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Marine microorganism activities are commonly assessed by bulk methods and assigned to the total cell count. The presence in significant amounts of ghost, dead, and damaged cells makes such as assignment a non-correct one. A Nucleic Acid Double Staining protocol (NADS) of fresh water bacteria (Barbesti et al., Cytometry 40 (2000) 214-218) has been adapted to resolve viable, damaged and dead cells in marine environments (Gregori et al., Appl. Environ. Microbiol. 67 (2001) 4662-4670). The present reports the first in situ application of this approach, conducted in the Bay of Marseilles in winter and spring periods at two sites with contrasted features.

13 pages, 7 figures, 3 tables, Two large-scale mass mortality events (MMEs) of unprecedented extent and severity affecting rocky benthic communities occurred during the summers of 1999 and 2003 along the coasts of the NW Mediterranean Sea. These mortality outbreaks were associated with positive thermal anomalies. In this study, we performed an analysis of inter-regional and inter-annual differences in temperature (T) conditions associated with MMEs of the red gorgonian Paramuricea clavata by analyzing high resolution T time series (hourly records for 3 to 8 years) from four regions of the NW Mediterranean with differing hydrological conditions and biological impacts. High resolution records allowed a detailed analysis using classical and new descriptors to characterize T anomalies. We were able to determine that the MMEs were triggered by two main types of positive thermal anomalies, with the first type being characterized by short periods (2 to 5 days) with high Mean T reaching more than 27°C in some regions and being associated with high intra-day and intra-period variability, while the second type of anomaly presented long duration (near one month) at warm T (24°C) with low intra-period variability. Inter-regional patterns arose; some regions displayed both types of anomalies, while others exhibited only one type. The results showed that T conditions should be considered as the main factor that explains the observed inter-regional and inter-annual differences in mortality impacts. In explaining these differences, the late timing of T anomalies, in addition to their magnitude was found to be determinant. Finally, by combining thermotolerance experimental data with the maximal T stress conditions observed in the four regions, we were able to determine the differential risk of mass mortality across regions. We conclude that expanding high resolution T series is important for the development of sound management and conservation plans to protect Mediterranean marine biodiversity in the face of climate change, Funding: This work was supported by the Alßan Program (C. Crisci doctoral fellowship, fellowship code: E06D103213UY; www.programalban.org); Parc National de Port-Cros; The IFB Foundation (Institut Français de la Biodiversite´ ); Agence Nationale pour la Recherche (ANR) (MEDCHANGE Project (http://www.agencenationale-recherche.fr; http://sudek.esil.univmed.fr/medchange/spip); Ministry of Science and Innovation (Biorock project ref. CTM2009-08045). These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dr. Nathaniel Bensoussan is employed by IPSO-FACTO SARL; he contributed to the analysis of the data and the writing of the manuscript

The respective roles of regular echinoids and scarid fishes in the transformation of turf algae, the main food resource for reef herbivores, were investigated on French Polynesian coral reefs. The role of one species of parrotfish (Scarus sordidus) was compared with that of four species of echinoids. The degree and ways of degradation of the algal matter were determined by the organic matter percentage, the composition of the sugar fraction, and the concentration and composition of chlorophylltype pigments as assayed by HPLC analysis. Chemical analyses were performed on anterior and posterior intestines for scarids, intestinal contents and faeces for echinoids, and on fresh algal turf as a control of initial food quality. A decrease in mean percentage of organic matter in gut content was observed from intestine (9.7%) to faeces (7%) in sea urchins, but not in parrotfishes. The total sugar fraction decreased from fresh algal turf (32% of total organic matter) to echinoid (28%) to scarid (18%) gut contents. The ratio of insoluble to soluble sugars (I/S ratios) was higher in echinoids (2.6) than in scarid gut contents (1.0). A decrease in the total pigment concentration was measured from fresh algal turf to echinoid and it was found to be even lower in scarid gut contents. Chromatograms showed that the composition of chlorophyll-type pigments in scarid intestines was very similar to fresh algal turf, with a dominance of native forms, mainly chlorophyll a and b. On the contrary, degraded pigment forms dominated in echinoids. The main degraded products were pheophorbides in sea urchins, and chlorophyllides in parrotfishes. These results provided evidence for differentiation in digestive processes occurring in the two types of grazers. Echinoids released higher degraded algal material than did scarids. Thus, these two types of grazers play different roles in the recycling of organic matter on coral reefs.

Experiments were conducted on the calcareous red alga, Corallina elongata, a species representative of shallow water vegetal cover in Mediterranean areas with biomass ranging from 820 to 2544 gDW.m-2, in order to estimate its productivity and calcification rates. Carbonate and oxygen budgets were estimated on samples incubated in situ under natural light cycles, by measuring initial and final dissolved oxygen, pH and alkalinity levels. In light conditions, oxygen concentrations and pH values increased as a consequence of oxygen production and carbon dioxide consumption due to the productivity process, and were a direct function of sample biomass. Strictly-reverse dynamics were recorded in dark conditions. A comparison of photosynthetic performances was conducted on a non-calcareous green alga, Ulva rigida, which showed higher rates of oxygen production and pH modification than C. elongata, but no significant change in total alkalinity. For C. elongata, a significant decrease in total alkalinity with incubation time was observed under light conditions, which was directly related to the algal sample biomass (R2 = 0.95; n=16). Light to dark calcification ratio (L/D) was about 3.6. In these experiments, the photosynthetic quotient of C. elongata was 0.89, its net carbon productivity was 2.5 g C.m-2.d-1, gross production to daily respiration (Pg/R) was about 4.9 and its calcification rate was estimated at 13.8 g CaCO3.m-2.d-1. Los experimentos se efectuaron sobre el alga calcárea roja, Corallina elongata, un representante de aguas someras en áreas mediterráneas con biomasa comprendida entre los límites de 820 GDW m-2, y 2544 GDW m-2para estimar su productividad y las tasas de calcificación. Las concentraciones de carbonato y oxígeno fueron estimados sobre muestras incubadas in situ bajo ciclos naturales de luz, midiendo el oxígeno disuelto al inicio y al final, así como los niveles de alcalinidad y pH. En condiciones de luz, las concentraciones de oxígeno y el pH aumentaron como consecuencia de la producción de oxígeno y el consumo de dióxido de carbono debido al proceso de productividad, y eran una función directa de la biomasa de la muestra. La dinámica estrictamente inversa fue registrada en condiciones de oscuridad. Una comparación del funcionamiento fotosintético se llevó a cabo sobre un alga nocalcárea verde, Ulva rigida, que presentó tasas más altas de producción de oxígeno y variación de pH que C. elongata, pero ningún cambio significativo en alcalinidad total. Para C. elongata, una disminución significativa en alcalinidad total con el tiempo de incubación fue observada en condiciones de luz, que directamente fueron relacionadas con la biomasa de muestra algal (R2 = 0.95; n=16). La relación entre la calcificación a la luz y a la oscuridad (L/D) fue aproximadamente 3.6. En estos experimentos, el cociente fotosintético de C. elongata fue 0.89, su productividad neta de carbono, 2.5 g C.m-2.d-1, la relación producción total respecto a la respiración diaria (Pg/R), 4.9 y su tasa de calcificación, 13.8 g CaCO3. C.m-2.d-1.

This study deals with the colonisation of experimental Porites blocks by epilithic and endolithic algal community in two atoll lagoons and two high-island lagoons in French Polynesia, exposed to different environmental conditions during a 5-year period. A qualitative analysis of the community (cyanobacteria and micro-algae) was carried out, and the relationship between the epilithic and endolithic strata observed. HPLC analysis of chlorophyll pigments was used to quantitatively determine the plant biomass, which was then compared to that of the algal turf of neighbouring reefs. The results show that these blocks are colonised according to two factors: the reef structure (atoll or high-island) and the degree of human disturbance. The epilithic and endolithic population dynamics differ. The density of the epilithic algal community and the species diversity increase with the degree of eutrophication and the amount of borers (sea-urchins, parrotfish), while the proportion of endolithic species remains low (34 %-59 %). This is especially marked in the high-island lagoons, where leaching has caused enhanced nutrient levels. An extreme case is the station at Faaa, a highly polluted harbour, where increased sea-urchin levels (44 ind. m(-2)) meant that the blocks disappeared completely in less than five years due to bioerosion. In the oligotrophic waters of the atoll lagoons, the losses of CaCO3 by bioerosion do not exceed 35 % per block, and the algal community is made up mainly of endolithic species (66 %-85 %). The algal biomass is lowest in the Takapoto atolls (10-11 mu g cm(-2)) and maximum in Tikehau atoll (23 and 56 mu g cm(-2)). The first microborers to settle in the ecological succession of the community are the cyanobacteria. These are gradually replaced by Ostreobium (Chlorophyceae borers), which become dominant in the blocks exposed to the lowest bioerosion rate. The richness and the density of the algal turf reflect the convergence of many ecological factors: it seems that the covering of epilithic algae or the richness of endolithic flora could be used as a global indicator of the quality and the health of a reef., Cette étude porte sur la colonisation par du feutrage algal épilithe et endolithe de bloes expérimentaux de Porites, mis en place dans deux atolls et deux îles hautes de Polynésie française et soumis à des contraintes environnementales différentes pendant cinq ans. La composition qualitative des peuplements (cyanobactéries et microalgues) et les relations entre «strateå épilithe et «strateå endolithe ont été observées. La quantification de la biomasse végétale a été effectuée par la mesure des pigments chlorophylliens analysés en HPLC. Des comparaisons ont étéétablies avec le feutrage algal des substrats récifaux avoisinants. Les résultats font apparaître des différences dans l'évolution de la colonisation de ces blocs selon deux facteurs: la structure récifale (atolls ou îles hautes) et le degré d'anthropisation des milieux. Par ailleurs, la dynamique de la colonisation est différente pour les populations épilithes et endolithes. La densité du feutrage algal épilithe et la diversité spécifique augmentent avec le degré d'eutrophisation des eaux et la quantité des organismes brouteurs (oursins, Scaridés), alors que la contribution des espèces endolithes dans ce feutrage reste faible (34 et 59 %). C'est le cas notamment dans les stations des îles hautes, enrichies en nutriments par le lessivage des sols. L'exemple extrême est représenté par la station de Faaa (ville de Papeete) où la forte pollution urbaine et le nombre élevé d'oursins bioérodeurs (> 44 ind. m2) ont entraîné la disparition des blocs en moins de cinq ans. Dans les atolls, les eaux sont oligotrophes, les pertes de CaCO3 par bioérosion ne dépassent pas 35 % par bloc, et le peuplement algal est constitué principalement d'espèces endolithes (66 % à 85 % de l'ensemble du peuplement). Sur les blocs de l'atoll de Takapoto, la biomasse du feutrage algal épilithe et endolithe est la plus faible et n'atteint que 10 à 11 μg chl a cm−2, alors qu'elle est maximale (23 et 56 μg chl a cm−2) aux deux stations de l'atoll de Tikehau. Dans la succession écologique, les premiers microperforants qui s'installent sont les cyanobactéries; celles-ci sont peu à peu remplacées par l'Ostreobium (Chlorophycée perforante) qui devient dominant dans les blocs soumis à la plus faible bioérosion. La richesse et la densité du feutrage algal reflétant l'intégration de nombreux facteurs écologiques, il apparaît que la couverture en épilithes ou la richesse en flore endolithe pourraient servir d'indicateur global de la qualité et de la santé d'un récif.

Chlorophyll and its breakdown product contents were determined by both high pressure liquid chromatography (HPLC) and the fluorimetric method in surface microlayer and related bulk water samples collected in the western Mediterranean Sea. Differences in chlorophyll α measurements between the two methods are enhanced in highly loaded organic matter water samples such as surface microlayers. In several samples, pheophytins, which are commonly reported to be present in surface microlayers, were detected by the fluorimetric method, but not by HPLC. In most cases, the accumulation processes of pigments can be recorded in surface microlayers, but with small differences in pigment composition when compared with that of related bulk waters. Enrichment factors for chlorophyll are often higher than those of its degradation products. On the other hand, the low reported values of phytoneustonic productivity provide evidence for an inhibition of phytoplankton organisms transported to the sea-air interface; these organisms should be stressed but alive. These results also support the hypothesis that pigments could be actively photo-oxidized by the high level of solar radiation prevailing in surface microlayers.

Discriminating approach of various microplanktonic-stage contributions to whole electron transport system (ETS) measurements in sea-water sampless. b. Bacateria Data obtained from cultures of natural bacterial populations were applied to various water samples in order to determine both the phytoplanktonic and bacterial contributions to whole ETS water-sample measurements. The bacterial part was estimated both by cell enumerations and HPLC muramic-acid measurements and the phytoplankton contribution by enumeration and chlorophyll levels. An appropriate first-order equation is adequate to low carbon content samples but must be corrested for highly organic loaded samples in order to obtain a better adjustment between the sum of the “estimated” phytoplanktonic and bacterial ETS and the really-measured ETS concentrations in the water samples.

Discriminating Approach of Various Microplanktonic-stage Contributions to Whole ETS Measurements in Sea-water Samples. a. Phytoplankton Data obtained from algal cell cultures were applied to various sea water samples in order to estimate the specific contribution of phytoplanktonic organisms to whole ETS (electron transport system) measurements of these samples which have been collected at eleven stations in the northwest Mediterranean Sea. When the phytoplankton ETS contributions were quantified by cell enumerations or by other indirect-biomass-indicator measurements, the results are in good accordance to the recorded ETS measurement values of the sample. More specific of the phytoplankton stage is the index used for such an estimation (as chlorophyll a), and better is the relationship between both estimated and measured ETS values.

Based on the information obtained from the Gulf of Fos, France, we propose herewith a generalized model to characterize the physical, chemical and biological parameters and their interrelationship in the eutrophic coastal system. This system is characterized by having high concentration of suspended materials, especially the mineral fraction (C/N > 30). Nutrients concentrations are very high, except in diluted basins. The N-NO3/P-P04 ratio is usually low, due to the preferential assimilation of nitrates and the presence of polyphosphates from sewage waters. The concentrations of phaeopigments are always high (more than 50%), due to the mortalities of freshwater originated phytoplankton, the resuspension of dead vegetal materials, and the grazing of zooplankton. We noted in diluted water of eutrophic system the biomass is high, but the diversity is relatived low, among adapted cells with low " energetic charge". Whereas in oceanic water, the biomass is less, the diversity and energetic charge are increasing. This may indicate that phytoplankton communities have to make an effort to grow and duplicate. In comparing zooplankton populations from eutrophic to oligotrophic waters, biochemical diferentiations were observed for a same species, which may due to the difference in salinities and the presence of chemical pollutants.

Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 � 10 −10 to 5.3 � 10 −10 μg per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 10 8 to 10 11 cells per liter. The muramic acid content in natural marine water decreased from 5.3 � 10 −10 to 1.6 � 10 −10 μg per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis.