Uncovering diversity and structure of bacteroidetes populations in marine environments

[EN]One of the main questions in microbial ecology is to understand what are the processes that structure and govern the species composition of communities. Answering to this question implies the need to detect bacterial species in environmental samples or at least to define ecologically meaningful “units of differentiation”. The dynamics and distribution of different broad taxonomic groups that constitute the bacterioplankton (at the phylum or class level) has been widely studied. The different large groups are known to follow different spatial distributions and seasonal cycles, but important variations also occur at lower levels (i.e. species or ecologically distinct populations). In this thesis we aimed to study some of these aspects for an important group of marine bacteria: the phylum Bacteroidetes.

Diversity of microbial communities at shallow-hydrothermal vents off Dominica, Lesser Antilles

Hydrothermal vents are often compared to desert oases, because of the presence of highly diverse and abundant biotic communities inhabiting these extreme environments. Nevertheless, the microbial communities associated with shallow-hydrothermal systems have been poorly studied. Hydrothermal activity at Dominica Island is quite well known under the geological and geochemical aspects, but no previous information existed about the microbial communities associated to this area. This thesis is therefore targeting the microbiology of hydrothermal sediments combining geochemical and molecular biological investigations, focusing on differences between hydrothermal vents and background (i.e. control) areas, and between hydrothermal sites. It was also intended to assess relationship between geochemical parameters and microbial diversity at the two hydrothermally impacted sites. Two shallow-sea hydrothermal vents located south-west off Dominica Island (Lesser Antilles) have been investigated in this study: Champagne Hot Springs and Soufriere Bay offshore vent. During this study, sediments for geochemical and molecular analyses were collected every 2 cm from the two impacted areas and from two control sites not associated with hydrothermal activity; in situ temperatures measurements were also taken every 5 cm deep in the sediment for all the sites. A geochemical characterization of the sediment porewater was performed through the analysis of several elements’ concentrations (i.e. H2S, Cl-, Br-, SO42-, Fe2+, Na+, K+, B+, Si+). Microbial communities at the different sites were studied by Automated Intergenic Spacer Analysis (ARISA). Inspection of the operational taxonomic units (OTUs) distribution was performed, as well as statistical analyses for communities’ structure and composition differences, and for changes of β-diversity along with sediment geochemistry. Data suggested that mixing between hydrothermal fluids and seawater results in distinct different environmental gradients and potential ecological niches between the two investigated hydrothermal vents, reflecting a difference in microbial community structures between them.

Polychaete fauna of the Northwest Portuguese Coastal Shelf: ecology, diversity and distribution

Polychaetes are one of the larger groups of macroinvertebrates with more than 9000 species recognised, distributed worldwide. Thanks to the broad ecological adaptability and high abundaces, this taxon plays a leading role and is considered an important component of all benthic assemblages. Our knowledge about the West Iberian Coast polychaete fauna are scarce, and the only studies are recent. In this sense, the aim of this work was to investigate the composition and the spatial distribution of the polychaete fauna along the NW Portuguese Coastal Shelf, focusing on their relationship to environmental factors (depth, grain size, longitude and latitude) and to add new data to the existing biological dataset. A total of 39 sites were analysed, collected in an area of about 5665 km², between 20 and 150 m depth, distributed in a way to cover the overall grain size gradient. A total of 9352 specimens belonging to 41 families were found, and the analysis based on the abundance of polychaete species revealed five affinity groups: (a) nearshore medium sand characterised by Pisione parapari and Hesionura elongata; (b) very coarse sand that showed the highest abundance of Syllidae and was characterised by Protodorvillea kefersteini and Syllis garciai; (c) fine sand dominated by Spiophanes bombyx and Glycera tridactyla; (d) very fine sand with Nepthys assimilis and Amage sp. and the highest abundance of Paraonidae; (d) mud characterised by Labioleanira yhleni and Ampharete finmarchica. The combination of the environmental variables and the biological data, done with BIOENV routine, demonstrated that depth, grain size and fine contents were the best related with the biological data (rho=0.598). In general, the results agree with the composition and the spatial distribution of the polychaete fauna in other parts of the world; further polychaete assemblages related to mud sediments were firstly recorded in the Northwestern Portuguese Coastal Shelf.

Ecology, sexual selection and speciation

The spectacular diversity in sexually selected traits among animal taxa has inspired the hypothesis that divergent sexual selection can drive speciation. Unfortunately, speciation biologists often consider sexual selection in isolation from natural selection, even though sexually selected traits evolve in an ecological context: both preferences and traits are often subject to natural selection. Conversely, while behavioural ecologists may address ecological effects on sexual communication, they rarely measure the consequences for population divergence. Herein, we review the empirical literature addressing the mechanisms by which natural selection and sexual selection can interact during speciation. We find that convincing evidence for any of these scenarios is thin. However, the available data strongly support various diversifying effects that emerge from interactions between sexual selection and environmental heterogeneity. We suggest that evaluating the evolutionary consequences of these effects requires a better integration of behavioural, ecological and evolutionary research.

A natural freshwater origin for two chlamydial species, Candidatus Piscichlamydia salmonis and Candidatus Clavochlamydia salmonicola, causing mixed infections in wild brown trout (Salmo trutta)

Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout (Salmo trutta) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site-specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected 'Candidatus Piscichlamydia salmonis' and 'Candidatus Clavochlamydia salmonicola' infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

IMPACTS OF CLIMATE CHANGE ON SOIL MICROORGANISMS IN NORTHERN HARDWOOD FORESTS

As global climate continues to change, it becomes more important to understand possible feedbacks from soils to the climate system. This dissertation focuses on soil microbial community responses to climate change factors in northern hardwood forests. Two soil warming experiments at Harvard Forest in Massachusetts, and a climate change manipulation experiment with both elevated temperature and increased moisture inputs in Michigan were sampled. The hyphal in-growth bag method was to understand how soil fungal biomass and respiration respond to climate change factors. Our results from phospholipid fatty acid (PLFA) analyses suggest that the hyphal in-growth bag method allows relatively pure samples of fungal hyphae to be partitioned from bacteria in the soil. The contribution of fungal hyphal respiration to soil respiration was examined in climate change manipulation experiments in Massachusetts and Michigan. The Harvard Forest soil warming experiments in Massachusetts are long-term studies with 8 and 18 years of +5 °C warming treatment. Hyphal respiration and biomass production tended to decrease with soil warming at Harvard Forest. This suggests that fungal hyphae adjust to higher temperatures by decreasing the amount of carbon respired and the amount of carbon stored in biomass. The Ford Forestry Center experiment in Michigan has a 2 x 2 fully factorial design with warming (+4-5 °C) and moisture addition (+30% average ambient growing season precipitation). This experiment was used to examine hyphal growth and respiration of arbuscular mycorrhizal fungi (AMF), soil enzymatic capacity, microbial biomass and microbial community structure in the soil over two years of experimental treatment. Results from the hyphal in-growth bag study indicate that AMF hyphal growth and respiration respond negatively to drought. Soil enzyme activities tend to be higher in heated versus unheated soils. There were significant temporal variations in enzyme activity and microbial biomass estimates. When microbial biomass was estimated using chloroform fumigation extractions there were no differences between experimental treatments and the control. When PLFA analyses were used to estimate microbial biomass we found that biomass responds negatively to higher temperatures and positively to moisture addition. This pattern was present for both bacteria and fungi. More information on the quality and composition of the organic matter and nutrients in soils from climate change manipulation experiments will allow us to gain a more thorough understanding of the mechanisms driving the patterns reported here. The information presented here will improve current soil carbon and nitrogen cycling models.

Microbial-immune cross-talk and regulation of the immune system

We are all born germ-free. Following birth we enter into a lifelong relationship with microbes residing on our body's surfaces. The lower intestine is home to the highest microbial density in our body, which is also the highest microbial density known on Earth (up to 10(12) /g of luminal contents). With our indigenous microbial cells outnumbering our human cells by an order of magnitude our body is more microbial than human. Numerous immune adaptations confine these microbes within the mucosa, enabling most of us to live in peaceful homeostasis with our intestinal symbionts. Intestinal epithelial cells not only form a physical barrier between the bacteria-laden lumen and the rest of the body but also function as multi-tasking immune cells that sense the prevailing microbial (apical) and immune (basolateral) milieus, instruct the underlying immune cells, and adapt functionally. In the constant effort to ensure intestinal homeostasis, the immune system becomes educated to respond appropriately and in turn immune status can shape the microbial consortia. Here we review how the dynamic immune-microbial dialogue underlies maturation and regulation of the immune system and discuss recent findings on the impact of diet on both microbial ecology and immune function.

Seasonal Variability in the Bacteriolytic Capacity of the Deposit Feeder Arenicola Marina: Environmental Correlates

Although deposit-feeding macrofauna consume and digest sedimentary bacteria, it is unclear whether feeding rates and digestion efficiencies are high enough to significantly impact the composition and abundance of bacteria in marine sediments. It is likely that both feeding rates and efficiency of digestion vary markedly through space and time. We used a turbidimetric assay to compare the rate of bacteriolysis by digestive fluids collected seasonally from the deposit-feeding polychaete Arenicola marina. Under standardized, experimental conditions, bacteriolytic rates represent concentrations of lytic agents. This concentration was found to vary significantly throughout the year (p = 0.001), showing greater than a 2x range. Lytic agent concentration was positively correlated with bioavailable amino acid concentrations in the surface sediment (r = 0.85, p = 0.03) but showed no apparent relationship to other proxies for food resources (e.g, chl a), sediment temperature, or gut throughput time. In vitro, temperature has been shown to have a strong positive influence on bacteriolytic rate. Temperature has no influence, however, on the in situ concentration of lytic agent in gut fluids, thus it appears that compensation for this temperature dependence is unimportant. These findings, combined with previous kinetics studies with A. marina gut fluids, predict that the quantitative influence of deposit feeding on the microbial ecology of sediments will exhibit clear seasonal variation.

Environmental and Endogenous Factors Influencing Emigration in Juvenile Anadromous Alewives

We analyzed juvenile anadromous alewife migration at Bride Lake, a coastal lake in Connecticut, during summer 2006 and found that migration on 24-hour and seasonal timescales was influenced by conditions of the environment and characteristics of the individual. To identify environmental cues of juvenile migration, we continuously video recorded fish at the lake outflow and employed information-theoretic model selection to identify the best predictors of daily migration rate. More than 80% of the approximately 320,000 juveniles that migrated from mid-June to mid-August departed in three pulses lasting one or two days. Pulses of migration were associated with precipitation events, transient decreases in water temperature and transient increases in stream discharge. Diel timing of migration shifted over the summer. Early in the season most migration occurred around dawn; late in the season migration occurred at night. To identify individual characteristics associated with migratory behavior, we compared migrating juveniles that we collected as they were exiting Bride Lake to non-migrating juveniles that we collected from the center of the lake. Migrants were a non-random subset of the population; they were on average 1 – 12 mm larger, 2 – 14 d older, had grown more rapidly (11% greater length-at-age), and were in better condition (14% greater mass-at-length) than non-migrant fish. We infer that the amount of accumulated energy has a positive effect on the net benefit of migration at any time in the migratory season.

Biphytane and stable carbon isotopic values of sugars and glycerol from ODP Hole 204-1245D and 201-1229A

The membrane lipids diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2G-GDGTs) in marine subsurface sediments are believed to originate from uncultivated benthic archaea, yet the production of 2G-GDGTs from subseafloor samples has not been demonstrated in vitro. In order to validate sedimentary biosynthesis of 2G-GDGTs, we performed a stable carbon isotope probing experiment on a subseafloor sample with six different 13C-labelled substrates (bicarbonate, methane, acetate, leucine, glucose and Spirulina platensis biomass). After 468 days of anoxic incubation, only glucose and S. platensis resulted in label uptake in lipid moieties of 2G-GDGTs, indicating incorporation of carbon from these organic substrates. The hydrophobic moieties of 2G-GDGTs showed minimal label incorporation, with up to 4 per mil 13C enrichment detected in crenarchaeol-derived tricyclic biphytane from the S. platensis-supplemented slurries. The 2G-GDGT-derived glucose or glycerol moieties also showed 13C incorporation (Dd13C = 18 - 38 per mil) in the incubations with glucose or S. platensis, consistent with a lipid salvage mechanism utilized by marine benthic archaea to produce new 2G-GDGTs. The production rates were nevertheless rather slow, even when labile organic matter was supplied. The 2G-GDGT turnover times of 1700 - 20 500 years were much longer than those estimated for subseafloor microbial communities, implying that sedimentary 2G-GDGTs as biomarkers of benthic archaea are cumulative records of past and present generations.

Heterotrophic bacteria, Synechococcus, Prochlorococcus and Virus in the eastern Mediterranean Sea in March and April 2008 during SES_GR1

The dataset is based on samples collected in the framework of the project SESAME, in the Ionian, Libyan and Aegean Sea during March- April 2008. The objectives were to measure the standing stocks and calculate the production of the microbial compartment of the food web, describe the vertical distribution pattern and characterize its structure and function through the water column. Heterotrophic bacteria, Synechococcus, Prochlorococcus and Virus abundance: Subsamples for virus, heterotrophic bacteria and cyanobacteria (Synechococcus spp. and Prochlorococcus spp.) counting were analyzed using a FACSCalibur (Becton Dickinson) flow cytometer equipped with a standard laser (488 nm) and filter set and using deionized water as sheath fluid. Fluorescent beads with a diameter of 0.97 µm (Polysciences) were added to each sample as an internal standard, and all parameters were normalized to the beads and expressed as relative units. SYBRGreen I stain (Molecular Probe) was used to stain viral and heterotrophic bacterial DNA. Viruses were counted according to (Brussaard 1984). In order to avoid bulk consentrations of viruses samples we dilluted to Tris-EDTA (pH=8,0) buffer to a final sollution of 1/5 to 1/100. Total abundance and nucleid content classes were calculated using the Paint-A-Gate software (Becton Dickinson).

Soil characteristics and abundance of archaea in samples from the Ross Sea Region, Antarctica

The objective of this study was to examine the presence and diversity of Archaea within mineral and ornithogenic soils from 12 locations across the Ross Sea region. Archaea were not abundant but DNA sufficient for producing 16S rRNA gene clone libraries was extracted from 18 of 51 soil samples, from four locations. A total of 1452 clones were analysed by restriction fragment length polymorphism and assigned to 43 operational taxonomic units from which representatives were sequenced. Archaea were primarily restricted to coastal mineral soils which showed a predominance of Crenarchaeota belonging to group 1.1b (>99% of clones). These clones were assigned to six clusters (A through F), based on shared identity to sequences in the GenBank database. Ordination indicated that soil chemistry and water content determined archaeal community structure. This is the first comprehensive study of the archaeal community in Antarctic soils and as such provides a reference point for further investigation of microbial function in this environment.

Microsensor measurements in photosynthetic microbial mats

Based on combined microsensor measurements of irradiance, temperature and O2, we compared light energy budgets in photosynthetic microbial mats, with a special focus on the efficiency of light energy conservation by photosynthesis. The euphotic zones in the three studied mats differed in their phototrophic community structure, pigment concentrations and thickness. In all mats, < 1% of the absorbed light energy was conserved via photosynthesis at high incident irradiance, while the rest was dissipated as heat. Under light-limiting conditions, the photosynthetic efficiency reached a maximum, which varied among the studied mats between 4.5% and 16.2% and was significantly lower than the theoretical maximum of 27.7%. The maximum efficiency correlated linearly with the light attenuation coefficient and photopigment concentration in the euphotic zone. Higher photosynthetic efficiency was found in mats with a thinner and more densely populated euphotic zone. Microbial mats exhibit a lower photosynthetic efficiency compared with ecosystems with a more open canopy-like organization of photosynthetic elements, where light propagation is not hindered to the same extent by photosynthetically inactive components; such components contributed about 40-80% to light absorption in the investigated microbial mats, which is in a similar range as in oceanic planktonic systems.