This work seeks to explore current ecological theory through application to communities inhabiting hydrothermal vents. This thesis aims to: (1) add to and synthesise knowledge of species and their distributions at the intra- and intersegment scale; and (2) evaluate vent community patterns and speculate on processes. Samples used are submersible grabs of low temperature (<60°C) tubeworm assemblages on basalt and sulphide surfaces. Species abundances and distributions on three segments of the Juan de Fuca Ridge (Axial, Cleft, and CoAxial) are described. Community descriptors such as species density, Simpson's and the Shannon-Wiener diversity indices, evenness, species richness, species abundance-distribution models, species percent-average relative abundance and density are used. Vent community structure is compared among segments using these descriptors, visual descriptions, pairwise correlations, Friedman tests of distributions, cluster and correspondence analysis, rarefaction, complementarity, a test for saturation, and Whittaker's beta diversity. Vent community composition on Axial, north Cleft, and CoAxial is similar at the segment and inter-segment scale. The limpet Lepetodrilus fucensis is the most abundant species at all sites. Differences among communities are best seen temporally, not spatially. Senescent communities can be distinguished from active vent assemblages. Pioneer communities, however, are statistically indistinguishable from intermediate communities when sampled two or more years post-eruption. Axial and Cleft species dispersion fits the core-satellite hypothesis. The exceptions are the polynoids Branchinotogluma sp., Lepidonotopodium piscesae, and Levensteiniella kincaidi, which are widespread and present in low local abundances. Both local and mesoscale regional mechanisms explain observed local diversity. Spatial isolation, not habitat differences, influences between-habitat diversity (beta diversity) on Axial, Cleft, and all three segments combined. Meiofauna are important for species richness estimates, identifying differences among structurally similar communities, and understanding input/output between vents and the deep-sea. Measurements such as species richness and diversity indices may be poor at distinguishing among vent communities because vents are species poor and uneven. The Michaelis-Menten, Jackknife 2, and Chao 2 nonparametric vent species richness estimators perform well with small samples. Vent communities should be compared to habitats of similar diversity and evenness as well as disturbance and productivity regimes. Candidate comparison communities include communities in early successional states, selected taxocenes such as carabid beetles on fungi, or high disturbance and/or low diversity systems like the rocky intertidal, organically polluted sediments and oxygen minimum zones below upwelling regions in the deep-sea. / Graduate

Deep-sea hydrothermal vents are some of the most biologically productive ecosystems on Earth, yet receive little to no input of photosynthetically derived organic matter. The trophic system at hydrothermal vents is based primarily on the reduction-oxidation (redox) of inorganic chemicals by Bacteria and Archaea. However, the distributional patterns of the microorganisms that colonize deep-sea hydrothermal vent deposits and their link to the geologic setting are still not deeply understood. The goal of the studies presented in this thesis was to quantify the abundance, and distribution of major and understudied vent colonizing archaeal groups from globally distributed and geochemically distinct hydrothermal vent fields. The archaeal community composition was analyzed using quantitative PCR with lineage specific functional gene primers that target methanogens, and 16S rRNA gene primers designed or optimized from this study for the Thermococcales, Archaeoglobus, Ignicoccus and marine Nanoarchaeota. Overall, a general relationship was demonstrated between the geochemical differences of the hydrothermal vent fields and the archaeal community structure. The archaeal community assemblage varied dramatically from hydrothermal vents with different vent host rocks along the Mid-Atlantic Ridge and Eastern Lau Spreading Center. In contrast, two vent fields in the East Pacific, 9°N on the EPR and Guaymas Basin that are basalt and basalt-sediment hosted were found to have similar community composition. These observed differences may be driven in part by the metabolically available chemical energy as hydrogen oxidizing lineages of the methanogens and Archaeoglobus were found in higher abundance in the samples from vent field that had a high concentration of end-member hydrogen and the heterotrophic Thermococcales constituted a higher proportion of the archaeal community at the less enriched vent fields. Interestingly, the Nanoarchaeota and the genus of its only confirmed symbiont, Ignicoccus, were found to have an inconsistent proportional relationship, with the Nanoarchaeota comprising a larger proportion of the archaeal community at the ultramafic and fast spreading basalt vent fields and Ignicoccus at the ultra-slow spreading basalt and andesite hosted vent fields. There was also a more localized pattern identified within the hydrothermal vent deposit. The chemosynthetic lineages of the methanogens and Archaeoglobus constituted a higher proportion of the archaeal community in chimney samples compared to Thermococcales that was found in a higher proportion at horizontal flange samples. This archaeal proportional shift could be driven by energetic micro-niches within the vent deposit, as the chemolithotrophic lineages colonize the area closest to the venting source, and the heterotrophic Thermococcales dominate in more mature structures further from the venting source. Quantitative assessments of the archaeal community composition from this study provided added insight into the dynamic geologic influence on the archaeal lineages that colonize deep-sea hydrothermal vents, on a global and local scale.

The discovery of deep-sea hydrothermal vents in 1977 revealed an ecosystem supported by chemosynthesis with a rich diversity of invertebrates, Archaea and Bacteria. While the invertebrate vent communities are largely composed of endemic species and exist in different biogeographical provinces, the possible factors influencing the distribution patterns of free-living Archaea and Bacteria are still being explored. In particular, how differences in the geologic setting of vent fields influence microbial communities and populations associated with active vent deposits remains largely unknown. The overall goal of the studies presented in this dissertation was to examine the links between the geologic setting of hydrothermal vent fields and microorganisms associated with actively venting mineral deposits at two levels of biological organization. At the community level, bar-coded pyrosequencing of a segment of the archaeal and bacterial 16S rRNA gene was employed to characterize and compare the microbial communities associated with numerous deposits from several geochemically different vent fields. Results from these studies suggest that factors influencing end-member fluid chemistry, such as host-rock composition and degassing of magmatic volatiles, help to structure the microbial communities at the vent field scale. At the population level, targeted cultivation-dependent and -independent studies were conducted in order to expand our understanding of thermoacidophily in diverse hydrothermal environments. Results of these studies expanded the phylogenetic and physiological diversity of thermoacidophiles in deep-sea vent environments and provided clues to factors that are influencing the biogeography of an important thermoacidophilic archaeal lineage. Overall, these studies have increased our understanding of the interplay between geologic processes and microorganisms in deep-sea hydrothermal environments.

As part of a large study aimed at searching for patterns of diversity in the genus Persephonella along the north to south geochemical gradient of the ELSC, ten novel strains of Alphaproteobacteria were isolated unexpectedly. Using defined media under microaerophilic conditions to enrich for Persephonella from chimney samples collected at the seven vent fields on the ELSC and the dilution to extinction by serial dilution method to purify cultures, a total of ten strains belonging to the Alphaproteobacteria were isolated. Two of these isolates, designate MN-5 and TC-2 were chosen for further characterization and are proposed as two new species of a novel genus to be namedThermopetrobacter. Both strains are aerobic, capable of chemoautotrophic growth on hydrogen and grow best at 55°C, pH 6 and 3.0% NaCl. Strain MN-5 is capable of heterotrophic growth on pyruvate and malate and TC-2 is only able to grow heterotrophically with pyruvate. The GC content of MN-5 is 69.1 and TC-2 is 67 mol%. GenBank BLAST results from the 16S rRNA gene reveal the most closely related sequence to MN-5 is 90% similar and the most closely related sequence to strain TC-2 is 89% similar. Sampling at a shallow marine vent on the coast of Vulcano Island, Italy in 2007 led to the isolation of a novel species of Hydrogenothermus, a genus within the Hydrogenothermaceae family. This isolate, designated NV1, represents the secondHydrogenothermusisolated from a shallow marine vent. NV1 cells are rod-shaped, approximately 1.5μm long and 0.7μm wide, motile by means of a polar flagellum and grow singularly or in short chains. Cells grow chemoautotrophically using hydrogen or thiosulfate as electron donors and oxygen as the sole electron acceptor. Growth was observed between 45 and 75°C with an optimum of 65°C (doubling time 140 min), pH 4.0-6.5 and requires NaCl (0.5-6.0% w/v). The G+C content of total DNA is 32 mol%.

<p>Reactive oxygen species (ROS) are produced endogenously in all aerobes and are induced by environmental stressors. ROS oxidize and disable essential cellular components such as DNA, proteins, and lipid membranes. Exposure to metals, polycyclic aromatic hydrocarbons (PAHs), and some pesticides can induce oxidative stress in marine invertebrates. All aerobic organisms have a network of antioxidants and enzymes to quench ROS and prevent oxidative damage. This dissertation examines antioxidant and oxidative stress biomarkers in endemic molluscs and crabs from two natural extreme environments: deep-sea hydrothermal vents in the Lau and North Fiji Basin, and cold seeps in the Gulf of Mexico. In addition, the acute toxicity and sub-lethal effects of four insecticides and an herbicide are examined in the estuarine blue crab, <italics>Callinectes sapidus</italics>. Blue crabs are North Carolina's most important fishery species and are frequently found in agricultural drainage ditches, an example of an anthropogenic extreme environment. </p> <p>Total glutathione, catalase, superoxide dismutase, and lipid peroxidation levels were of the same respective order of magnitude in the two vent gastropods, <italics>Alviniconcha</italics> sp. and <italics>Ifremeria nautilei</italics>, and vent mussel, <italics>Bathymodiolus brevior</italics>. These biomarkers activities were similar to those from previous reports on Mid-Atlantic Ridge mussels, except for ~100-fold higher lipid peroxidation levels among Lau molluscs. Principal component analysis (PCA) of mollusc tissue-specific biomarker levels grouped individuals by species rather than by site. </p> <p>Biomarker levels in the seep mussels <italics>Bathymodiolus childressi, B. brooksi</italics>, and <italics>B. heckerae</italics> were similar across species except for elevated foot and gill cytosolic SOD in mussels from MC-640 compared to those from AC-645. PCA of seep mussel biomarker levels differentiated by species with <italics>B. childressi</italics> isolated from <italics>B. brooksi</italics> and <italics>B. heckerae</italics>. The addition of <italics>B. brevior</italics> biomarker data to the PCA showed them grouping around <italics>B. brooksi</italics> and <italics>B. heckerae</italics>. <italics>Bathymodiolus childressi</italics> is ancestral to the other species and contains only methanotrophic endosymbionts. Whether symbionts play a role in alleviating possible toxic conditions remains unknown.</p> <p>Pesticides were acutely toxic to blue crabs in the order of Lambda-cyhalothrin > imidacloprid &#8776; aldicarb > acephate &#8776; Roundup® (glyphosate). Megalopae were almost always more sensitive to pesticides than early stage juveniles. Commercial formations of pesticides generally showed similar toxicity to active ingredients alone. Exposure to LC₂₀ levels of acephate, aldicarb, imidacloprid and Roundup significantly increased the frequency of juvenile mortality after molting. There was no significant change in total glutathione or lipid peroxidation of exposed megalopae. Lambda-cyhalothrin-, imidacloprid-, and aldicarb-based products have the potential to cause acute toxicity and molting-related mortality in shallow creeks and ditches.</p> / Dissertation

Abstract The gammaproteobacterium Thiomicrospira crunogena XCL–2 is a hydrothermal vent chemolithoautotroph that has a carbon concentrating mechanism (CCM), which is functionally similar to that of cyanobacteria. At hydrothermal vents, dissolved inorganic carbon (DIC) concentrations and pH values fluctuate over time, with CO2 concentrations ranging from 20 μM to greater than 1 mM, therefore having a CCM would provide an advantage when CO2 availability is very low as CCMs generate intracellular DIC concentrations much higher than extracellular, thereby providing sufficient substrate for carbon fixation. The CCM in T. crunogena includes α–carboxysomes (intracellular inclusions containing form IA RubisCO and carbonic anhydrase), and also presumably requires at least one active HCO3 µ transporter to generate the elevated intracellular concentrations of DIC. To determine whether RubisCO itself might be adapted to low CO2 concentrations, the KCO2 for purified carboxysomal RubisCO was measured (250 μM SD ±; 40) and was much greater than that of whole cells (1.03 μM). This finding suggests that the primary adaptation by T. crunogena to low–DIC conditions has been to enhance DIC uptake, presumably by energy–dependent membrane transport systems that are either ATP–dependent and/or dependent on membrane potential (δ ψ). To determine the mechanism for active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis andδ ψ. After separate incubations with the ATP synthase inhibitor DCCD and the protonophore CCCP, intracellular ATP was diminished, as was the concentration of intracellular DIC and fixed carbon, despite the absence of an inhibitory effect on δ ψ in the DCCD–incubated cells. In some organisms, DCCD inhibits the NDH–1 and bc1 complexes so it was necessary to verify that ATP synthase was the primary target of DCCD in T. crunogena. Both electron transport complex activities were assayed in the presence and absence of DCCD and there was no significant difference between inhibited (309.0 μmol/s for NDH–1 and 3.4 μmol/s for bc1) and uninhibited treatments (271.7 μmol/s for NDH–1 and 3.6 μmol/s for bc1). These data support the hypothesis that an ATP–dependent transporter is primarily responsible for HCO3 µ transport in T. crunogena. The ATP–dependent transporter solute–binding protein gene (cmpA) from Synechococcus elongatus PCC 7942, was used to perform a BLAST query. Tcr_1153 was the closest match in the T. crunogena genome. However, the gene neighborhood and the result of a maximum likelihood tree suggest that Tcr_1153 is a nitrate transporter protein. Work is underway to find the genes responsible for this ATP–dependent transporter.

Univ., Diss.--Sydney. / Literaturverz. S. [237] - 268.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 102-114).

Three gastropods occupy a range of habitats along gradients in hydrothermal flux at Juan de Fuca Ridge vents. I examined how these species co-exist and identified mechanisms driving their abundances. First, I measured temperatures and spatial patterns in adult densities of the three species at three distances from vents to test if thermal regime relates to their habitat selection. Lepetodrilus fucensis and Depressigyra globulus were most dense in-vent (0-25 cm) at variable temperatures (10+5°C): 2100 and 240 incl. dm -2 (respectively). Provanna variabilis was most abundant far-vent (51-75 cm: 60 ind. dm 2) at stable temperatures (3±0.5°C). Thermal conditions are key in their habitat selection: behavioural experiments showed that these gastropods select fluid temperatures<18°C. L.fucensis and D. globulus preferred 5-15°C, while P. variabilis preferred 4-12°C. The next studies sought to explain how Lepetodrilus fucensis reaches order of magnitude higher densities in comparison to other gastropods. First. I quantified L. fucensis recruitment and sex ratio patterns to identify innovative life history traits. I meaured size structure and density at in- and far-vent locations. Early postlarval juveniles occupied far-vent at remarkable densities (2419 ind. dm-2). To test for sex ratio biases, I sexed animals from different habitats and sizes. Populations nearest vents hosted the largest females (>6.0 mm), while peripheral habitats were male-biased. A transplant experiment showed that female survivorship and gonad fullness were significantly lower than males in far-vent locations. Sex ratio biases are driven by two mechanisms: females maximize their reproductive output by selecting optimal habitats and suffer relatively higher mortality in low flux. Next, I hypothesized that the Lepetodrilus fucensis gill symbiosis is a key adaptation. I used multiple approaches to determine if the prevalence of the association and relationship to the limpets condition support this hypothesis. FISH probes specific to the 16S rRNA molecule of a gamma-Proteobacteria hybridized where bacteria were present. Direct sequencing using symbiont-specific primers gave a single unambiguous sequence. indicating high specificity. Light and TEM micrographs of gill tissue from a range of species also showed that the symbiosis is ubiquitous. In addition, the gills of in-vent animals had high surface area. dense symbiont populations and healthy tissues, while far-vent animals showed the reverse trend, suggesting that the symbiosis benefits L. fucensis. Carbon fixation by gill tissues was stimulated by inorganic sulfide and related to the abundance of bacteria on the gill. These data indicate a persistent and specific symbiosis that is dependent on access to sulphide. I further examined feeding by Lepetodrilus fucensis to determine if the bacteria contribute to their host's nutrition. The morphology of feeding structures were compared among Lepetodrilus species. L. fucensis exhibited specialized features: the gill is enlarged. the lamellae are free of the mantle. do not narrow and are stabilized by ciliary junctions. The radula and stomach of L. fucensis are also reduced. Shipboard observations confirmed suspension feeding by L. fucensis. In addition, the symbiont may be ingested because its phylotype was well-represented in food material on the gill. The limpet's morphological specializations are consistent with dependence on suspension feeding and/or symbiont farming; however, L. fucensis also grazes. a mechanism likely important in peripheral locations. Lepetodrilus fucensis populations are partitioned by size and sex along environmental gradients near vents. Peripheral populations are dominated by recruits and adults tend to be male: grazing is likely their primary feeding mode. Larger animals form stacks in venting fluids and are female-biased. These populations access suspended particles for food and sulphide, which generates dense symbiont populations for ingestion. Multiple feeding modes sustain high L. fucensis densities in a space-limited environment and may be an innovative strategy that drives its remarkable abundances.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24679号 / 農博第2562号 / 新制||農||1100(附属図書館) / 学位論文||R5||N5460(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 澤山 茂樹, 教授 吉田 天士, 准教授 中川 聡 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM