Thesis (PhD)--University of South Australia, 2001.

The discovery of deep-sea chemosynthesis-based ecosystems including hydrothermal vents and cold seeps has greatly expanded our view of life on Earth. Nevertheless, for many benthic organisms in these ecosystems, little is known about where they come from, how scattered populations are connected by larval dispersal, and how they adapt to the local environments. Mussels of Bathymodiolus platifrons (Bivalvia: Mytilidae) are one of the dominant and foundation species in deep-sea chemosynthesis-based ecosystems. They are known to have a wide geographic distribution, and are also one of the few deep-sea species capable of living in both hydrothermal vents [in Okinawa Trough (OT)] and methane seeps [in the South China Sea (SCS) and Sagami Bay (SB)]. Previous population genetics studies of B. platifrons mostly relied on one to several genes, which suffered from the lack of sensitivity required to resolve their fine-scale genetic structure, and were unable to reveal their adaptation to the local environments. With the repaid development of molecular techniques, it is now possible to address their demographic mechanisms and local adaptation from a genome-wide perspective. Therefore, in the first part of my thesis, I aimed to generate genome-wide single nucleotide polymorphisms (SNPs) for B. platifrons via a combination of genome survey sequencing and the type IIB endonuclease restriction-site associated DNA (2b-RAD) approach, assess the potential use of SNPs in detecting fine-scale population genetic structure and signatures of diversifying selection, as well as their cross-species application in other bathymodioline mussels. Genome survey sequencing was conducted for one individual of B. platifrons. De novo assembly resulted in 781 720 sequences with a scaffold N50 of 2.9 kb. Using these sequences as a reference, 9307 genome-wide SNPs were identified from 28 B. platifrons individuals collected from a methane seep in the SCS and a hydrothermal vent in the middle OT (M-OT), with nine outlier SNPs showed significant evidence of diversifying selection. The small FST value (0.0126) estimated based on the neutral SNPs indicated high genetic connectivity between the two populations. However, the permutation test detected significant differences (P < 0.00001), indicating the two populations having clearly detectable genetic differentiation. The Bayesian clustering analyses and principle component analyses (PCA) performed based on either the neutral or outlier SNPs also showed that the two populations were genetically differentiated. This initial study successfully demonstrated the applicability of combining genome sequencing and 2b-RAD in population genomics studies of B. platifrons. Besides, using the survey genome of B. platifrons as a reference, a total of 10 199, 6429, and 3811 single nucleotide variants (SNVs) were detected from three bathymodioline mussels Bathymodiolus japonicus, Bathymodiolus aduloides, and Idas sp. These results highlighted the potential of cross-species and cross-genus applications of the B. platifrons genome for SNV/SNP identification among different bathymodioline lineages, which can be further used in various evolutionary and genetic studies. To have a deeper understanding of how individuals of B. platifrons are connected among and adapt to their habitats, in the second part of my thesis, I used both mitochondrial genes and genome-wide SNPs to conduct a more comprehensive population genetics/genomics study of B. platifrons. Three mitochondrial genes (i.e. atp6, cox1, and nad4) and 6398 SNPs generated by 2b-RAD were obtained from 110 B. platifrons individuals from six representative locations along their known distribution range in the Northwestern Pacific. The small FST values based on both types of genetic markers all revealed high genetic connectivity of B. platifrons, which may have been driven by the strong ocean currents (i.e. Kuroshio Current, North Pacific Intermediate Water). However, when using SNP datasets rather than mitochondrial genes, individuals in the SCS were identified as a distinct genetic group, indicating the Luzon Strait may serve as a dispersal barrier that limits their larval exchange between the SCS and the open area in the Northwestern Pacific. Moreover, a genetic subdivision of B. platifrons in the southern OT (S-OT) from those in M-OT and SB was observed when using 125 outlier SNPs for data analyses. The outlier-associated proteins were found to be involved in various biological processes, such as DNA and protein metabolism, transcription and translation, and response to stimulus, indicating local adaptation of B. platifrons even they are confronted with extensive gene flow in the OT-SB region. Furthermore, by using SNP datasets, populations in S-OT were revealed to be the source of gene flow to those in the SCS, M-OT, and SB. Overall, these results offered novel perspectives on the potential forces that may have led to the genetic differentiation and local adaptation of B. platifrons, which can serve as an example for other deep-sea species with high dispersal potential, and contribute to the designation of marine protected areas and conservation of deep-sea chemosynthesis-based ecosystems. Molluscan shell formation is one of the most common and abundant biomineralization processes in metazoans. Although composed of less than 5 wt% of the molluscan shells, shell matrix proteins (SMPs) are known to play multiple key roles during shell formation, such as providing a gel-like micro-environment to favour mineral precipitation, promoting crystal nucleation, as well as guiding and inhibiting crystal growth. To date, all studies on SMPs have focused on molluscs in terrestrial and shallow-water ecosystems with no reports for those living in the deep ocean. Herein, the third part of my thesis was to study the shell proteomes of B. platifrons and its shallow-water relative Modiolus philippinarum with the aim to bridge such knowledge gaps in biomineralization studies. A total of 94 and 55 SMPs were identified from the shell matrices of B. platifrons and M. philippinarum, respectively, with 31 SMPs shared between two species. These SMPs can be assigned into six broad categories, comprising calcium binding, polysaccharide interaction, enzyme, extracellular matrix-related proteins, immunity-related proteins, and those with uncharacterized functions. Many of them, such as tyrosinases, carbonic anhydrases, collagens, chitin-related proteins, peroxidases, as well as proteinase and proteinase inhibitor domain-containing proteins, have been widely found in molluscan shell matrices and other metazoan calcified tissues (e.g. exoskeletons of corals, tubes of tubeworms), whereas some others, such as cystatins, were found for the first time in molluscan shell matrices, and ferric-chelate reductase-like proteins and heme-binding proteins were to be detected for the first time in metazoan calcified tissues. This is the first report of the shell proteome of deep-sea molluscs, which will support various follow-up studies to better understand the functions of these SMPs, especially in relation to environmental adaptation. Overall, my population genetics/genomics studies have improved our understanding of the population dynamics, genetic connectivity, fine-scale genetic structure, and local adaptation of B. platifrons in the Northwestern Pacific, and my proteomics study has shed light on the biomineralization processes of molluscs in the deep ocean.

M.Sc. / Abalone are among the world’s leading shellfish consumed by human populations. Harvesting in California began in the late 1800s from intertidal zones and in the early 1900s wild abalone were collected by diving. Popular demand for abalone products in the Far East then led to extensive harvesting of wild abalone and a drastic decline in population numbers. This problem was overcome to a degree by the development of land-based abalone farms. At these farms it was possible to breed abalone on a large scale. Currently twelve abalone farms operate in South Africa and the estimated production for 2006 was 537 tons of meat, worth R 80 mil. Parasites and diseases pose threats to the production of abalone, especially under farmed conditions, and can cause considerable financial loss. Labyrinthuloides haliotidis, Haplosporidium nelsoni and Terebrasabelle heterouncinata are a few parasites that contribute to the above mentioned problems. Lately, a new protozoan parasite was discovered in the digestive glands of Haliotis midae farmed in the Western Cape Province, during routine health assessments. For the purposes of this dissertation it is designated an unidentified digestive gland parasite (UDP). The aims of this study are thus to undertake a comprehensive literature review of parasites infecting wild and farmed abalone, as well other shellfish species, describe and characterise the UDP infecting the digestive gland of Haliotis midae based on its structure and ultrastructure, evaluate the role of this parasite in disease by analysing data from histological studies, provide a preliminary indication of the life cycle of this parasite, attempt analysis of DNA from the UDP, and identify potential areas for further research into control of the parasite. A total of 180 abalone, (Haliotis midae) were collected from three abalone farms in the Western Cape during May 2005, October 2005, January 2006 and January 2007. To establish whether this parasite also occurs in wild abalone, a single sampling (six H. midae and 28 H. spadicea) took place during 2006 in Tsitsikamma National Park. Collected farmed and wild abalone were weighed and measured, removed from their shells and then killed according to accepted methods before their digestive glands were removed.

Les dinoflagellés sont des microorganismes marins (phytoplanctons) qui ont démontrés être source importante de toxines. Une attention particulière a été donnée au genre Amphidinium, qui a la particularité de vivre en symbiose avec des vers plats marins (plathelminthe) présents dans la baie d’Okinawa. L’équipe du professeur Kobayashi a isolé différentes espèces d’Amphidinium et élucidé les structures de ces métabolites secondaires: les amphidinolides. Kobayashi et ses collaborateurs ont ensuite déterminé l’activité cytotoxique de ces molécules. En particulier, nous nous sommes tournés vers la synthèse de l’amphidinolide N car il a montré une puissante activité cytotoxique : CI50= 0.08nM sur cellules L1210 (lymphome), CI50= 0.09 nM sur cellules KB (carcinome). Afin d’obtenir cette molécule, nous avons développé une stratégie convergente basée sur l’assemblage de quatre fragments principaux : C1-C5, C6-C12, C13-C17 et C18-C29. Le fragment C1-C5 a été synthétisé en suivant deux stratégies différentes : la première nous a donné le composé souhaité en six étapes avec un rendement global de 8,4%, la deuxième stratégie a permis d’obtenir ce fragment en sept étapes avec un rendement global de 3,8%. Les deux procédures ont permis la formation de ce synthon en contrôlant la configuration des centres stéréogènes, néanmoins la deuxième stratégie a permis l’obtention d’intermédiaires plus stables. Le synthon C6-C12 a pu être formé au bout de dix étapes avec un rendement global satisfaisant de 9,4%. La chimie développée est robuste et les réactions sont répétables avec des bons rendements. Le fragment C13-C17 a été formé en trois étapes avec un rendement global de 40,6% ; la configuration absolue a été assurée par le produit naturel utilisé au cours de la synthèse. Le dernier fragment, C18-C29, a pu être synthétisé au bout de neuf étapes avec un rendement global de 19,4% à partir de l’acide D-glutamique, produit d’origine naturelle. L’assemblage des fragments C1-C5 et C6-C12 a été réalisé grâce au couplage de Stille. Malgré tout, il faudrait envisager une solution alternative pour l’obtention du fragment C1-C12 à cause de la mauvaise reproductibilité du couplage de Stille. La partie C13-C29 a pu être couplée avec succès grâce à des réactions d’ouverture d’époxydes. D’autres études et essais seraient requis pour coupler les deux macro-fragments (C1-C12 et C13-C29) et terminer ainsi la synthèse totale de l’amphidinolide N. / The dinoflagellates are marine organisms (phytoplancton) that have shown to be a major source of toxins. Special attention was given to gender Amphidinium, which has the particularity of living in symbiosis with marine flatworms present in the bay of Okinawa (Japan). The team of professor Kobayashi has isolated different species of Amphidinium and elucidated the structures of their secondary metabolites: the amphidinolides. Kobayashi and colleagues then determined the cytotoxic activity of these molecules. In particular we decided to synthesize amphidinolide N because of his cytotoxic activity: IC50 = 0.08 nM on L1210 cells (lymphoma), IC50 = 0.09 nM on KB cells (carcinoma). To achieve this molecule we have developed a strategy based on a convergent assembly of four main fragments: C1-C5, C6-C12, C13-C17 and C18-C29. C1-C5 fragment has been synthesized following two different strategies: the first one gave us the desired compound in six steps and with a global yield of 8.4%, the second strategy allowed us to obtain this fragment in seven steps with a global yield of 3.8%. The two procedures gave us the C1-C5 portion controlling the configurations of the stereogenic centres, although the second strategy gave us the possibility to obtain more stables intermediates. C6-C12 fragment has been obtained in ten steps with a good global yield of 9.4%. The chemistry developed is strong; the reactions are repeatable with good results. C13-C17 fragment has been synthesized in three steps with a global yield of 40.6%; the absolute configuration has been assured by the natural compound used as starting material. The last fragment, C18-C29, has been obtained from natutal D-glutamic acid in nine steps, with a global yield of 19.4%. The assembly of C1-C5 fragment and C6-C12 has been realized with Stille’s coupling, although an alternative strategy is needed because of the lack of reproducibility. C13-C29 fragment has been successfully obtained by epoxydes opening reactions. Further studies and trial are needed to couple the two macro-fragments (C1-C12 and C13-C29) and finish the total synthesis of amphidinolide N.

Philosophiae Doctor - PhD / Marine organisms including algae and bacteria are known to produce chemically diverse secondary metabolites for survival purposes in the marine environment. Scientists have identified some of these natural products as therapeutic agents including some antibiotics. Given the increase in the resistance of pathogenic microorganisms especially methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis to commonly prescribed antibiotics, researchers have turned towards exploiting marine natural products for new antibacterial compounds. Due to the proven success of finding bioactive compounds in the marine environment this study therefore aims to discover lead compounds against MRSA and Mycobacterium tuberculosis from two marine sources, the marine algae and the bacteria associated with marine invertebrates referred to as bacterial isolates. / 2024

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Briareum asbestinum, a soft coral, is a rich source of diterpenoid natural products. The secondary metabolites of B. asbestinum fall into four classes : asbestinins, briarellins, briareolate esters, and briaranes. Briareolate esters have been shown to possess biological activity and were previously only reported from Tobago. Our group recently isolated briareolate esters from a specimen collected off the coast of Boca Raton, Florida. To determine whether location has an impact on the chemistry produced by the organism, a method to discern between chemotypes was sought. Several techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and sclerite analysis were employed, with NMR being the most successful method. By utilizing both 1H and COSY NMR experiments, it is possible to differentiate between the chemotypes of B. asbestinum. Application of this method allowed analysis of chemical variability with respect to location. / by Melody D. Rondeau. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / System requirements: Adobe Reader. / Mode of access: World Wide Web.

O consumo de peixes e frutos do mar está associado à manutenção de uma dieta saudável em função da sua proteína de elevado valor biológico, da presença de ácidos graxos poli-insaturados e de importantes nutrientes como selênio, ferro, cálcio e fósforo. No entanto, esses organismos representam uma importante fonte de elementos-traço contaminantes para os seres humanos em função da bioacumulação desses elementos que também podem biomagnificar ao longo da cadeia alimentar. Apesar da ocorrência natural de elementos-traço no meio ambiente, sua concentração nos ecossistemas costeiros tem aumentado drasticamente em função de atividades antropogênicas, fazendo com que os organismos marinhos atuem como um elo da contaminação ambiental à contaminação humana. O presente trabalho avalia a concentração de elementos-traço em diferentes tecidos de organismos marinhos sob dois aspectos: (1) o risco alimentar associado ao consumo das espécies analisadas e (2) a bioacumulação dos elementos em diferentes tecidos. Para isso foram analisadas três espécies de peixes comercializadas na cidade de Santos (SP): Sphyraena guachancho (bicuda), Priacanthus arenatus (olho de cão) e Genidens genidens (bagre) e três espécies coletadas em diferentes pontos do Sistema Estuarino de Santos (SP): Callinectes danae (siri azul), Mytella guyanensis (mexilhão) e Mugil curema (parati). O hábito alimentar foi o principal critério para seleção das espécies. O risco alimentar, calculado pelo Índice Provisório de Risco (PHI) e pelo Índice Provisório de Risco Total (PTHI), apresentou valores elevados para Sphyraena guachancho (espécie piscívora) e para peixes e siris coletados no Sistema Estuarino de Santos. A bioacumulação foi associada à espécie, tecido e tipo de elemento avaliado. A espécie piscívora apresentou elevada concentração de Ba, Hg e Ag no tecido muscular, indicando a potencial biomagnificação desses elementos ao longo da cadeia alimentar. Genidens genidens, espécie detritívora, apresentou elevada concentração de Ba, Cd e Pb na nadadeira, possivelmente associado às estratégias de detoxificação da espécie, por meio da imobilização desses contaminantes no esporão ósseo. / The consumption of fish and seafood is associated with the maintenance of a healthy diet due to its protein of high biological value, the presence of polyunsaturated fatty acids and important nutrients as selenium, iron, calcium and phosphorus. However, these organisms represent an important source of contaminant trace elements for humans due to the bioaccumulation of these elements in tissues that can also biomagnify along the food chain. Despite the natural occurrence of trace elements in the environment, its concentration in coastal ecosystems has increased dramatically as a result of anthropogenic activities, causing marine organisms to act as a link between environmental and human contamination. The present work evaluates the concentration of trace elements in different tissues of marine organisms under two aspects: (1) the food risk associated with the consumption of the species analyzed and (2) the bioaccumulation of the elements in different tissues. Three species of fish commercialized in the city of Santos (SP) were analyzed: Sphyraena guachancho (bicuda), Priacanthus arenatus (dog eye) and Genidens genidens (catfish) and three species collected at different points of the Santos Estuarine System (SP): Callinectes danae (blue crab), Mytella guyanensis (mussel) and Mugil curema (parati). The eating habit was the main criteria for selection of the species. The food risk, calculated by means of the Provisional Hazard Index (PHI) and the Provisional Total Hazard Index (PTHI), presented high values for the piscivorous species Sphyraena guachancho and for fish and crabs collected in Santos Estuarine System. The bioaccumulation was associated with the species, tissue and type of element evaluated. The piscivorous species had a high concentration of Ba, Hg and Ag in muscle tissue, indicating the potential biomagnification of these elements along the food chain. Genidens genidens, a detivorous species, presented a high concentration of Ba, Cd and Pb in the fin, possibly associated with the detoxification strategies of the species, through the immobilization of these contaminants in the bone spur.

by Rian J. Meginley. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader. / The gorgonian Briareum asbestinum is widely studied because it possesses highly oxygenated novel structures, many of which exhibit useful biological activities. Recently, two new briarane diterpenoids, briareolate esters J and K, together with two known briareolate esters have been isolated from a specimen of Briareum asbestinum collected off the coast of Boca Raton, Florida. The method used was a 96-well plate real-time cell electronic sensing (RT-CES) system to discover compounds that impact human embryonic stem cell growth. The compounds were isolated using reversed phase polystyrene divinylbenzene chromatographic support HP20ss followed by normal phased HPLC using a luna silica column. The structures of the compounds were established though the interpretation of spectroscopic data. Activity testing was conducted against hESCs (BG02) with briareolate ester J showing no inhibition activity and briareolate ester K showing mild activity with an EC50 value of 25 (So(BM. These results confirm that the exact confirmation and existence of the (E,Z)-dienone is related to the activity that was observed with the previously isolated briareolate esters L and M.

Com o objetivo de aplicar e avaliar a viabilidade de uso dos métodos disponíveis com organismos marinhos, no controle da toxicidade de efluentes líquidos que são lançados em ambientes estuarinos, foram realizados testes de toxicidade aguda com os crustáceos Mysidopsis juniae, Artemia sp, Temora stylifera e Acartia IiIljeborgi e testes de toxicidade crônica de curta duração com o equinodermo Lytechinus variegatus, utilizando-se os efluentes industriais de uma indústria siderúrgica, COSIPA e uma fábrica de fertilizantes, ULTRAFÉRTIL/JARDIM SÃO MARCOS, ambos lançados no estuário do Rio Cubatão. Dentre os organismos-testes utilizados, para avaliação do efeito tóxico agudo, o misidáceo M. juniae foi o mais sensível para ambos os efluentes, sendo que Artemia sp foi o menos sensível. Testes de toxicidade crônica com L. variegatus também se mostraram bastante úteis para avaliação de efeitos subletais. Os efluentes analisados apresentaram grande variabilidade durante o período de estudo, o que foi evidenciado através do cálculo do coeficiente de variação para testes com M. juniae. Foi avaliado, também, o efeito da salinidade sobre a sensibilidade dos crustáceos M. juniae e Artemia sp a agentes químicos (zinco e DSS) e aos efluentes industriais. A salinidade não interferiu significativamente nos resultados observados, com exceção de um experimento realizado a 15x10-3 com Artemia sp, com o efluente da COSIPA. Verificou-se, ainda, o possível efeito da utilização de salmoura obtida através dos processos de congelamento e evaporação da água do mar, sendo que o primeiro processo foi indicado para salinização de efluentes. / Acute toxicity tests with the crustaceans Mysidopsis juniae, Anemia sp, Temora stylifera e Acartia lilljeborgi and short-term chronic toxicity test with the echinoderm Lytechinus variegatus were conducted with the objective of evaluating the appropriateness of their application for the control of liquid effluents disposed off in estuarine environments. Effluents of a siderurgic industry, COSIPA, and of a fertilizer plant, ULTRAFÉRTIL/JARDIM SÃO MARCOS, were used. Both are discharged into the estuary of Cubatão river. The test with M. juniae was the most sensitive of the acute methods, for both kinds of effluents, while that with Artemia sp was the least sensitive. Chronic toxicity test with L. variegatus showed to be very useful for the evaluation of sublethal effects. The different effluent samples of both industries had great variability in their toxic effects, along the different sampling periods, as was evidenced by the high coefficient of variation for tests with M. juniae. The effects of salinity on the sensitivity of M. juniae and Artemia sp to chemicals (zinc and DSS) and to the industrial effluents, was evaluated. Salinity did not signifieantly affect the test results, except for the experiment conducted at 15x10-3 with Artemia sp, with the effluent from the siderurgic industry. The possible effect of the use of brine, obtained by freezing or evaporating sea water, was also analyzed. Freezing was the recommended procedure to salinize effluents.