Marc Troussellier

Microbial Biodiversity: Approaches to Experimental Design and Hypothesis Testing in Primary Scientific Literature from 1975 to 1999

SUMMARY Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.

Protected and Threatened Components of Fish Biodiversity in the Mediterranean Sea

The Mediterranean Sea (0.82% of the global oceanic surface) holds 4%-18% of all known marine species (~17,000), with a high proportion of endemism [1, 2]. This exceptional biodiversity is under severe threats [1] but benefits from a system of 100 marine protected areas (MPAs). Surprisingly, the spatial congruence of fish biodiversity hot spots with this MPA system and the areas of high fishing pressure has not been assessed. Moreover, evolutionary and functional breadth of species assemblages [3] has been largely overlooked in marine systems. Here we adopted a multifaceted approach to biodiversity by considering the species richness of total, endemic, and threatened coastal fish assemblages as well as their functional and phylogenetic diversity. We show that these fish biodiversity components are spatially mismatched. The MPA system covers a small surface of the Mediterranean (0.4%) and is spatially congruent with the hot spots of all taxonomic components of fish diversity. However, it misses hot spots of functional and phylogenetic diversity. In addition, hot spots of endemic species richness and phylogenetic diversity are spatially congruent with hot spots of fishery impact. Our results highlight that future conservation strategies and assessment efficiency of current reserve systems will need to be revisited after deconstructing the different components of biodiversity.

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry

A bstractA new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 μm3). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.

Responses of enteric bacteria to environmental stresses in seawater

Abstract The effects of different environmental factors (nutrient deprivation, hyperosmotic shock, exposure to light) on enteric bacteria which have been transferred into the marine environment, have been studied experimentally (microcosms) by considering demographic, physiological and genetic responses in Escherichia coli or Salmonella typhimurium populations. Short-term experiments (≤ 48 h) showed that nutrient deprivation induced limited changes in measured bacteriological variables, but when combined with hyperosmotic shock, it results in an energy charge decrease and inactivation of membrane transport. Light exposure mainly affects the colony-forming capacity of bacterial populations. Combining different stress factors confirmed the rapid appearance of a viable, but nonculturable state (VBNC) in populations of E. coli and S. typhimurium. It has been shown that cellular forms other than those previously described in the literature can be generated following incubation in seawater. It was also established that pre-adaptation phenomena may occur, leading to better survival (e.g. pre-incubation in seawater in darkness enhanced survival under light exposure). An explanation concerning these phenomena can be found by looking at the rpoS gene which controls the expression of numerous genes and can trigger a general anti-stress response under different adverse conditions. Although the results provide better comprehension of the fate of enteric bacteria in the marine environment, they also raise numerous questions related to fundamental and applied problems, given in the conclusion of this paper.

PHYLOGENETIC ANALYSIS AND GENOME SIZE OF OSTREOCOCCUS TAURI (CHLOROPHYTA, PRASINOPHYCEAE)

Ostreococcus tauri Courties et Chrétiennot‐Dinet is the smallest described autotrophic eukaryote dominating the phytoplanktonic assemblage of the marine Mediterranean Thau lagoon (France). Its taxonomic position was partly elucidated from ultrastructure and high‐pressure liquid chromatography (HLPC) pigment analysis. The sequence analysis of the 18S rDNA gene of O. tauri measured here is available in EMBL Nucleotide Sequence Database (accession number: Y15814) and allowed to clarify its phylogenetic position. O. tauri belongs to the Prasinophyceae and appears very close to Mantoniella, a typical scaly Prasinophyceae, morphologically very different from the naked and coccoid Ostreococcus. An electrophoretic analysis of O. tauri shows that the nucleus contains 10.20 mbp. This small genome, fragmented into 14 chromosomes ranging in size from 300 to 1500 kbp, confirms the minimalist characteristics of Ostreococcus tauri.

Relationships among Bacterial Cell Size, Productivity, and Genetic Diversity in Aquatic Environments using Cell Sorting and Flow Cytometry.

A bstractThe study of relationships between cell size and productivity is of key importance in microbial ecology to understand which members of natural aquatic communities are responsible for the overall activity and/or productivity. Flow sorting of microorganisms from different environmental samples was used to analyze the activity of bacterial cells depending on their biovolume. Bacterial cells from five different natural samples taken along the Mediterranean coast including fresh- and seawaters were incubated with tritiated leucine, then stained with SYTO 13 and sorted by flow cytometry according to their average side-angle-scattered (SSC) light. In all samples, a bell-shaped relationship was found between cell biovolume and activity, whereas activity of a given cell-size class varied between samples. In contrast, an inverse relationship was found between biovolumes and abundances. These results suggest that medium-sized cells with highest growth rates are probably submitted to intense grazing. For one sample, bacteria within five different size classes were sorted and the genetic diversity of cells within each sorted size class and that of the whole community were analyzed by the denaturing gradient gel electrophoresis (DGGE) method. The genetic diversity, as determined at the community level was highly represented into the pool of small cells, whereas only few species were present into larger cell subpopulations. The results suggest that only a few genotypes may be dominant within the largest and most productive cells. Furthermore, cell size polymorphism as well as heterogeneous cellular activities were found within some species.

Evaluation of simple statistical criteria to qualify a simulation

Statistical and deterministic simulation modelling rely on a complex process made of trials, errors, and gradual improvement of the simulations. The major problem is to be able to quantify the quality of the simulations in order to know if a modification of the concepts, the laws simulating the processes, or the parameters improve it. To try to quantify the quality of simulations using a mathematical criterion we focus on simple linear regression parameters: the values of the slope (a) and the y-intercept (b). The estimated values of these parameters differ depending on which kind of regression model (model I or II) is used. An artificial dataset illustrates that ordinary least-squares regression (OLS; model I regression) leads to results that are not those expected; but using major axis regression (MA; model II regression) instead of OLS leads to the correct answer. The value of a, when it significantly differs from 1, indicates a difference between observed and simulated values proportional to the values of the variable. The value of b, when it significantly differs from 0, indicates a systematic and constant difference between observations and simulations. Taking into account the values of a and b, we define four possible outcomes which allow, at first, to define the quality of a simulation without considering the coefficient of determination, r2: (i) a n.s.d. (not significantly different from) 1 and b n.s.d. 0 (perfect agreement between observations and simulations), (ii) a n.s.d. 1 and b s.d. 0 (significant constant difference between observations and simulations) or a s.d. 1 and a s.d. 0 and b n.s.d. 0 (differences proportional to the values of the variable), (iii) a s.d. 1 and a s.d. 0 and b s.d. 0 (superimposition of a constant difference and a proportional difference), and (iv) a n.s.d. 0 (no relation between simulations and observations). The value of r 2 is used to rank two simulations pertaining to the same group. That classification of the quality of the simulations is applied to a real-data example: a simulation of the temporal change in chlorophyll a in a high-rate algal pond.

Flow cytometry in oceanography 1989–1999: Environmental challenges and research trends

BACKGROUND The present review is based on the identification of four major environmental crises that have been approached from a biological oceanographic viewpoint. These crises are the release of contaminants in near shore marine waters, the collapse of marine resources that were renewable until recently, the loss of biodiversity, and global climate change METHODS The review examines the contribution of cytometry-based biological oceanography to the resolution of the four environmental crises. Using a database of 302 papers, flow cytometric (FCM) studies in biological oceanography over the 1989--1999 decade are examined. Future biological oceanographic applications of FCM are discussed. RESULTS Most of the published FCM oceanographic studies focus on phytoplankton and bacterioplankton. Analysis of our 1989-1999 database shows the predominance of studies dedicated to phytoplankton (77%), followed by heterotrophic bacteria (21%). The latter progressively increased over the last decade, together with the improved understanding of the biogeochemical and trophic roles of marine bacteria. Most studies on these two microorganisms were conducted in vitro until 1996, after which the trend reversed in favor of in situ research. The most investigated areas were those with major international sampling efforts, related to the changing climate. Concerning environmental topics, 62% of papers on phytoplankton and bacterioplankton focused on the structure of microbial communities and fluxes (e.g., production, grazing); this provides the basis for biological oceanographic studies on resources and climate change. CONCLUSIONS Future progress in the biological oceanographic use of FCM will likely fall into two categories, i.e., applications where FCM will be combined with the development of other methods and those where FCM will be the main analytical tool. It is expected that FCM and other cytometric approaches will improve the ability of biological oceanography to address the major environmental challenges that are confronting human societies.

A microcosm study of the survival of Escherichia coli and Salmonella typhimurium in brackish water

Abstract The study of the survival of Escherichia coli and Salmonella typhimurium in sewage treated by stabilization ponds, and then mixed more or less rapidly with coastal marine water, has been experimentally tested in microcosms (membrane diffusion chambers). The results obtained show that under the same experiment conditions (slow saline, marine and rapid saline stress) the survival time of the two bacteria varies considerably. In sewage treated by stabilization ponds, Salmonella typhimurium and Escherichia coli exhibit the similar survival rates. T 90 evaluated for Escherichia coli and Salmonella typhimurium in the winter period was, respectively, 125 and 132 h. In brackish water the survival rate of Salmonella typhimurium was generally higher than that of Escherichia coli . When these two bacteria were suspended in stabilization ponds effluent and rapidly mixed with brackish water, survival time was particularly short, whereas it was prolonged when the bacteria were submitted to a gradual increase in salinity. Biological factors, and in particular predation, can play an important role in eliminating enteric bacteria during pond system treatment, but their efficiency is supposed to diminish significantly when there is an environmental change such as the discharge of pond system effluent into brackish water. This can explain the increase of enteric bacteria survival.

Bacterial contamination of Mediterranean coastal seawater as affected by riverine inputs: simulation approach applied to a shellfish breeding area (Thau lagoon, France)

Consequences of short-term changes in thermotolerant coliform loads on their spatio-temporal distribution in a Mediterranean lagoon with large-scale mollusk farming (Thau lagoon, France) were explored using a simulation approach. Simulations were based on bacterial transport and survival coupled models forced by the input of bacterial loads from the two main rivers (Vène and Pallas) that flow into the lagoon. Different flow types (reference, sudden and constant), bringing the same bacterial load, were considered and subsequent spatial and temporal bacterial contamination of lagoon surface water and shellfish was estimated. Simulation results showed that as long as loads were high, hydrodynamical processes governed the distribution of bacterial abundance in receiving areas. As soon as loads decreased or when time supply increased, biological die-off processes became dominant. Bacterial contamination of shellfish induced by the different flow types appeared to depend on the receiving area. In the case of Pallas River area, a sudden input of bacteria led to a high bacterial contamination of shellfish but only during a short period ( approximately 1 day). A constant input of the same amount of bacteria induced a lower but significant contamination during all the simulation period (10 days). On the contrary, bacterial inputs from the Vène River led to shellfish contamination only when bacteria were delivered through a flood event. Exposure time of bacteria to adverse environmental conditions appeared to be the main explanation to the above-mentioned differences. Consequences of our results in terms of environmental management strategy were discussed.