoons of southeastern Australia. Consequently, a large part of this thesis was devoted to quantifying basic patterns of distribution. I started with a pilot study to develop an effective sampling design, followed by large-scale comparisons among different types of lake, comparisons among habitats within lakes and an environmental impact study. I concluded with a manipulative experiment to examine processes responsible for smallscale patterns of distribution of sponges in seagrass meadows.In the pilot study, distributions of sponges and ascidians were quantified at a hierarchy of three spatial scales in each of two coastal lakes. Nested analyses of variance were then used to identify spatial scales at which variation was significant. Most sponges and ascidians were very patchily distributed at a range of spatial scales from 10s of metres up to 100s of kilometres. Unlike other published examples of cost– benefit analyses, very few taxa were widespread over the larger spatial scales. Cost– benefit analyses done to determine the optimal sampling design revealed inclusion of patchily distributed taxa in analyses improved the overall precision of sampling for comparisons of assemblages among lakes.Large-scale comparisons of assemblages of sponges and ascidians were made among lakes of different size (big versus small), opening regime (mostly open to the ocean versus mostly closed) and level of environmental modification (extensively modified versus more pristine). Similar to other taxa studied in coastal lakes, in general there were more species in lakes mostly open to the ocean compared to the mostly closed lakes, and importantly, no sponges and only one species of ascidian was found in the small closed lakes. There also appeared to be an effect of the level of modification of a lake with relatively smaller abundances of ascidians in extensively modified lakes, and a complete absence of sponges from one of the extensively modified lakes.Habitat-associated patterns were examined at smaller spatial scales by comparing the distributions of sponges and ascidians with the species composition and percentage cover of seagrass and macroalgae within two lakes; St Georges Basin and Wallis Lake. Several patterns of association were observed, but these varied among species of sponge and ascidian. In St Georges Basin, the most common sponge, Aplysinella cf. rhax and the native ascidian Pyura stolonifera were positively correlated with the seagrass, Posidonia australis. In contrast, the introduced ascidian, Styela plicata was more abundant in areas dominated by the seagrass, Zostera capricorni. In Wallis Lake, sponges were most diverse and some species most abundant in large beds of the macroalga, Lamprothamnion sp., while other sponges were found only on the holdfasts of brown macroalgae. In both lakes, sponges were generally less common in areas dominated by dense meadows of the seagrass, Zostera capricorni.Among the many anthropogenic impacts threatening the ecology of coastal lakes, the discharge of cooling water from coal-fired power stations represents an almost ideal case study from which to develop appropriate sampling regimes for detecting impacts on sponges and ascidians. Using reference locations both within and outside Lake Macquarie which has two cooling water outlets, I found assemblages of sponges and ascidians were often more diverse, more abundant and less temporally variable near to the outlets compared to reference locations.Based on the observation that the sponge, Suberites sp. which contains photosynthetic symbionts was absent from meadows of dense Zostera capricorni, I used in situ manipulative experiments in Smiths Lake to investigate processes which maybe affecting their distribution. Individual Suberites sp. were shaded, had water flow reduced and were transplanted into areas of dense Z. capricorni. There were no measurable short-term effects of shading or reduced water flow, but transplanted sponges were quickly eaten and I concluded predation by fish was likely to be a key process determining small-scale patterns of distribution of Suberites sp. in seagrass meadows. This result was in stark contrast to the majority of previous studies of the effects of seagrass habitat complexity on predation which have found predation to decrease with increasing density or complexity.In conclusion, I have sought to provide sound scientific information to aid in the management of these systems. A simple, but nevertheless key finding was that sponges and ascidians are indeed present and widespread in coastal lakes and lagoons of southeastern Australia and should not be continually overlooked in the management and conservation of these systems. Conservation will be complex and requires an understanding of environmental impacts and the consequences of management on the full range of biodiversity. The distributions of sponges and ascidians at large ‘lakewide’ scales appear to behave similarly to other taxa. Management strategies which change the characteristics of a lake at these large spatial scales such as artificial openings of entrances could therefore be predicted to have similar effects across a range of taxa including sponges and ascidians. In contrast, at smaller spatial scales such as the complexity of seagrass meadows, some species of sponges and ascidians may behave very differently from other taxa. At present, our understanding of these naturally variable and complex systems is incomplete and will require ongoing scientific investigation to identify natural patterns of distribution, environmental impacts, important natural processes and the ecological consequences of management strategies.

This thesis investigates the chemistry of 25 ascidian species collected from Algoa Bay, South Africa with a concerted focus on metal accumulation by these ascidians and the possible interaction of these metals with ascidian metabolites. Chapter 2 details the screening techniques employed to establish the presence of nitrogenous metabolites (1H- 15N HMBC), hyper-accumulated metal ions (ICP-MS) and potential metal ion/ ascidian metabolite complexes (LC-ICP-MS/ESI-MS). Unfortunately, exhaustive attempts to detect intact metal ion/ascidian metabolite complexes through the use of liquid chromatography with parallel inductively coupled plasma mass spectrometry/electrospray mass spectrometry (LC-ICPMS/ ESI-MS) were unsuccessful. However, the LC-ICP-MS/ESI-MS data obtained for the crude organic extracts of six of the Algoa Bay ascidian species, Distaplia skoogi, Aplidium monile, Aplidium sp., Didemnum sp., Leptoclindines sp. and Polycitor sp. enabled identification of a number of ten halogenated metabolites, namely the indoles 2.28-2.30, and the tyramine and tyrosine derivatives (2.31-2.33, 2.41, 2.43, 2.44 and 2.46), within the ascidian extracts. This study confirmed that LC-ICP-MS/ESI-MS is a powerful tool for the dereplication of halogenated metabolites in complex mixtures especially where these compounds are present in very small amounts. This study is also the first report of these compounds (eight of which are known) in African ascidians. Compounds 2.32 and 2.46 have not been reported before from a marine source. Compounds 2.28-2.30 and 2.33 were present in sufficient amounts in the respective ascidian extracts to allow their isolation and structure elucidation using standard spectroscopic techniques Chapter 3 explores the ability of ascidians to accumulate a wide range of metal ions at concentrations which are often orders of magnitude higher than those of the surrounding sea water. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the total ion concentrations of 24 metals in 25 Algoa Bay ascidian species. To the best of our knowledge this is the largest and most extensive investigation of metal concentrations in a group of different ascidians occurring in the same area. Hypotheisizing that the metal ion concentrations for each ascidian specimen screened may represent a unique fingerprint for each specimen principal component analysis (PCA) was used in an attempt to establish whether there were spatial, temporal or phylogenetic relationships associated with the metal concentration fingerprints of the ascidians that formed part of this study. The PCA results showed that there were no statistically significant relationships between ascidian metal ion concentrations and either the collection year or the collection site of the ascidians. However, species from the family Didemnidae provided the clearest statistical evidence supporting a phylogenetic relationship between these ascidians and their hyperaccumulated metal ion profiles. Furthermore, these results suggested that ascidian species are indeed actively concentrating metal ions from the surrounding sea water and are not simply sinks for passively accumulated metal ions. Interestingly, the concentration of vanadium in the set of ascidians studied did not appear to correlate with any of the other metals accumulated by these ascidians suggesting that there is possibly a unique method employed for the accumulation of vanadium by ascidians. Chapter 4 investigated this possibility further after the nucleosides 4.10, 4.11, 4.13, 4.15, 4.17 and 4.40 were isolated from the vanadium accumulating ascidian Aplidium monile. Studies into the interactions between nucleosides and vanadyl are unfortunately rare and usually qualitative in nature with limited information provided about the stability or structures of the complexes formed. The vanadyl accumulating aplousobranch ascidians e.g. Aplidium monile dominated our study of Algoa Bay ascidians therefore providing us with the rationale to investigate the relatively little studied binding ability and stability of vandyl-nucleoside complexes. Potentiometric studies were conducted to determine the stability constants of complexes formed between the oxovanadium ion vanadyl (VO2+) and the commercially available nucleosides 4.10-4.14. The data afforded by this analysis clearly confirmed the complexity of the vanadyl/nucleoside complexation and suggested that guanosine (4.12) formed the most stable complex with oxovanadium ions. We were also able to establish a third protonation constant for the hydroxyl moiety in 4.12 with a logK 8.87 which has not been previously reported. Finally, Chapter 5 revisited the cytoxicity two Algoa Bay ascidians, Clavelina sp. and Atriolum marinense the extracts from which produced promising bioactivity results in previous studies against oesophageal cancer cells. The HP-20 fractionated extracts of Clavelina sp. and Atriolum marinense proved to be similalrly cytotoxic to breast cancer cells. With the exception for the 100% acetone(aq)fractions the NMR data for both species suggested that most active non polar fractions were dominated by what appeared to be structurally unremarkable fatty acid glycerides and as such were not pursued further. Purification of the 100% acetone(aq)fraction of A. marinense resulted in the isolation of a styrene trimer, 5.1, common to both ascidian extracts. The NMR simulation software WIN-DAISY was employed to confirm the structure of 5.1. Attempts to establish if 5.1 was an isolation artefact or a product of marine pollution were inconclusive

Invasive species constitute a major threat to global biodiversity and cause important economic losses and ecological impacts. In the marine realm, ascidians include several aggressive invasive species, some of which have worldwide colonisation ranges, such as Didemnum vexillum. In this thesis, some biological and ecological characteristics implicated in the invasiveness of the species are assessed. First, we performed a 20 month monitoring to determine settlement and growth preferences of invasive ascidian species in the Ebro Delta aquaculture facility, including D. vexillum. Our results indicated that D. vexillum has a preference for complex substrates. To minimise fouling on bivalves, spat immersion during fall and below 1 m depth is recommended. To detect new introduced species, a follow-up program based on occurrences would be sufficient. Second, a protocol for small DNA samples combining whole genome amplification (WGA) and genotyping-by-sequencing (GBS) was developed and applied to D. vexillum using a single zooid per colony to determine patterns of genomic diversity and differentiation, describe the colonisation history of the species, and study its capability to form chimeras. Our results confirmed that Japan is in the native area of the species and only one clade spread worldwide. We found that the two main mitochondrial clades are strongly differentiated at the genomic level suggesting reproductive isolation, we determined that three independent colonisation events shaped the global distribution of the species, and we found that populations are diverse and well differentiated indicating a high expansion potential of D. vexillum. Third we detected high prevalence of chimerism, and fusion was unlinked to global genetic relatedness. Finally, we analysed the microbiome of D. vexillum that showed markedly different composition than a congeneric species and water. The invasive clade had a small but abundant core and a highly diverse variable microbiome component with a strong capacity to enrich the symbionts from the environment. The microbiome structure correlated to host genetic distance, temperature and geographical distances, pointing to vertical and horizontal transmission. In conclusion, D. vexillum is an aggressive invasive species with a high adaptive capacity that may contribute to the invasiveness of this global pest.

Marine non-native species threaten economic and environmental health, making it crucial to understand factors that make them successful. Research on these species, therefore, allows for greater preparedness and informed management of biological invasions and increases understanding of elements structuring biological communities. Among the marine non-native species, and particularly the fouling community, non-native ascidians are a taxon of particular concern because they can crowd out native benthic species and smother mariculture products. This thesis addresses management for ascidians and other fouling organisms and includes research on the invasiveness of this taxon in addition to the invasibility of recipient fouling communities. On the West Coast of the U.S., limited efforts have been made to coordinate biofouling management across states, despite the myriad vectors increasing propagule pressure over time along coastal states. Building on recent state and local efforts, I developed a Pacific Regional Biofouling Plan for the states of Oregon and Washington to help start a consensus-driven process by which these states could create a forum for more comprehensive coordination efforts, following California's lead. As states address authority gaps, the biofouling management framework I've written is meant to be used to guide the conversation between managers as various stages of coastal management are realized. To better inform the scope and efficacy of management and regulatory efforts, the study of invasions ecology asks and aims to answer questions regarding recipient community interactions and characteristics of the non-native species themselves. Studies that identify characteristics that make ascidians successful (invasiveness) and determine the influence native communities have on their success (invasibility) are important for assessing overall risk of establishment and spread from non-native ascidians. Therefore, I aimed to: 1) explore the hypothesis that fouling communities on suspended, artificial structures are more invasible than benthic habitats; and 2) identify characteristics influencing predation patterns on the native Distaplia occidentalis versus non-native ascidian species using mensurative and experimental studies in Charleston Marina, Oregon. I conducted a series of feeding assays, surveys, and a caloric content analysis. Feeding assays were conducted with a suite of predators. The flatworm predator (Eurylepta leoparda) was found to be highly selective on the native ascidian Distaplia occidentalis, and only preyed on whole colony samples. Feeding assay data suggest that test (tunic) structure or thickness may be an influential factor affecting nudibranch (Hermissenda crassicornis) predation rates on native versus non-native ascidians, with greater predation on the native ascidian species. Non-native ascidians may escape predation in floating but not benthic environments on the Oregon coast due to their palatability characteristics, likely tunic structure and low caloric content. In this case, this suite of predators may indirectly facilitate the invasion of docks but provide at least partial resistance to the invasion of natural benthic areas. The chapters herein address gaps in management and scientific knowledge regarding non-native species of the marine fouling community. Future work enhanced by my efforts could include the development of the coastal biofouling management plan, coordinated by the Western Regional Panel on Aquatic Invasive Species Coastal Committee, and broadening the geographic and taxonomic scope of my research with a more comprehensive study of predator-prey interactions involving non-native ascidians and a diverse suite of predators. These interactions may be an important factor in explaining the success of ascidians and other fouling organisms on floating structures and lack of success on nearby benthic substrata.

This thesis describes the chemical investigations of several ascidian species collected from the Great Barrier Reef, Queensland, Australia. The thesis is divided into two separate components, Part A focuses on the isolation and structure elucidation of 11 previously undescribed ascidian metabolites. All structures were assigned using a combination of spectroscopic and/or chemical methods. Part B relates to the isolation and chemical conversion of a natural product to a combinatorial template. The natural product template was subsequently used in the generation of a solution-phase combinatorial chemistry library. A further two combinatorial libraries were generated from a synthesised model compound that was related to the natural product template. Part A. Investigation of Aplidium longithorax collected from the Swains Reefs resulted in the isolation of two new para-substituted cyclofarnesylated quinone derived compounds, longithorones J (30) and K (31). The former compound had its absolute stereochemistry determined by the advanced Mosher method. From an Aplidium longithorax collected from Heron Island, two new cyclofarnesylated hydroquinone compounds, longithorols C (46) and D (47) and a novel macrocyclic chromenol, longithorol E (48) were isolated. Longithorol C (46) had its absolute stereochemistry determined by the advanced Mosher method. Chemical investigation of the deep-purple colonial ascidian, Didemnum chartaceum collected from Swains Reefs led to the isolation of five new lamellarin alkaloids, which included the 20-sulfated derivatives of lamellarins B (94), C (95) and L (96), the 8-sulfated derivative of lamellarin G (97) and the non-sulfated compound, lamellarin Z (98). The known lamellarins A (63), B (80), C (64), E (65), G (67), and L (71) plus the triacetate derivatives of lamellarin D (82) and N (83) were also isolated. An aberration in the integration of signals in the 1H NMR spectra of the 20-sulfated derivatives (94-96) led to NMR relaxation studies. T1 values were calculated for all protons in the sulfated lamellarins (94-97) and their corresponding non-sulfated derivatives (80, 64, 71, 67). The protons ortho to the sulfate group in compounds (94-97) had T1 values up to five times larger than the corresponding protons in their non-sulfated derivatives (80, 64, 71, 67). A specimen of Eudistoma anaematum collected from Heron Island was shown to contain a new b-carboline alkaloid, eudistomin V (130), in addition to the two known metabolites, eudistomin H (105) and I (106). Part B. The known natural products, 1,3-diphenethylurea (29), 1,3-dimethylxanthine (30), 1,3-dimethylisoguanine (31) and the salts of tambjamine C (16), E (18) and F (19) were isolated from the ascidian, Sigillina signifera collected in Blue Lagoon, Lizard Island. Base hydrolysis on mixtures of the salts of tambjamine C (16), E (18) and F (19) resulted in the production of 4-methoxy-2,2-bipyrrole-5-carbaldehyde (26). This natural product template (26) was used in the generation of an enamine combinatorial chemistry library (98, 103-111) using solution-phase parallel synthesis. The biaryl compound, 4-(2-thienyl)-1H-pyrrole-2-carbaldehyde (59) was successfully synthesised using Suzuki-Miyaura coupling conditions and subsequently used as a template in the generation of an amine (67, 77, 80-87) and imine (78, 92-95) combinatorial library using solution-phase parallel synthesis.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
Full Text

Nous proposons dans cette thèse de caractériser quantitativement la variabilité à différentes échelles au cours de l'embryogenèse. Pour ce faire, nous utilisons une combinaison de modèles mathématiques et de résultats expérimentaux. Dans la première partie, nous utilisons une petite cohorte d'oursins digitaux pour construire une représentation prototypique du lignage cellulaire, reliant les caractéristiques des cellules individuelles avec les dynamiques à l'échelle de l'embryon tout entier. Ce modèle probabiliste multi-niveau et empirique repose sur les symétries des embryons et sur les identités cellulaires; cela permet d'identifier un niveau de granularité générique pour observer les distributions de caractéristiques cellulaires individuelles. Le prototype est défini comme le barycentre de la cohorte dans la variété statistique correspondante. Parmi plusieurs résultats, nous montrons que la variabilité intra-individuelle est impliquée dans la reproductibilité du développement embryonnaire. Dans la seconde partie, nous considérons les mécanismes sources de variabilité au cours du développement et leurs relations à l'évolution. En nous appuyant sur des résultats expérimentaux montrant une pénétrance incomplète et une expressivité variable de phénotype dans une lignée mutante du poisson zèbre, nous proposons une clarification des différents niveaux de variabilité biologique reposant sur une analogie formelle avec le cadre mathématique de la mécanique quantique. Nous trouvons notamment une analogie formelle entre l'intrication quantique et le schéma Mendélien de transmission héréditaire. Dans la troisième partie, nous étudions l'organisation biologique et ses relations aux trajectoires développementales. En adaptant les outils de la topologie algébrique, nous caractérisons des invariants du réseaux de contacts cellulaires extrait d'images de microscopie confocale d'épithéliums de différentes espèces et de différents fonds génétiques. En particulier, nous montrons l'influence des histoires individuelles sur la distribution spatiales des cellules dans un tissu épithélial
We propose in this thesis to characterize variability quantitatively at various scales during embryogenesis. We use a combination of mathematical models and experimental results. In the first part, we use a small cohort of digital sea urchin embryos to construct a prototypical representation of the cell lineage, which relates individual cell features with embryo-level dynamics. This multi-level data-driven probabilistic model relies on symmetries of the embryo and known cell types, which provide a generic coarse-grained level of observation for distributions of individual cell features. The prototype is defined as the centroid of the cohort in the corresponding statistical manifold. Among several results, we show that intra-individual variability is involved in the reproducibility of the developmental process. In the second part, we consider the mechanisms sources of variability during development and their relations to evolution. Building on experimental results showing variable phenotypic expression and incomplete penetrance in a zebrafish mutant line, we propose a clarification of the various levels of biological variability using a formal analogy with quantum mechanics mathematical framework. Surprisingly, we find a formal analogy between quantum entanglement and Mendel’s idealized scheme of inheritance. In the third part, we study biological organization and its relations to developmental paths. By adapting the tools of algebraic topology, we compute invariants of the network of cellular contacts extracted from confocal microscopy images of epithelia from different species and genetic backgrounds. In particular, we show the influence of individual histories on the spatial distribution of cells in epithelial tissues

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015
Exotic species have been considered by several authors as the worst threat that can occur to native biodiversity. Due to the lack of information on sea squirts and barnacle invaders in southern Portugal, the first step of this study aims to detect and identify their presence in the ports of the region. A comparison reporting differences between these ports was performed: i) fishing and recreational; ii) port location; iii) sheltered versus unsheltered facilities. The localities of Lagos, Quarteira-Vilamoura, Faro and Olhão were the study areas where seasonal sampling was done. A case study to obtain the total recruitment for the invasive species was also carried out, comparing surfaces and removal of invaders. Higher percentages of invasive species were found (60% for barnacles and 58.3% for sea squirts). The appearance of one invasive gastropod, originating in the West African coast, was reported in European waters the first time. Besides, for barnacle and sea squirt counting, there were significant differences in all comparisons except between seasons for barnacles and type of port for sea squirts. The main differences in the abundance, frequency of occurrence and communities seems to be related with the transit of international vessels, the settlement period and the conditions presented between ports in terms of exposure to the sea. Differences between surfaces during the case study were found, showing wallets as the best surface for sea squirts and cement plates for barnacles in general. Cement can be considered as the best surface for recruitment for the invasive Amphibalanus amphitrite in relation with the huge abundance found. The other two invasive Amphibalanus cf. eburneus and Styela plicata found during the experiment, did not present any preference between surfaces. So in general for the three invasive species of this area the use of cement plate guarantees a high rate of recruitment.

碩士
國立中山大學
海洋生物科技暨資源學系研究所
97
Complete DNA sequences were determined for the mitochondrial (mt) genomes of the sea anemones, Mesactinia genesis （20,544 bp）, Heteractis aurora (19,800 bp) and partial mtDNA between cox2 and nad4 of the Calliactis sp. (3,713 bp). In addition, complete mtDNA sequences were determined for the sea squirt, Eudistoma gilboviride (14,203 bp). The circular, sea anemones genomes contain the genes for 13 energy pathway proteins and two ribosomal RNAs and two transfer RNAs. H. aurora contains a previously undescribed ORF between the cox2 and nad4 genes encoding a putative protein of 646 amino acids. In M. geneisis and Calliactis sp. encodes two separate smaller ORFs of 259 and 243 as well as 269 and 345 in the corresponding regions, respectively. Possible control region of the mitochondrial genomes of M. geneisis and H. aurora were identified in the intergenic region 13. The sea squirt genomes contain the genes for 11 energy pathway proteins and two rRNAs and 22 tRNAs. All genes are encoded by the heavy strand, except for trnM, trnK, and trnV, which are encoded by the light strand. The ascidians showed frequent and extensive gene rearrangement. The gene order in E. gilboviride are very much different from the other ascidians mt-genome. The E. gilboviride mtDNA does not encode the nad6 and a tpyical atp8. Molecular phylogenetic analyses based on nucleic acid and amino acid sequences of the deuterostome (echinoderms, chordate and Xenoturbella), and cnidaria coincide with the morphological characters.

Tese de mestrado em Ecologia Marinha, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
O oceano constitui grande parte do planeta, acomodando cerca de 1 368 milhões Km3 de água, providencia mais espaço que todos os habitats terrestres. Apesar da maioria da biodiversidade marinha se encontrar na zona fótica e menos profundas, uma grande variedade vive nas partes mais profundas e sem luz. Em algumas partes do oceano, entre os 10 e os 1 300m, existem zonas de oxigénio mínimo (ZOM). Estes habitats pelágicos apresentam condições estáveis de níveis baixos e persistentes de oxigénio e baixas temperaturas ao longo de vastas áreas. Estes habitats resultam de uma combinação de uma fraca ventilação assim como pouca circulação da água. A maioria das ZOM exibe um perfil de oxigénio semelhante, mas os níveis de oxigénio, espessura e a profundidade de ocorrência podem variar regionalmente. No Atlântico Tropical Oriental, a uma profundidade entre os 200m a 800m, existe uma zona mínima de oxigénio localizada entre o sistema da Corrente Equatorial (a Sul) e a Corrente Equatorial Norte (a Norte). Aqui, o perfil de oxigénio tem dois mínimos a cerca de 70m e 400m de profundidade, com o mínimo mais proeminente entre o Senegal e a Ilha de Cabo Verde. Os cefalópodes (classe Cephalopoda) podem ser encontrados em todos os oceanos do mundo, desde águas costeiras ao mar profundo, e algumas espécies conseguem viver em condições extremas, como as fontes hidrotermais e as ZOM. A maioria destes moluscos são predadores oportunistas com altas taxas de crescimento, uma única época de reprodução e uma esperança média de vida curta nas zonas costeiras. No mar profundo, estas características biológicas são bastante diferentes, dado que as suas capacidades locomotoras e requisitos metabólicos são mais reduzidos. De uma forma geral, os cefalópodes desempenham um papel fundamental nos ecossistemas marinhos, uma vez que são uma fonte primária de alimento para muitos predadores marinhos, como peixes, mamíferos marinhos ou aves marinhas. Os cranchídeos (família Cranchiidae) estão entre as lulas mais abundantes do mundo, de grande diversidade morfológica em ambientes pelágicos. A maioria delas têm uma aparência transparente (por isso designadas também por lulas de vidro) dado possuírem grandes cavidades celómicas cheias de fluido amoniacal neutro e de baixa densidade, que lhes confere flutuabilidade neutra. Embora a sua morfologia e anatomia estejam bem estudadas, e de serem muito abundantes, pouco se sabe sobre a sua biologia e ecologia. Muitas das espécies de cranchídeos passam grande parte de sua vida em águas onde quase não há luz ou em completa escuridão. A maioria desses pequenos animais não depende da velocidade para escapar de predadores, mas sim de outras estratégias defensivas, - a sua transparência, que sob condições de iluminação adequadas, as tornam praticamente invisíveis. A poluição marinha tornou-se um problema muito grave durante as últimas décadas, devido aos seus impactos na biodiversidade e no funcionamento dos ecossistemas. A presença de concentrações acima do normal de contaminantes já foi detetada em vários tipos de habitats marinhos, nomeadamente no mar profundo, onde a preocupação tem vindo a aumentar devido à possibilidade de este, devido à sua dimensão, se poder tornar no maior depósito para tais elementos. A bioacumulação tem sido amplamente estudada em muitos organismos marinhos e demonstrou-se que os moluscos, como os cefalópodes, têm a capacidade de acumular altos níveis de contaminantes. As maiores concentrações determinadas são o carbono, hidrogénio, azoto, oxigénio e enxofre, que são elementos estruturais. Outros elementos encontrados podem ser essenciais, cujo papel biológico é conhecido, como ferro, cobre, zinco, iodo, manganês, selénio ou flúor. Os não essenciais, não têm um papel conhecido nas funções fisiológicas, como ocorre com mercúrio, chumbo e cádmio. Embora existam estudos sobre a acumulação de elementos em cefalópodes, a maioria está focada em espécies comerciais. Entre essas espécies, foram geralmente registados altos níveis de metais, como o cádmio, o cobre e o zinco, tornando os cefalópodes uma potencial via de transporte de contaminantes para níveis tróficos superiores. A absorção de contaminantes por organismos marinhos pode ocorrer a partir da água, incluindo partículas em suspensão, alimentos e sedimentos. Outros fatores, como a disponibilidade de elementos na água, período de exposição, temperatura, tamanho, sexo, estágio de maturidade e local de captura, também são importantes na acumulação de metais nos tecidos destes organimsos. É importante referir que dado que os cefalópodes apresentam uma vida curta e uma alta acumulação de contaminantes, estes organismos podem refletir as condições actuais do ambiente onde vivem e serem utilizados como indicadores de contaminação ambiental. Embora não haja informações sobre contaminantes nos cranchídeos, existe informação sobre outras espécies de cefalópodes, incluindo outras lulas oceânicas. A maioria dos estudos mostrou a capacidade dos cefalópodes de concentrar Zn, Cu e Cd na glândula digestiva mesmo em ambientes com baixa disponibilidade. Neste contexto, o objetivo desta dissertação, foi determinar, pela primeira vez, as concentrações de alguns elementos (V, Cr, Co, Ni, Cu, Zn, As, Se, Cd e Pb) nos tecidos (glândula digestiva, manto e tentáculos) de nove espécies de cranchídeos, da ZOM de Cabo Verde. As lulas foram apanhadas na zona económica exclusiva de Cabo Verde, em oito estações de amostragem, através da utilização de redes pelágicas MOCNESS e IKMT em diferentes profundidades. Os indivíduos capturados foram identificados até à espécie (quando possível) e preservados em azoto líquido abordo do RV Maria S. Merian e depois transferidas para -80ºC no laboratório. Os indivíduos foram dissecados de modo a separar o manto, os tentáculos e a glândula digestiva. Neste trabalho, foram detectadas diferenças significativas entre espécies de cranchídeos e entre os diferentes tecidos analisados. De uma forma geral, os elementos na glândula digestiva variaram entre os 0,070 e 17 μg g-1 peso seco, com as concentrações variando da seguinte forma decrescente: Zn > Cd > As > Cu > V > Se > Co > Cr > Ni > Pb. No manto variaram entre os 0,070 e 28 μg g-1 peso seco, com as concentrações variando da seguinte forma decrescente: Zn > Cu > Cr > As > V > Ni > Se > Cd > Pb > Co. Nos tentáculos, os contaminantes variaram entre os 0,13 e os 24 μg g-1 peso seco, com as concentrações variando da seguinte forma decrescente: Se > Zn > As > Cu > Cr> Ni > V > Cd > Co > Pb. Estes resultados em comparação com os de outros grupos de cefalópodes (nomeadamente chocos, polvos e outras lulas pelágicas) mostraram que as espécies pelágicas (lulas) apresentavam menores concentrações de Cd do que as espécies bentónicas (polvos) ou nectobentónicas (chocos). Essas diferenças podem ser atribuídas às diferentes concentrações de Cd nas suas presas, uma vez que as presas bentônicas (crustáceos e bivalves) têm maiores concentrações de Cd, do que os peixes que são presas preferenciais de espécies pelágicas. Em relação às espécies nectobentónicas e bentónicas, os sedimentos também podem ser uma via de acumulação - embora a transferência direta seja menor, podem atuar como uma fonte indireta de contaminantes. Por último, as diferenças com os nautilus podem estar relacionadas com diferentes tempos de vida, uma vez que os nautilus vivem entre os 10 e os 15 anos, enquanto os Coleoids (choco, lulas e polvos) vivem entre 1 a 3 anos. Embora a expectativa de vida da maioria dos cranchídeos não seja conhecida, não é provável que os indivíduos estudados atinjam essas idades. É importante notar que os músculos dos cranchídeos apresentaram maior variação e uma maior concentração do que as outras espécies de cefalópodes. Estes resultados não eram esperados e levanta a questão porque é que os cranchídeos concentram elevados níveis de alguns elementos nestes tecidos. A capacidade dos cefalópodes para concentrar altos níveis de metais sem um sinal de toxicidade, está associada a eficientes processos de desintoxicação que permitem reter os elementos de forma não metabolicamente disponível, limitando assim a sua toxicidade. Neste contexto, a glândula digestiva é um órgão complexo envolvido em várias funções, como digestão, secreção e desintoxicação (entre outros). Por ser considerado um tecido de armazenamento, é também um órgão-chave para a desintoxicação. Não surpreendentemente, mas com algumas exceções (por exemplo, Hg e As), a maioria dos contaminantes são encontrados em concentrações mais elevadas neste tecido. Pensa-se que inicialmente os contaminantes estão ligados a proteínas solúveis, o que implica que as células da glândula digestiva provavelmente estarão envolvidas na desintoxicação dos contaminantes. Esta associação com proteínas citosólicas, inibe as interações tóxicas de iões metálicos com locais de ligação sensíveis (proteínas, moléculas ou estruturas celulares). Além disso, uma vez que os metais como Ag, Cd, Cu, Hg e Zn têm uma alta afinidade para as metalotioninas, pensa-se que estas desempenham um papel fundamental na homeostasia dos metais essenciais, bem como um papel importante na tolerância dos organismos a elementos não-essenciais. Estudos anteriores mostraram que não é só o Cd, mas também outros contaminantes, como Co, Cr, Ni e V, são encontrados em concentrações mais elevadas na glândula digestiva do que no músculo. No entanto, nos cranchídeos, e apesar de o Co ter apresentado concentrações mais elevadas na glândula digestiva, o V, Cr e Ni apresentaram valores mais elevados no manto. Quanto às diferenças entre manto e tentáculos, estas podem ser devido a diferentes composições proteicas e respectivos sistemas enzimáticos, podendo explicar os resultados obtidos com os cranchídeos Em suma, o fato das lulas de vidro, que prosperam em ambientes pelágicos profundos, exibirem concentrações de contaminantes semelhantes aos encontrados nos cefalópodes costeiros (cujos habitats estão sujeitos a uma maior intervenção humana), é inesperado. Estes dados corroboram a ideia corrente de que esses ambientes remotos estão-se a tornar o principal acumulador de contaminantes no planeta.
Trace elements are persistent and have been detected in a wide range of environments, including the deep-sea, where the concern is increasing, as it might act as a global sink for them. One group of cephalopods that thrive in such harsh environments, including the mesopelagic oxygen minimum zones, are the cranchiid (glass) squids. Although their anatomy and morphology is well understood, little is known about their biology and ecology. Elemental bioaccumulation has been greatly studied in many marine organisms and it has been shown that cephalopods have the ability to accumulate high levels of elements. The uptake of trace elements can occur from water, including suspended particulate matter, food and sediments. Because cephalopods display a short life span and high accumulation of trace elements, these characteristics may clearly reflect the ambient life conditions and indicators of environmental contamination. Yet, up to our knowledge, there is no information about trace elements in the deep cranchiid squids. Within this context, the aim of the present dissertation was to determined, for the first time, the concentrations of V, Cr, Co, Ni, Cu, Zn, As, Se, Cd and Pb in the digestive gland, mantle and tentacles of nine different cranchiid squids from the Cape Verde Exclusive Economic Zone in the Eastern Atlantic Ocean. Concomitantly, a comprehensive comparison with trace elements obtained in shallow-living cephalopod species (including cuttlefish, octopods and other squids) was also conducted. In general, trace elements in cranchiid digestive gland ranged between 0.07 and 17 μg-1 dw, with the following average concentrations in descending order: Zn > Cd > As > Cu > V > Se > Co > Cr > Ni > Pb. In the mantle ranged between 0.07 and 28 μg g-1 dw, with the following average concentrations in descending order: Zn > Cu > Cr > As > V > Ni > Se > Cd > Pb > Co. In the tentacles, trace elements ranged between 0.13 and 24 μg g-1 dw, with the following average concentrations in descending order: Se > Zn > As > Cu > Cr> Ni > V > Cd > Co > Pb. The high Cd levels in cranchiid squids may be related to the enriched environment around Cape Verde islands, with dissolved Cd being reinjected in the water column by the upwelling of subsurface waters. Additionally, another route of contamination could be from the Saharian dust deposition, which represents an important source of trace elements (Co, Ni, Zn and Cd) into the Atlantic Ocean. In the present work, it also became evident that benthic cephalopod species showed higher concentrations of Cd than the pelagic conspecifics. These differences could be attributed to distinct trophic ecologies (different trace elements concentrations on their preys). Previous studies have also shown that not only the Cd but also other trace elements, like Co, Cr, Ni and V are found at higher concentrations in the digestive gland (the major storage site and a key organ for detoxification) than in the muscle. These findings were not corroborated here, since while Cd and Co levels were higher concentrations in the digestive gland, V, Cr and Ni have higher concentration values in the mantle. Last, it is worth noting that the fact that glass (transparent) squids that thrive in deep pelagic environments display trace elements concentrations as high as those found in coastalcephalopods, which live in habitats exposed to enhanced anthropogenic forcing, is quite surprising. These findings corroborate the on-going notion that such remote environments are now the major global sink for contaminants in the planet.