Reactive Oxygen Species (ROS) constitute important intracellular signaling molecules. Mitochondria are admitted sources of ROS, especially of superoxide anions through the electron transport chain. Here the mitochondria-targeted ratiometric pericam (RPmt) was used as a superoxide biosensor, by appropriate choice of the excitation wavelength. RPmt was transfected in vivo into mouse muscles. Confocal imaging of isolated muscle fibers reveals spontaneous flashes of RPmt fluorescence. Flashes correspond to increases in superoxide production, as shown by simultaneous recordings of the fluorescence from MitoSox, a mitochondrial superoxide probe. Flashes occur in all subcellular populations of mitochondria. Spatial analysis of the flashes pattern over time revealed that arrays of mitochondria work as well-defined superoxide-production-units. Increase of superoxide production at the muscle fiber level involves recruitment of supplemental units with no increase in per-unit production. Altogether, these results demonstrate that superoxide flashes in muscle fibers correspond to physiological signals linked to mitochondrial metabolism. They also suggest that superoxide, or one of its derivatives, modulates its own production at the mitochondrial level.

This study examined the gametogenic cycle of Crassostrea gigas in controlled conditions over one year, with a focus on the initiation of gametogenesis. This work analysed also the role of temperature and photoperiod in the regulation of oyster reproduction. Broodstock were maintained in natural (NC), accelerated (AC) and perpetual winter (WC) conditions of temperature and photoperiod, with feeding ad libitum. Qualitative and quantitative analyses of the reproductive pattern were performed using biometric measurement approach, sex ratio determination, histology and a gonad filling index. Each experimental treatment led to different strategies for growth and resource allocation. The gametogenic cycle, appeared entirely modulated, accelerated or delayed, by coupled temperature/photoperiod parameters. Temperature played a key role in gonial mitosis regulation. Gonia proliferation was set off and sustained by winter temperature (8-11 °C) whatever the physiological state of oysters. Maturation of germ cells appeared to be a function of temperature and could proceed at low temperature, while ripe oysters were obtained at 8 °C in winter conditioning. The three conditioning methods used in this study, allowed the production of gametes throughout the year, including in the autumnal resting period. Moreover, stocks of ripe oysters could be maintained at low temperature during several months to produce spat when desired for aquaculture production.

The Dynamic Energy Budget (DEB) model (Kooijman, S.A.L.M., 1986. Energy budgets can explain body size relations. pp.) has been adapted to describe the dynamics of growth and reproduction of the Pacific oyster (Crassostrea gigas) reared in different areas under conditions ranging from controlled to natural. The values of the model parameters were estimated from available physiological data and from published information. The sets of data used to validate the model came from three long-term growth experiments (> 5 months) performed on Pacific oysters reared under different conditions of food and environment. The forcing variables were temperature and phytoplankton densities, the latter being assessed from in vivo fluorescence and chlorophyll-a concentration measurement. The successful validation of the model on the three data sets demonstrated its ability to capture the dynamics of the energy budget in the Pacific oyster in various environments with the same set of parameters. The only parameter that varied between simulations was the half-saturation coefficient (XK), because of a different diet composition between the three environments under test. The model successfully reproduced quantitatively the growth and reproduction and the timing of spawning. These first simulation data led us to propose several promising perspectives of application for this model in shellfish ecosystems.

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International audienceSummer mortality of Pacific oysters is known in several countries. However no specific pathogen has been systematically associated with this phenomenon. A complex combination of environmental and biological parameters has been suggested as the cause and is now starting to be identified. A high genetic basis was found for survival in oysters when a first generation (G1) was tested in three sites during summer. This paper presents a synthesis on physiological characteristics of two selected groups (‘R' and ‘S', from families selected for resistance and susceptibility to summer mortality respectively), of the second and third generations. R and S showed improvement or reduction of survival compared with the control in both field and laboratory trials confirming the high heritability of survival of juveniles <1 year old. Interestingly, no correlation was observed between growth and survival. Comparison between the two selected groups showed that S oysters invested more energy in reproduction and stayed a longer time without spawning than R oysters which had high synchronous spawning. This was mainly shown with high rather than low dietary rations (respectively 12% and 4% DW algae/DW oyster) in a controlled experiment. Moreover, early partial spawning was detected in S oysters and not R ones in the high dietary ration. S showed a higher respiration rate and an earlier decrease in absorption efficiency than R during gametogenesis, but they were not significantly different in glycogen or ATP utilisation. Two months before a mortality episode, hemocytes from S oysters had a higher adhesive capacity than R hemocytes and significantly higher reactive oxygen species production capacity. One month before mortality, S oysters had the highest hyalinocyte concentration and their expression of genes coding for glucose metabolism enzymes (Hexokinase, GS, PGM, PEPCK) was significantly lower in the labial palps. After a thermal increase from 13 °C to 19 °C, during 8 days in normoxia, S oysters showed a large HSP70 increase under hypoxia contrary to R oysters, suggesting their high susceptibility to stress. Their catalase activity was lower than in R oysters and showed no further change to subsequent hypoxia and pesticide stresses, in contrast to R oysters. These observations suggest possible links between higher reproductive effort in S oysters, their specific stress response to temperature and hypoxia, ROS production, partial spawning, hyalinocyte increase and the infection process. To compare R and S oysters in a more integrated way, a suppression subtractive hybridisation (SSH) library and a micro-array strategy are being undertaken

Efforts to restore the native oyster Ostrea edulis and its associated habitats are gaining momentum across Europe. Several projects are currently running or being planned. To maximize the success of these, it is crucial to draw on existing knowledge and experience in order to design, plan and implement restoration activities in a sustainable and constructive approach. For the development of best practice recommendations and to promote multidimensional knowledge and technology exchange, the Native Oyster Restoration Alliance (NORA) was formed by partners from science, technology, nature conservation, consultancies, commercial producers and policy-makers. The NORA network will enhance scientific and practical progress in flat oyster restoration, such as in project planning and permitting, seed oyster production, disease management and monitoring. It also focuses on joint funding opportunities and the potential development of national and international regulatory frameworks. The main motivation behind NORA is to facilitate the restoration of native oyster habitat within its historic biogeographic range in the North Sea and other European seas along with the associated ecosystem services; services such as enhancing biodiversity, including enhanced fish stocks, nutrient cycling and sediment stabilization. NORA members agreed on a set of joint recommendations and strongly advise that any restoration measure should respect and apply these recommendations: The Berlin Oyster Recommendation is presented here. It will help guide the development of the field by developing and applying best practice accordingly. NORA also aims to combine the outreach activities of local projects for improved community support and awareness and to provide educational material to increase knowledge of the key ecological role of this species and increase awareness among regulators, permit providers and stakeholders. A synthesis of O. edulis restoration efforts in Europe is provided and underlines the general significance in the field.

As in all previous determinations in mammals and non-mammals, release flux consisted of an early peak, relaxing to a lower level from which it continued to decay more slowly. Decay of flux in this second stage, which has been attributed largely to depletion of SR Ca 2+ , was studied in detail. A simple depletion mechanism without change in release permeability predicts an exponential decay with time. In contrast, flux decreased non-exponentially, to a finite, measurable level that could be maintained for the longest pulses applied (1.8 s). An algorithm on the flux record allowed us to define a quantitative index, the normalized flux rate of change (NFRC), which was shown to be proportional to the ratio of release permeability P and inversely proportional to Ca 2+ buffering power B of the SR, thus quantifying the 'evacuability' or ability of the SR to empty its content. When P and B were constant, flux then decayed exponentially, and NFRC was equal to the exponential rate constant. Instead, in most cases NFRC increased during the pulse, from a minimum reached immediately after the early peak in flux, to a time between 200 and 250 ms, when the index was no longer defined. NFRC increased by 111% on average (in 27 images from 18 cells), reaching 300% in some cases. The increase may reflect an increase in P, a decrease in B, or both. On experimental and theoretical grounds, both changes are to be expected upon SR depletion. A variable evacuability helps maintain a constant Ca 2+ output under conditions of diminishing store Ca 2+ load. To allow contractile activation of skeletal muscle upon action potential depolarization, Ca 2+ is rapidly released from the sarcoplasmic reticulum (SR) by ryanodine-receptor channels (RyR) that open under the influence of voltage sensors of the transverse (t) tubule membrane. The response of the channels to a single action potential consists of a single peak or spike of release flux. The magnitude and evolution of this flux is determined by a number of mechanisms, which presumably evolved to optimize the signalling function at the activation frequencies that occur during physiological muscle contraction.A classical approach to the study of the mechanisms that control Ca 2+ release is the examination of Ca 2+ transients and release flux caused by prolonged depolarization. These studies were initiated in frog muscle (Baylor et al. 1983;Melzer et al. 1984) and the methods developed for the frog were later adapted to rat (Garcia & Stefani, 1990;

Ingestion, growth and metamorphosis of Pacific oyster, Crassostrea gigas, larvae were studied under controlled conditions of food density and temperature using a combination of a flow-through rearing system and a hydrobiological monitoring device. In a first experiment larvae were exposed to three different phytoplankton densities (12, 20 and 40 cells µl-1) while in a second trial larvae were reared at five different temperatures (17, 22, 25, 27 and 32°C). Both food concentration and temperature significantly affected the larval physiology throughout the entire development from D-veliger to young spat. Larvae survived over a wide range of both environmental parameters with high survival at the end of experiments. The feeding functional response provided the maximal ingestion rate (50 000 cells larva-1 day-1) which occurred at an algal density of 20 cells µl-1 surrounding the larvae and 25 °C. At the highest temperature (32 °C), maximal growth and metamorphosis performances were reached in less than 2 weeks while the lowest temperature (17°C) consistently inhibited ingestion and growth over the entire larval period. The estimate of the Arrhenius temperature (TA) was 11000 K for C. gigas larvae. Larval development could be divided on the basis of feeding activity into an initial mixotrophic period with a lower and constant ingestion over the first days (from D-stage to early umbonate larva of ≈110 µm length) followed by an exotrophic phase characterized by a sharp increase in ingestion (umbonate to eyed of ≈ 300 µm length) and, finally, a third period for larvae 300 µm during which ingestion decreased suddenly because of metamorphosis. Optimum larval development and settlement of the oyster C. gigas occurred at 27 °C and an increasing food supply as the larvae were growing. A food density of ≥20 cells µl-1 of T-ISO + CP or CG (1:1 cells number) in the culture water was required to maximise growth and metamorphosis success.