Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Stephen Craig Cary, College of Earth, Ocean, & Environment. Includes bibliographical references.

Thesis (Ph.D.)--Aberdeen University, 2008. / "Oceanlab" Includes bibliographical references.

The deep-sea hypersaline lakes in the Red Sea are among the most challenging, extreme, and unusual environments on the planet Earth. Despite their harshness to life, they are inhabited by diverse and novel members of prokaryotes. Methanogenesis was proposed as one of the main metabolic pathways that drive microbial colonization in similar habitats. However, not much is known about the identities of the methane-producing microbes in the Red Sea, let alone the way in which they could adapt to such poly extreme environments. Combining a range of microbial community assessment, cultivation and omics (genomics, transcriptomics, and single amplified genomics) approaches, this dissertation seeks to fill these gaps in our knowledge by studying archaeal composition, particularly methanogens, their genomic capacities and transcriptomic characteristics in order to elucidate their diversity, function, and adaptation to the deep-sea brines of the Red Sea. Although typical methanogens are not abundant in the samples collected from brine pool habitats of the Red Sea, the pilot cultivation experiment has revealed novel halophilic methanogenic species of the domain Archaea. Their physiological traits as well as their genomic and transcriptomic features unveil an interesting genetic and functional adaptive capacity that allows them to thrive in the unique deep-sea hypersaline environments in the Red Sea.

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.

Organismos de mar profundo encontram limitações na disponibilidade de alimentos e exploram enriquecimentos orgânicos esporádicos que chegam ao assoalho oceânico. O objetivo deste trabalho foi descrever a diversidade das comunidades bacterianas associadas a parcelas sintéticas e orgânicas (vértebras de baleia e blocos de madeira) no Espírito Santo, Rio de Janeiro e São Paulo a 3.300 m de profundidade, avaliando a influência dos substratos e da localização geográfica sobre essas comunidades. Foi realizada a extração de DNA e amplificação do gene RNAr 16S para sequenciamento por Illumina Miseq e análises estatísticas pelo Qiime. Os Gêneros dominantes nos substratos sintéticos, madeira e vértebras foram Psychroserpens (Flavobacteriia), Phaeobacter, (Alphaproteobacteria), Desulfobacter, (Deltaproteobacteria), respectivamente. Com base nos resultados obtidos, afirma-se que o tipo de substrato teve maior influência do que a localização geográfica sobre a estrutura das comunidades bacterianas. / Deep sea organisms found limitations in the availability of food and exploit sporadic organic enrichments that reach the ocean floor. The aim of this study was to describe the diversity of bacterial communities associated with synthetic and organic substrate (whale bone and wood blocks) in Espírito Santo, Rio de Janeiro and Sao Paulo to 3,300 m deep, assessing the influence of substrates and location geographical about these communities. 16S rRNA sequencing was performed by Illumina Miseq and statistical analysis by Qiime. The dominant genera in synthetic substrates, wood and vertebrae were Psychroserpens (Flavobacteriia), Phaeobacter (Alphaproteobacteria) and Desulfobacter, (Deltaproteobacteria), respectively. Based on these results, it is stated that the substrate type had greater influence than geographic location on the structure of bacterial communities.

Thesis (MTech (Mechanical Engineering))--Cape Peninsula University of Technology, 2017. / In South Africa, squid fishing vessels need to find and then anchor above benthic squid egg beds to effect viable catches. However, waves acting on the vessel produce a dynamic response on the anchor line. These oscillatory motions produce impact forces of the chain striking the seabed. It is hypothesised that this causes damage to the squid egg bed beneath the vessels. Different mooring systems may cause more or less damage and this is what is investigated in this research. The effect of vessel mooring lines impact on the seabed during squid fishing is investigated using a specialised hydrodynamic tool commercial package ANSYS AQWA models. This study analysed the single-point versus the two-point mooring system’s impact on the seabed. The ANSYS AQWA models were developed for both mooring systems under the influence of the wave and current loads using the 14 and 22 m vessels anchored with various chain sizes. The effect of various wave conditions was investigated as well as the analysis of three mooring line configurations. The mooring chain contact pressure on the seabed is investigated beyond what is output from ANSYS AQWA using ABAQUS finite element analysis. The real-world velocity of the mooring chain underwater was obtained using video analysis. The ABAQUS model was built by varying chain sizes at different impact velocities. The impact pressure and force due to this velocity was related to mooring line impact velocity on the seabed in ANSYS AQWA. Results show the maximum impact pressure of 191 MPa when the 20 mm diameter chain impacts the seabed at the velocity of 8 m/s from video analysis. It was found that the mooring chain impact pressure on the seabed increased with an increase in the velocity of impact and chain size. The ANSYS AQWA impact pressure on the seabed was found to be 170.86 MPa at the impact velocity of 6.4 m/s. The two-point mooring system was found to double the seabed mooring chain contact length compared to the single-point mooring system. Both mooring systems showed that the 14 m vessel mooring line causes the least seabed footprint compared to the 22 m vessel.

abstract: This thesis research focuses on phylogenetic and functional studies of microbial communities in deep-sea water, an untapped reservoir of high metabolic and genetic diversity of microorganisms. The presence of photosynthetic cyanobacteria and diatoms is an interesting and unexpected discovery during a 16S ribosomal rRNA-based community structure analyses for microbial communities in the deep-sea water of the Pacific Ocean. Both RT-PCR and qRT-PCR approaches were employed to detect expression of the genes involved in photosynthesis of photoautotrophic organisms. Positive results were obtained and further proved the functional activity of these detected photosynthetic microbes in the deep-sea. Metagenomic and metatranscriptomic data was obtained, integrated, and analyzed from deep-sea microbial communities, including both prokaryotes and eukaryotes, from four different deep-sea sites ranging from the mesopelagic to the pelagic ocean. The RNA/DNA ratio was employed as an index to show the strength of metabolic activity of deep-sea microbes. These taxonomic and functional analyses of deep-sea microbial communities revealed a `defensive' life style of microbial communities living in the deep-sea water. Pseudoalteromonas sp.WG07 was subjected to transcriptomic analysis by application of RNA-Seq technology through the transcriptomic annotation using the genomes of closely related surface-water strain Pseudoalteromonas haloplanktis TAC125 and sediment strain Pseudoalteromonas sp. SM9913. The transcriptome survey and related functional analysis of WG07 revealed unique features different from TAC125 and SM9913 and provided clues as to how it adapted to its environmental niche. Also, a comparative transcriptomic analysis of WG07 revealed transcriptome changes between its exponential and stationary growing phases. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2013

Hydrostatic pressure (HP) and low temperature are among the main parameters that affect the microbial activity in the deep sea. Especially in the event of an oil spill, the natural microbial degradation of hydrocarbons in the harsh conditions of the deep sea can be significantly impaired. In the Red Sea, the temperature in the deep (22°C) is much higher than in other oceans and may favor hydrocarbon degradation. Bacteria of the genus Alcanivorax, which are prominent and ubiquitous alkane degrading bacteria, have been extensively studied because of their high abundance in oil-contaminated shallow water, but have been shown to be absent in the deep sea because of their piezo-sensitivity. In the present thesis, the novel species Alcanivorax marisrubri isolated at 1000 m from the Southern Red Sea has been evaluated for its piezo-adaptation under different combinations of temperature, and HP. A. marisrubri showed a piezotolerance different from other Alcanivorax species. Furthermore, a positive compensation of growth inhibition was observed when the cells were exposed to mild HP (10 MPa) in combination with a relatively high temperature of 38°C. While growth was inhibited at lower temperatures (20 and 26°C) under mild-HP (5 and 10 MPa), the metabolic activity was triggered, possibly in response to cellular stress. This study showed that the growth and metabolic activity of A. marisrubri under HP depend on temperature, which exerts a positive compensation effect and may extend the growth of this bacterium to the depths of the Red Sea.

Sponges play an important role in many marine habitats and are crucial for maintaining the deep-sea marine ecosystems. However, there is a knowledge gap in the field of sponge biodiversity due to insufficient exploration of the deep-sea, and the probability of finding new species is fairly high. The deep waters of the Indian and Pacific Oceans are particularly poorly explored, and their sponge diversity is barely known. During the KANADEEP 2 expedition in the south of New Caledonia a large collection of Demospongiae and Hexactinellida was gathered to assess the sponge biodiversity. In this project 110 Demosponges were investigated morphologically and identified to the order level. Twenty-one of these were selected for further identification and description in terms of shape and geometry of spicules using light microscopy. The DNA was extracted from eight specimens for the confirmation of new species. Amplification of CO1 Folmer fragment was conducted using PCR. The resulting PCR-products were analyzed using gel electrophoresis and DNA-sequencing. The twenty-one specimens were assigned to the genera Tethya, Stupenda and Geodia belonging to the order Tetractinellida, which was found to be the dominant order in the deep waters in the New Caledonia region. There are potentially six new species amongst the studied specimens. However, it has to be confirmed with molecular analysis of specific markers. The morphological analysis of 21 specimens collected during the KANADEEP 2 expedition demonstrated that 17 specimens belonged to Geodia, two specimens to Tethya, and two specimens to Stupenda.

Chemosynthetic ecosystems are habitats whose food webs rely on chemosynthesis, a process by which bacteria fix carbon using energy from chemicals, rather than sunlight-driven photosynthesis for primary production, and they are found all over the world on the ocean floor. Although these deep-sea habitats are remote, they are increasingly being impacted by human activities such as oil and gas exploration and the imminent threat of deep-sea mining. My dissertation examines deep-sea chemosynthetic ecosystems at several ecological scales to answer basic biology questions and lay a foundation for future researchers studying these habitats. There are two major varieties of chemosynthetic ecosystems, hydrothermal vents and cold seeps, and my dissertation studies both. My first chapter begins at cold seeps and at the population level by modeling the population dynamics and lifespan of a single species of tubeworm, Escarpia laminata, found in the Gulf of Mexico. I found that this tubeworm, a foundation species that forms biogenic habitat for other seep animals, can reach ages over 300 years old, making it one of the longest-lived animals known to science. According to longevity theory, its extreme lifespan is made possible by the stable seep environment and lack of extrinsic mortality threats such as predation. My second chapter expands the scope of my research from this single species to the entire cold seep community and surrounding deep-sea animals common to the Gulf of Mexico. The chemicals released at cold seeps are necessary for chemosynthesis but toxic to non-adapted species such as cold-water corals. Community studies in this area have previously shown that seeps shape community assembly through niche processes. Using fine-scale water chemistry samples and photographic mapping of the seafloor, I found that depressed dissolved oxygen levels and the presence of hydrogen sulfide from seepage affect foundation taxa distributions, but the concentrations of hydrocarbons released from these seeps did not predict the distributions of corals or seep species. In my third chapter I examine seep community assembly drivers in the Costa Rica Margin and compare the macrofaunal composition at the family level to both hydrothermal vents and methane seeps around the world. The Costa Rica seep communities have not previously been described, and I found that depth was the primary driver behind community composition in this region. Although this margin is also home to a hybrid “hydrothermal seep” feature, this localized habitat did not have any discernible influence on the community samples analyzed. When vent and seep communities worldwide were compared at the family-level, geographic region was the greatest determinant of community similarity, accounting for more variation than depth and habitat type. Hydrothermal vent and methane seeps are two chemosynthetic ecosystems are created through completely different geological processes, leading to extremely different habitat conditions and distinct sets of related species. However, at the broadest spatial scale and family-level taxonomic resolution, neutral processes and dispersal limitation are the primary drivers behind community structure, moreso than whether the habitat is a seep or a vent. At more local spatial scales, the abiotic environment of seeps still has a significant influence on the ecology of deep-sea organisms. The millennial scale persistence of seeps in the Gulf of Mexico shapes the life history of vestimentiferan tubeworms, and the sulfide and oxygen concentrations at those seeps determine seep and non-seep species’ distributions across the deep seafloor. / Biology