A new colorimetrically-calibrated automated video-imaging protocol for day-night fish counting at the OBSEA coastal cabled observatory.

Field measurements of the swimming activity rhythms of fishes are scant due to the difficulty of counting individuals at a high frequency over a long period of time. Cabled observatory video monitoring allows such a sampling at a high frequency over unlimited periods of time. Unfortunately, automation for the extraction of biological information (i.e., animals' visual counts per unit of time) is still a major bottleneck. In this study, we describe a new automated video-imaging protocol for the 24-h continuous counting of fishes in colorimetrically calibrated time-lapse photographic outputs, taken by a shallow water (20 m depth) cabled video-platform, the OBSEA. The spectral reflectance value for each patch was measured between 400 to 700 nm and then converted into standard RGB, used as a reference for all subsequent calibrations. All the images were acquired within a standardized Region Of Interest (ROI), represented by a 2 × 2 m methacrylate panel, endowed with a 9-colour calibration chart, and calibrated using the recently implemented "3D Thin-Plate Spline" warping approach in order to numerically define color by its coordinates in n-dimensional space. That operation was repeated on a subset of images, 500 images as a training set, manually selected since acquired under optimum visibility conditions. All images plus those for the training set were ordered together through Principal Component Analysis allowing the selection of 614 images (67.6%) out of 908 as a total corresponding to 18 days (at 30 min frequency). The Roberts operator (used in image processing and computer vision for edge detection) was used to highlights regions of high spatial colour gradient corresponding to fishes' bodies. Time series in manual and visual counts were compared together for efficiency evaluation. Periodogram and waveform analysis outputs provided very similar results, although quantified parameters in relation to the strength of respective rhythms were different. Results indicate that automation efficiency is limited by optimum visibility conditions. Data sets from manual counting present the larger day-night fluctuations in comparison to those derived from automation. This comparison indicates that the automation protocol subestimate fish numbers but it is anyway suitable for the study of community activity rhythms.

Ten years after the prestige oil spill: seabird trophic ecology as indicator of long-term effects on the coastal marine ecosystem.

Major oil spills can have long-term impacts since oil pollution does not only result in acute mortality of marine organisms, but also affects productivity levels, predator-prey dynamics, and damages habitats that support marine communities. However, despite the conservation implications of oil accidents, the monitoring and assessment of its lasting impacts still remains a difficult and daunting task. Here, we used European shags to evaluate the overall, lasting effects of the Prestige oil spill (2002) on the affected marine ecosystem. Using δ ¹5N and Hg analysis, we trace temporal changes in feeding ecology potentially related to alterations of the food web due to the spill. Using climatic and oceanic data, we also investigate the influence of North Atlantic Oscillation (NAO) index, the sea surface temperature (SST) and the chlorophyll a (Chl a) on the observed changes. Analysis of δ ¹5N and Hg concentrations revealed that after the Prestige oil spill, shag chicks abruptly switched their trophic level from a diet based on a high percentage of demersal-benthic fish to a higher proportion of pelagic/semi-pelagic species. There was no evidence that Chl a, SST and NAO reflected any particular changes or severity in environmental conditions for any year or season that may explain the sudden change observed in trophic level. Thus, this study highlighted an impact on the marine food web for at least three years. Our results provide the best evidence to date of the long-term consequences of the Prestige oil spill. They also show how, regardless of wider oceanographic variability, lasting impacts on predator-prey dynamics can be assessed using biochemical markers. This is particularly useful if larger scale and longer term monitoring of all trophic levels is unfeasible due to limited funding or high ecosystem complexity.

A new laboratory radio frequency identification (RFID) system for behavioural tracking of marine organisms.

Radio frequency identification (RFID) devices are currently used to quantify several traits of animal behaviour with potential applications for the study of marine organisms. To date, behavioural studies with marine organisms are rare because of the technical difficulty of propagating radio waves within the saltwater medium. We present a novel RFID tracking system to study the burrowing behaviour of a valuable fishery resource, the Norway lobster (Nephrops norvegicus L.). The system consists of a network of six controllers, each handling a group of seven antennas. That network was placed below a microcosm tank that recreated important features typical of Nephrops' grounds, such as the presence of multiple burrows. The animals carried a passive transponder attached to their telson, operating at 13.56 MHz. The tracking system was implemented to concurrently report the behaviour of up to three individuals, in terms of their travelled distances in a specified unit of time and their preferential positioning within the antenna network. To do so, the controllers worked in parallel to send the antenna data to a computer via a USB connection. The tracking accuracy of the system was evaluated by concurrently recording the animals' behaviour with automated video imaging. During the two experiments, each lasting approximately one week, two different groups of three animals each showed a variable burrow occupancy and a nocturnal displacement under a standard photoperiod regime (12 h light:12 h dark), measured using the RFID method. Similar results were obtained with the video imaging. Our implemented RFID system was therefore capable of efficiently tracking the tested organisms and has a good potential for use on a wide variety of other marine organisms of commercial, aquaculture, and ecological interest.

The new Seafloor Observatory (OBSEA) for remote and long-term coastal ecosystem monitoring.

A suitable sampling technology to identify species and to estimate population dynamics based on individual counts at different temporal levels in relation to habitat variations is increasingly important for fishery management and biodiversity studies. In the past two decades, as interest in exploring the oceans for valuable resources and in protecting these resources from overexploitation have grown, the number of cabled (permanent) submarine multiparametric platforms with video stations has increased. Prior to the development of seafloor observatories, the majority of autonomous stations were battery powered and stored data locally. The recently installed low-cost, multiparametric, expandable, cabled coastal Seafloor Observatory (OBSEA), located 4 km off of Vilanova i la Gertrú, Barcelona, at a depth of 20 m, is directly connected to a ground station by a telecommunication cable; thus, it is not affected by the limitations associated with previous observation technologies. OBSEA is part of the European Multidisciplinary Seafloor Observatory (EMSO) infrastructure, and its activities are included among the Network of Excellence of the European Seas Observatory NETwork (ESONET). OBSEA enables remote, long-term, and continuous surveys of the local ecosystem by acquiring synchronous multiparametric habitat data and bio-data with the following sensors: Conductivity-Temperature-Depth (CTD) sensors for salinity, temperature, and pressure; Acoustic Doppler Current Profilers (ADCP) for current speed and direction, including a turbidity meter and a fluorometer (for the determination of chlorophyll concentration); a hydrophone; a seismometer; and finally, a video camera for automated image analysis in relation to species classification and tracking. Images can be monitored in real time, and all data can be stored for future studies. In this article, the various components of OBSEA are described, including its hardware (the sensors and the network of marine and land nodes), software (data acquisition, transmission, processing, and storage), and multiparametric measurement (habitat and bio-data time series) capabilities. A one-month multiparametric survey of habitat parameters was conducted during 2009 and 2010 to demonstrate these functions. An automated video image analysis protocol was also developed for fish counting in the water column, a method that can be used with cabled coastal observatories working with still images. Finally, bio-data time series were coupled with data from other oceanographic sensors to demonstrate the utility of OBSEA in studies of ecosystem dynamics.

Activity rhythms in the deep-sea: a chronobiological approach.

Ocean waters deeper than 200 m cover 70% of the Earth's surface. Light intensity gets progressively weaker with increasing depth and internal tides or inertial currents may be the only remaining zeitgebers regulating biorhythms in deep-sea decapods. Benthopelagic coupling, exemplified by vertically moving shrimps within the water column, may also act as a source of indirect synchronisation to the day-night cycle for species living in permanently dark areas. At the same time, seasonal rhythms in growth and reproduction may be an exogenous response to spring-summer changes in upper layer productivity (via phytoplankton) or, alternatively, may be provoked by the synchronisation mediated by an endogenous controlling mechanism (via melatonin). In our review, we will focus on the behavioural rhythms of crustacean decapods inhabiting depths where the sun light is absent. Potential scenarios for future research on deep-sea decapod behaviour are suggested by new in situ observation technologies. Permanent video observatories are, to date, one of the most important tools for marine chronobiology in terms of species recognition and animals' movement tracking.

Deep-sea biodiversity in the Mediterranean Sea: the known, the unknown, and the unknowable.

Deep-sea ecosystems represent the largest biome of the global biosphere, but knowledge of their biodiversity is still scant. The Mediterranean basin has been proposed as a hot spot of terrestrial and coastal marine biodiversity but has been supposed to be impoverished of deep-sea species richness. We summarized all available information on benthic biodiversity (Prokaryotes, Foraminifera, Meiofauna, Macrofauna, and Megafauna) in different deep-sea ecosystems of the Mediterranean Sea (200 to more than 4,000 m depth), including open slopes, deep basins, canyons, cold seeps, seamounts, deep-water corals and deep-hypersaline anoxic basins and analyzed overall longitudinal and bathymetric patterns. We show that in contrast to what was expected from the sharp decrease in organic carbon fluxes and reduced faunal abundance, the deep-sea biodiversity of both the eastern and the western basins of the Mediterranean Sea is similarly high. All of the biodiversity components, except Bacteria and Archaea, displayed a decreasing pattern with increasing water depth, but to a different extent for each component. Unlike patterns observed for faunal abundance, highest negative values of the slopes of the biodiversity patterns were observed for Meiofauna, followed by Macrofauna and Megafauna. Comparison of the biodiversity associated with open slopes, deep basins, canyons, and deep-water corals showed that the deep basins were the least diverse. Rarefaction curves allowed us to estimate the expected number of species for each benthic component in different bathymetric ranges. A large fraction of exclusive species was associated with each specific habitat or ecosystem. Thus, each deep-sea ecosystem contributes significantly to overall biodiversity. From theoretical extrapolations we estimate that the overall deep-sea Mediterranean biodiversity (excluding prokaryotes) reaches approximately 2805 species of which about 66% is still undiscovered. Among the biotic components investigated (Prokaryotes excluded), most of the unknown species are within the phylum Nematoda, followed by Foraminifera, but an important fraction of macrofaunal and megafaunal species also remains unknown. Data reported here provide new insights into the patterns of biodiversity in the deep-sea Mediterranean and new clues for future investigations aimed at identifying the factors controlling and threatening deep-sea biodiversity.

Light intensity determines temporal niche switching of behavioral activity in deep-water Nephrops norvegicus (Crustacea: Decapoda).

The temporal distribution of behavioral programs throughout the 24-h day, known as temporal niche of a species, is determined by ecological factors that directly affect the adaptive value of the timing of specific behaviors. Temporal niche switching has been described in several species and is likely adaptive in habitats where the daily timing of those factors changes. Benthic species whose habitats span a wide range of water depths are exposed to considerable depth-dependent environmental changes. Temporally scheduled trawl surveys of the Norway lobster, Nephrops norvegicus, reveal that animals emerge from burrows at night on the shallow shelf (10-50 m deep), at crepuscular hours on the lower shelf (50-200 m), and at daytime on the slope (200-400 m). The mechanisms underlying nocturnality/diurnality switches are chiefly unknown, and Nephrops offers a unique model for their study. The depth-dependent decrease in luminance is a likely candidate determining the temporal distribution of behavior. The authors explored this possibility in the laboratory by exposing Nephrops to light:dark (LD) cycles of 470-nm monochromatic lighting that mimic conditions at the 100-m-deep shelf (10 lux) or the 300-m slope (0.1 lux). Two groups of animals were respectively exposed to each light intensity according to the following protocol: an initial 12:12 LD stage followed by constant darkness (DD), followed in turn by a second 12:12 LD stage. Activity at the burrow opening (door-keeping = DK), as well as full emergence (E), was continuously monitored. Under 10-lux LD cycles, most animals showed nocturnal DK activity-with some being crepuscular or diurnal-and all animals showed nocturnal E activity. In contrast, both behaviors were clearly diurnal in animals under 0.1-lux LD cycles. The phase of the nocturnal and diurnal DK rhythms detected respectively at 10 and 0.1 lux upon release into DD revealed that these rhythms are entrained circadian rhythms. The present data indicate that nocturnality/diurnality switches in Nephrops in its natural habitat, evidenced by captures at different depths, are likely determined by light intensity. This temporal niche switching involves different patterns of photic entrainment, leading to bona fide circadian diurnal or nocturnal phenotypes, as well as exogenous masking of behavioral outputs.

A new morphometric implemented video-image analysis protocol for the study of social modulation in activity rhythms of marine organisms.

Video-image analysis can be an efficient tool for microcosm experiments portraying the modulation of individual behaviour based on sociality. The Norway lobster, Nephrops norvegicus is a burrowing decapod the commercial capture of which occurs by trawling only when animals are engaged in seabed excursions. Emergence behaviour is modulated by the day-night cycle but a further modulation occurs upon social interaction in a still unknown fashion. Here, we present a novel automated protocol for the tracking of the movement of different animals at once based on a multivariate morphometric approach. Four black and white tags were customized according to a precise geometric design. Shape Matching and Complex Fourier Descriptors analyses were used to track tag displacement through consecutive frames in a 7-day experiment under monochromatic blue light (480 nm)-darkness conditions. Shape Matching errors were evaluated in relation to tag geometry. Time series of centroid coordinates in pixels were transformed in centimetres. The FD analysis was slightly less efficient than the Shape Matching, although more rapid (i.e. up to 20 times faster). Nocturnal rhythms were reported for all animals. Waveform analysis indicated marked differences in the amplitude of activity phases as proof of interindividual interaction. Total diel activity presented a decrease in the rate of out of burrow locomotion as the testing progressed. N. norvegicus is a nocturnal species and present observations sustain the efficiency and fidelity of our automated tracking system.

A new tracking system for the measurement of diel locomotor rhythms in the Norway lobster, Nephrops norvegicus (L.).

The Norway lobster, Nephrops norvegicus (L.), is a deep-water burrowing decapod of high commercial value. Diel variations in trawl captures are produced by population rhythms of burrow emergences related to day-night cycles. Rhythms seem to be different in males and females since catches show variations in sex ratios depending on the season. Our hypothesis is that the diel rhythm of activity in this species can be distinguished in three different behavioural sets, the durations of which show gender-related modulation: door-keeping, proximal-, and distal-emergence from the burrow. Our aim is to detail the functioning of a new tracking system allowing the durations of these three behavioural components to be determined. Movement of animals was detected by subdividing aquaria into different zones by means of three rows of infrared-emitting and -receiving photodiodes in which blue light emitters were also integrated for the generation of light cycles. We recorded movement patterns in adult males and females (n=20) exposed to a standard photoperiod regime (i.e., 12 h; monochromatic at 480 nm of 5 lx) over 12 days. Marked diel nocturnal rhythms were reported at all barriers, with activity peaks diffused over the night at the burrow entrance and located at the day-night transition at other barriers (i.e., crepuscular peaks that decreased in the next few hours of darkness). Mean total activity was significantly higher for females than males at the burrow entrance (i.e., door-keeping behaviour). Males had significantly higher activity at other locations (proximal- and distal-emergence behaviours).

Climate influence on deep sea populations.

Dynamics of biological processes on the deep-sea floor are traditionally thought to be controlled by vertical sinking of particles from the euphotic zone at a seasonal scale. However, little is known about the influence of lateral particle transport from continental margins to deep-sea ecosystems. To address this question, we report here how the formation of dense shelf waters and their subsequent downslope cascade, a climate induced phenomenon, affects the population of the deep-sea shrimp Aristeus antennatus. We found evidence that strong currents associated with intense cascading events correlates with the disappearance of this species from its fishing grounds, producing a temporary fishery collapse. Despite this initial negative effect, landings increase between 3 and 5 years after these major events, preceded by an increase of juveniles. The transport of particulate organic matter associated with cascading appears to enhance the recruitment of this deep-sea living resource, apparently mitigating the general trend of overexploitation. Because cascade of dense water from continental shelves is a global phenomenon, we anticipate that its influence on deep-sea ecosystems and fisheries worldwide should be larger than previously thought.