Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA.

The naturally occurring ectoparasite salmon lice (Lepeophtherirus salmonis) poses a great challenge for the salmon farming industry, as well as for wild salmonids in the Northern hemisphere. To better control the infestation pressure and protect the production, there is a need to provide fish farmers with sensitive and efficient tools for rapid early detection and monitoring of the parasitic load. This can be achieved by targeting L. salmonis DNA in environmental samples. Here, we developed and tested a new L. salmonis specific DNA-based assay (qPCR assay) for detection and quantification from seawater samples using an analytical pipeline compatible with the Environmental Sample Processor (ESP) for autonomous water sample analysis of gene targets. Specificity of the L. salmonis qPCR assay was demonstrated through in-silico DNA analyses covering sequences of different L. salmonis isolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages of L. salmonis to investigate the relationship with the number of marker gene copies (MGC). Finally, field samples collected at different times of the year in the vicinity of a salmon production farm in Western Norway were analyzed for L. salmonis detection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages of L. salmonis was established in the laboratory conditions. In the field, L. salmonis DNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that only L. salmonis tissue or eDNA residues were detected. This novel study opens for a fully automatized L. salmonis DNA quantification using ESP robotic to monitor the parasitic load, but challenges remain to exactly transfer information about eDNA quantities to decisions by the farmers and possible interventions.

Exploring time series of hyperspectral images for cold water coral stress response analysis.

Hyperspectral imaging (HSI) is a promising technology for environmental monitoring with a lot of undeveloped potential due to the high dimensionality and complexity of the data. If temporal effects are studied, such as in a monitoring context, the analysis becomes more challenging as time is added to the dimensions of space (image coordinates) and wavelengths. We conducted a series of laboratory experiments to investigate the impact of different stressor exposure patterns on the spectrum of the cold water coral Desmophyllum pertusum. 65 coral samples were divided into 12 groups, each group being exposed to different types and levels of particles. Hyperspectral images of the coral samples were collected at four time points from prior to exposure to 6 weeks after exposure. To investigate the relationships between the corals' spectral signatures and controlled experimental parameters, a new software tool for interactive visual exploration was developed and applied, the HypIX (Hyperspectral Image eXplorer) web tool. HypIX combines principles from exploratory data analysis, information visualization and machine learning-based dimension reduction. This combination enables users to select regions of interest (ROI) in all dimensions (2D space, time point and spectrum) for a flexible integrated inspection. We propose two HypIX workflows to find relationships in time series of hyperspectral datasets, namely morphology-based filtering workflow and embedded driven response analysis workflow. With these HypIX workflows three users identified different temporal and spatial patterns in the spectrum of corals exposed to different particle stressor conditions. Corals exposed to particles tended to have a larger change rate than control corals, which was evident as a shifted spectrum. The responses, however, were not uniform for coral samples undergoing the same exposure treatments, indicating individual tolerance levels. We also observed a good inter-observer agreement between the three HyPIX users, indicating that the proposed workflow can be applied to obtain reproducible HSI analysis results.

Identification of tolerance levels on the cold-water coral Desmophyllum pertusum (Lophelia pertusa) from realistic exposure conditions to suspended bentonite, barite and drill cutting particles.

Cold-water coral (CWC) reefs are numerous and widespread along the Norwegian continental shelf where oil and gas industry operate. Uncertainties exist regarding their impacts from operational discharges to drilling. Effect thresholds obtained from near-realistic exposure of suspended particle concentrations for use in coral risk modeling are particularly needed. Here, nubbins of Desmophyllum pertusum (Lophelia pertusa) were exposed shortly (5 days, 4h repeated pulses) to suspended particles (bentonite BE; barite BA, and drill cuttings DC) in the range of ~ 4 to ~ 60 mg.l-1 (actual concentration). Physiological responses (respiration rate, growth rate, mucus-related particulate organic carbon OC and particulate organic nitrogen ON) and polyp mortality were then measured 2 and 6 weeks post-exposure to assess long-term effects. Respiration and growth rates were not significantly different in any of the treatments tested compared to control. OC production was not affected in any treatment, but a significant increase of OC:ON in mucus produced by BE-exposed (23 and 48 mg.l-1) corals was revealed 2 weeks after exposure. Polyp mortality increased significantly at the two highest DC doses (19 and 49 mg.l-1) 2 and 6 weeks post-exposure but no significant difference was observed in any of the other treatments compared to the control. These findings are adding new knowledge on coral resilience to short realistic exposure of suspended drill particles and indicate overall a risk for long-term effects at a threshold of ~20 mg.l-1.

Abundance and diversity of n-alkane and PAH-degrading bacteria and their functional genes - Potential for use in detection of marine oil pollution.

Monitoring environmental status through molecular investigation of microorganisms in the marine environment is suggested as a potentially very effective method for biomonitoring, with great potential for automation. There are several hurdles to that approach with regards to primer design, variability across geographical locations, seasons, and type of environmental pollution. Here, qPCR analysis of genes involved in the initial activation of aliphatic and aromatic hydrocarbons were used in a laboratory setup mimicking realistic oil leakage at sea. Seawater incubation experiments were carried out under two different seasons with two different oil types. Degenerate primers targeting initial oxygenases (alkane 1-monooxygenase; alkB and aromatic-ring hydroxylating dioxygenase; ARHD) were employed in qPCR assays to quantify the abundance of genes essential for oil degradation. Shotgun metagenomics was used to map the overall community dynamics and the diversity of alkB and ARHD genes represented in the microbial community. The amplicons generated through the qPCR assays were sequenced to reveal the diversity of oil-degradation related genes captured by the degenerate primers. We identified a major mismatch between the taxonomic diversity of alkB and ARHD genes amplified by the degenerate primers and those identified through shotgun metagenomics. More specifically, the designed primers did not amplify the alkB genes of the two most abundant alkane degraders that bloomed in the experiments, Oceanobacter and Oleispira. The relative abundance of alkB sequences from shotgun metagenomics and 16S rRNA-based Oleispira-specific qPCR assay were better signals for oil in water than the tested qPCR alkB assay. The ARHD assay showed a good agreement with PAHs degradation despite covering only 25% of the top 100 ARHD genes and missing several abundant Cycloclasticus sequences that were present in the metagenome. We conclude that further improvement of the degenerate primer approach is needed to rely on the use of oxygenase-related qPCR assays for oil leakage detection.

Effects of chronic exposure to the water-soluble fraction of crude oil and in situ burn residue of oil on egg-bearing Northern shrimp (Pandalus borealis).

Oil spill clean-up measures using in situ burning can potentially result in seafloor contamination affecting benthic organisms. To mimic realistic exposure and measure effects, ovigerous Northern shrimp were continuously exposed for two weeks to the water-soluble fraction of oil coated on gravel followed by two weeks in clean seawater. North Sea crude oil (NSC) and field generated in situ burn residue (ISBR) of NSC were used (Low: 3 g/kg gravel, Medium: 6 g/kg gravel and High: 12 g/kg gravel). The concentrations of polyaromatic hydrocarbons (PAHs) in the water resulting from NSC were higher compared to ISBR. No mortality was observed in any treatment and overall moderate sublethal effects were found, mostly after exposure to NSC. Feeding was temporarily reduced at higher concentrations of NSC. PAH levels in hepatopancreas tissue were significantly elevated following exposure and still significantly higher at the end of the experiment in NSCHigh and ISBRHigh compared to control. Mild inflammatory response reactions and tissue ultrastructural alterations in gill tissue were observed in both treatments. Signs of necrosis occurred in ISBRHigh. No change in shrimp locomotory activity was noted from NSC exposure. However, ISBR exposure increased activity temporarily. Larvae exposed as pleopod-attached embryos showed significant delay in development from stage I to stage II after exposure to NSCHigh. Based on this study, oil-contaminated seafloor resulting from in situ burning clean-up actions does not appear to cause serious effects on bottom-living shrimp.

A simple and selective electrochemical magneto-assay for sea lice eDNA detection developed with a Quality by Design approach.

Environmental DNA (eDNA) is a novel, non-invasive sampling procedure that allows the obtaining of genetic material directly from environmental samples without any evidence of biological sources. The eDNA methodology can greatly benefit from coupling it to reliable, portable and cost-effective tools able to perform decentralized measurements directly at the site of need and in resource-limited settings. Herein, we report a simple method for the selective analysis of eDNA using a magneto-assay with electrochemical detection. The proposed method involves the polymerase chain-reaction (PCR) amplification of mitochondrial eDNA of parasitic Salmon lice (Lepeophtheirus salmonis), extracted from seawater samples. The eDNA sequence was targeted via sandwich hybridization onto magnetic beads and enzymatic labeling was performed to obtain an electroactive product measured by differential pulse voltammetry. Quality by Design (QbD), a recent concept of science- and risk-oriented quality paradigm, was used for the optimization of the different parameters of the assay. Response surface methodology and Monte Carlo simulations were performed to define the method operable design region. The optimized electrochemical magneto-assay attained a limit of detection of 2.9 amol µL-1 of the short synthetic sea louse DNA analogue (43 bp). In addition, robustness testing using a further experimental design approach was performed for monitoring eDNA amplicons. Seawater samples spiked with individuals of free-swimming L. salmonis copepodite stages and seawater collected from tanks with sea lice-infested fish were analyzed.

Effects of crude oil and field-generated burned oil residue on Northern shrimp (Pandalus borealis) larvae.

In situ burning (ISB) is an oil spill clean-up option used by oil spill responders to mitigate impacts on the marine environment. Despite advantages such as high efficiency and potential applicability for challenging areas such as the Arctic, the actual environmental side effects are still uncertain. Acute and sublethal effects of the water accommodated fractions (WAFs from 25 g oil/L seawater) of a pre-weathered North Sea crude (Oseberg Blend 200 °C+) and field generated ISB residue were evaluated on Northern shrimp (Pandalus borealis) larvae. The larvae were first exposed for 96 h to a serial dilution of seven concentrations, and then maintained for two weeks in clean seawater post-exposure. No acute (mortality) or sublethal effects (feeding, development, or growth) were detected in any of the ISB residue concentrations. Significant larvae mortality was found in the three highest concentrations of crude oil (96-h LC50:469 µg/L total petroleum hydrocarbon) but no sublethal effects were found in the surviving larvae post-exposure. This study indicates that applying ISB could mitigate acute impacts of spilled oil on shrimp larvae.

qPCR-based assessment of microfaunal indicators of oil for monitoring benthos around oil and gas platforms.

Today's benthic offshore biological monitoring of oil & gas (O&G) activities relies on macrofauna taxa enumeration. For the future, analysis of DNA isolated directly from sediments holds great potential for multi-trophic biodiversity surveys and the monitoring of a larger spectrum of benthic taxa, including micro-fauna. Here, we evaluate more specifically the potential of microfauna-specific gene quantification in relation to both petroleum-related discharge compounds and other seafloor environmental properties. We carried out this evaluation using sediment samples collected at drilling Region III on the Norwegian continental shelf where DNA metabarcoding of eukaryotic diversity was already performed. Generally, the quantification of microfauna indicator taxa related well to the gradient of contamination on the seafloor. Contrary to eukaryotic Euplotida, metabarcoding data and qPCR numbers for indicative prokaryotic taxa showed the same relationship to offshore contaminants (both showed positive relationship). We found absolute numbers of SSU rRNA gene copies of (1) Dinophyceae, Bacillariophyceae and Alcanivorax were correlated with the level of petroleum-related compounds but not with other environmental variables, (2) bacteria closely related to Shewanella were correlated with the concentration of Ba, PAH, as well to percent of gravel, (3) Desulfobacteriales correlated with petroleum-related contaminants, but as well with percent of gravel and grain size. Findings from our study suggest that biomonitoring surveys of O&G activities on benthos could benefit from quantification of specific micro-fauna indicators that is simpler and faster than the methods currently used for impact assessment of benthos.

Metatranscriptomic Analysis of Oil-Exposed Seawater Bacterial Communities Archived by an Environmental Sample Processor (ESP).

The use of natural marine bacteria as "oil sensors" for the detection of pollution events can be suggested as a novel way of monitoring oil occurrence at sea. Nucleic acid-based devices generically called genosensors are emerging as potentially promising tools for in situ detection of specific microbial marker genes suited for that purpose. Functional marker genes are particularly interesting as targets for oil-related genosensing but their identification remains a challenge. Here, seawater samples, collected in tanks with oil addition mimicking a realistic oil spill scenario, were filtered and archived by the Environmental Sample Processor (ESP), a fully robotized genosensor, and the samples were then used for post-retrieval metatranscriptomic analysis. After extraction, RNA from ESP-archived samples at start, Day 4 and Day 7 of the experiment was used for sequencing. Metatranscriptomics revealed that several KEGG pathways were significantly enriched in samples exposed to oil. However, these pathways were highly expressed also in the non-oil-exposed water samples, most likely as a result of the release of natural organic matter from decaying phytoplankton. Temporary peaks of aliphatic alcohol and aldehyde dehydrogenases and monoaromatic ring-degrading enzymes (e.g., ben, box, and dmp clusters) were observed on Day 4 in both control and oil-exposed and non-exposed tanks. Few alkane 1-monooxygenase genes were upregulated on oil, mostly transcribed by families Porticoccaceae and Rhodobacteraceae, together with aromatic ring-hydroxylating dioxygenases, mostly transcribed by Rhodobacteraceae. Few transcripts from obligate hydrocarbonoclastic genera of Alcanivorax, Oleispira and Cycloclasticus were significantly enriched in the oil-treated exposed tank in comparison to control the non-exposed tank, and these were mostly transporters and genes involved in nitrogen and phosphorous acquisition. This study highlights the importance of seasonality, i.e., phytoplankton occurrence and senescence leading to organic compound release which can be used preferentially by bacteria over oil compounds, delaying the latter process. As a result, such seasonal effect can reduce the sensitivity of genosensing tools employing bacterial functional genes to sense oil. A better understanding of the use of natural organic matter by bacteria involved in oil-biodegradation is needed to develop an array of functional markers enabling the rapid and specific in situ detection of anthropogenic pollution.

Dispersant application increases adverse long-term effects of oil on shrimp larvae (Pandalus borealis) after a six hour exposure.

The application of chemical dispersants is one option of oil spill response (OSR). Here, Northern shrimp (Pandalus borealis) larvae were experimentally exposed for short periods (6 h and 1 h) to a realistic concentration of chemically dispersed oil (CDO) (~10 mg L-1 THC), mechanically dispersed oil (MDO) (~7 mg L-1 THC), and dispersant only (D). A control (C) with seawater served as reference. Short-term effects on survival and feeding were examined right after exposure and longer-term consequences on survival, feeding, growth and development following 30 days of recovery. Both exposure durations provoked long lasting effects on larval fitness, with 1 h exposure leading to minor effects on most of the selected endpoints. The 6 h exposure affected all endpoints with more adverse impacts after exposure to CDO. This study provides important data for assessing the best OSR option relevant to NEBA (Net Environmental Benefit Analysis).

Exposure to chemically-dispersed oil is more harmful to early developmental stages of the Northern shrimp Pandalus borealis than mechanically-dispersed oil.

Knowledge of key species sensitivity for oil spill response (OSR) options is needed to support decision-making and mitigate impact on sensitive life stages of keystone species. Here, Northern shrimp (Pandalus borealis) larvae were exposed for 24 h to a gradient (H-High, M-Medium: 10 times dilution and L-Low: 100 times dilution) of mechanically- (MDO) (H < 6 mg/L total hydrocarbon content) and chemically- (CDO) dispersed oil (Slickgone NS, H < 20 mg/L total hydrocarbon content), followed by a recovery period. Larval mortality, feeding rate and development were evaluated. Overall, the results show that 24 h exposure to field-realistic concentrations of CDO lead to lower survival, reduced feeding rate and slower larval development in P. borealis larvae compared to MDO. These effects persisted during recovery, indicating a higher vulnerability with dispersant use and the need for longer observation periods post-exposure to fully evaluate the consequences for sensitive life-stages from OSR.

Identification of microbial key-indicators of oil contamination at sea through tracking of oil biotransformation: An Arctic field and laboratory study.

In this paper, a molecular analytical approach for detecting hydrocarbonoclastic bacteria in water is suggested as a proxy measurement for tracking petroleum discharges in industrialized or pristine aquatic environments. This approach is tested for general application in cold marine regions (freezing to 5 °C). We used amplicon sequencing and qPCR to quantify 16S rRNA and GyrB genes from oleophilic bacteria in seawater samples from two different crude oil enrichments. The first experiment was conducted in a controlled environment using laboratory conditions and natural North Sea fjord seawater (NSC) at a constant temperature of 5 °C. The second was performed in the field with natural Arctic seawater (ARC) and outdoor temperature conditions from -7 °C to around 4 °C. Although the experimental conditions for NSC and ARC differed, the temporal changes in bacterial communities were comparable and reflected oil biotransformation processes. The common bacterial OTUs for NSC and ARC had the highest identity to Colwellia rossensis and Oleispira antarctica rRNA sequences and were enriched within a few days in both conditions. Other typical oil degrading bacteria such as Alcanivorax (n-alkane degrader) and Cycloclasticus (polycyclic aromatic hydrocarbons degrader) were rapidly enriched only in NSC conditions. Both the strong correlation between Oleispira SSU gene copies and oil concentration, and the specificity of the Oleispira assay suggest that this organism is a robust bioindicator for seawater contaminated by petroleum in cold water environments. Further optimization for automation of the Oleispira assay for in situ analysis with a genosensing device is underway. The assay for Colwellia quantification requires more specificity to fewer Colwellia OTUs and a well-established dose-response relationship before those taxa are used for oil tracking purposes.

Effects of oil and global environmental drivers on two keystone marine invertebrates.

Ocean warming (OW) and acidification (OA) are key features of global change and are predicted to have negative consequences for marine species and ecosystems. At a smaller scale increasing oil and gas activities at northern high latitudes could lead to greater risk of petroleum pollution, potentially exacerbating the effects of such global stressors. However, knowledge of combined effects is limited. This study employed a scenario-based, collapsed design to investigate the impact of one local acute stressor (North Sea crude oil) and two chronic global drivers (pH for OA and temperature for OW), alone or in combination on aspects of the biology of larval stages of two key invertebrates: the northern shrimp (Pandalus borealis) and the green sea urchin (Strongylocentrotus droebachiensis). Both local and global drivers had negative effects on survival, development and growth of the larval stages. These effects were species- and stage-dependent. No statistical interactions were observed between local and global drivers and the combined effects of the two drivers were approximately equal to the sum of their separate effects. This study highlights the importance of adjusting regulation associated with oil spill prevention to maximize the resilience of marine organisms to predicted future global conditions.

Implementing Morpholino-Based Nucleic Acid Sensing on a Portable Surface Plasmon Resonance Instrument for Future Application in Environmental Monitoring.

A portable surface plasmon resonance (SPR) instrument was tested for the first time for the detection of oligonucleotide sequences derived from the 16S rRNA gene of Oleispira antarctica RB-8, a bioindicator species of marine oil contamination, using morpholino-functionalized sensor surfaces. We evaluated the stability and specificity of morpholino coated sensor surfaces and tested two signal amplification regimes: (1) sequential injection of sample followed by magnetic bead amplifier and (2) a single injection of magnetic bead captured oligo. We found that the sensor surfaces could be regenerated for at least 85 consecutive sample injections without significant loss of signal intensity. Regarding specificity, the assay clearly differentiated analytes with only one or two mismatches. Signal intensities of mismatch oligos were lower than the exact match target at identical concentrations down to 200 nM, in standard phosphate buffered saline with 0.1 % Tween-20 added. Signal amplification was achieved with both strategies; however, significantly higher response was observed with the sequential approach (up to 16-fold), where first the binding of biotin-probe-labeled target oligo took place on the sensor surface, followed by the binding of the streptavidin magnetic beads onto the immobilized targets. Our experiments so far indicate that a simple coating procedure in combination with a relatively cost-efficient magnetic-bead-based signal amplification will provide robust SPR based nucleic acid sensing down to 0.5 nM of a 45-nucleotide long oligo target (7.2 ng/mL).

Effects of suspended drill cuttings on the coral Lophelia pertusa using pulsed and continuous exposure scenarios.

This study aimed at providing confidence in the predictability of the impacts of drill cuttings (DC) discharge on the cold-water coral Lophelia pertusa. L. pertusa was exposed to DC from offshore exploration in the lab with the goal to assess precautionary thresholds of effects. Two exposure scenarios with DC were tested: a long-term (LT) pulsed exposure (12 weeks, peak concentrations: 2-50 mg/L, mean concentrations: 1-25 mg/L) and a short-term (ST) continuous exposure (2.5 weeks, mean concentrations: 4-42 mg/L). After exposure, a recovery period of 16 and 4 weeks was maintained in LT and ST, respectively. While there was an assumption that DC might result in an increase in respiration, decrease in growth, enhanced mucus production, reduced fatty acid content, only a significant rise was noted in skeleton growth at DC 4 mg/L and a significant increase of mucus particulate organic carbon at 25 mg/L at end of the exposure. DC did not markedly reduce prey capture rate consecutive to DC exposure. However, the effect of DC produced an increase of coral polyp activity during exposure and a return to pre-exposure conditions after cessation of DC, and coenosarc was smothered from DC even after a long recovery period (4 weeks). Overall, a DC concentration of 10 mg/L seems to represent a threshold above which changes in coral conditions were observed however with no apparent physiological consequences for the coral within the experimental time scale.

Capturing Early Changes in the Marine Bacterial Community as a Result of Crude Oil Pollution in a Mesocosm Experiment.

The results of marine bacterial community succession from a short-term study of seawater incubations at 4°C to North Sea crude oil are presented herein. Oil was used alone (O) or in combination with a dispersant (OD). Marine bacterial communities resulting from these incubations were characterized by a fingerprinting analysis and pyrosequencing of the 16S rRNA gene with the aim of 1) revealing differences in bacterial communities between the control, O treatment, and OD treatment and 2) identifying the operational taxonomic units (OTUs) of early responders in order to define the bacterial gene markers of oil pollution for in situ monitoring.After an incubation for 1 d, the distribution of the individual ribotypes of bacterial communities in control and oil-treated (O and OD) tanks differed. Differences related to the structures of bacterial communities were observed at later stages of the incubation. Among the early responders identified (Pseudoalteromonas, Sulfitobacter, Vibrio, Pseudomonas, Glaciecola, Neptunomonas, Methylophaga, and Pseudofulvibacter), genera that utilize a disintegrated biomass or hydrocarbons as well as biosurfactant producers were detected. None of these genera included obligate hydrocarbonoclastic bacteria (OHCB). After an incubation for 1 d, the abundances of Glaciecola and Pseudofulvibacter were approximately 30-fold higher in the OD and O tanks than in the control tank. OTUs assigned to the Glaciecola genus were represented more in the OD tank, while those of Pseudofulvibacter were represented more in the O tank. We also found that 2 to 3% of the structural community shift originated from the bacterial community in the oil itself, with Polaribacter being a dominant bacterium.

Physiological responses and lipid storage of the coral Lophelia pertusa at varying food density.

Despite the importance of the cold-water coral Lophelia pertusa to deep-sea reef ecosystem functioning, current knowledge of key physiological responses to available food resources is scarce. Scenarios with varying food density may help to understand how corals deal with seasonal variations in the dark ocean and might be used to study consequences of anthropogenic activities potentially affecting food availability. Thus, the physiological responses of L. pertusa to varying food (Artemia salina nauplii) concentration, ranging from 20% to 300% of carbon equivalent turned over by basal coral respiration, were investigated. A starvation group was also included. Measurements of respiration, growth, mucus production, and energy reserves (storage fatty acids) were performed at several time intervals over 26 weeks. In general, data showed a stronger effect of experimental time on measured responses, but no significant influence of food density treatment. In starved corals, respiration rate declined to 52% of initial respiration, while skeleton growth rate was maintained at the same rate as Artemia-fed corals throughout the investigation. Mucus production measured as the sum of dissolved organic carbon (DOC) and particulate organic carbon (POC) was also similar across food treatments, but POC production exceeded that of DOC at the highest food density. No marked effect was observed on storage fatty acids. These results confirm that L. pertusa is highly resilient to environmental conditions with suboptimal food densities over a time scale of months. Regulation of several physiological processes, including respiration and mucus production, possibly in combination with an opportunistic feeding strategy, contributed to this tolerance to maintain viable corals. Thus, it appears that L. pertusa is well adapted to life in the deep sea.

An evaluation of coral lophelia pertusa mucus as an analytical matrix for environmental monitoring: A preliminary proteomic study.

For the environmental monitoring of coral, mucus appears to be an appropriate biological matrix due to its array of functions in coral biology and the non-intrusive manner in which it can be collected. The aim of the present study was to evaluate the feasibility of using mucus of the stony coral Lophelia pertusa (L. pertusa) as an analytical matrix for discovery of biomarkers used for environmental monitoring. More specifically, to assess whether a mass-spectrometry-based proteomic approach can be applied to characterize the protein composition of coral mucus and changes related to petroleum discharges at the seafloor. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) screening analyses of orange and white L. pertusa showed that the mucosal protein composition varies significantly with color phenotype, a pattern not reported prior to this study. Hence, to reduce variability from phenotype difference, L. pertusa white individuals only were selected to characterize in more detail the basal protein composition in mucus using liquid chromatography, mass spectrometry, mass spectrometry (LC-MS/MS). In total, 297 proteins were identified in L. pertusa mucus of unexposed coral individuals. Individuals exposed to drill cuttings in the range 2 to 12 mg/L showed modifications in coral mucus protein composition compared to unexposed corals. Although the results were somewhat inconsistent between individuals and require further validation in both the lab and the field, this study demonstrated preliminary encouraging results for discovery of protein markers in coral mucus that might provide more comprehensive insight into potential consequences attributed to anthropogenic stressors and may be used in future monitoring of coral health.

Direct Visualization of Mucus Production by the Cold-Water Coral Lophelia pertusa with Digital Holographic Microscopy.

Lophelia pertusa is the dominant reef-building organism of cold-water coral reefs, and is known to produce significant amounts of mucus, which could involve an important metabolic cost. Mucus is involved in particle removal and feeding processes, yet the triggers and dynamics of mucus production are currently still poorly described because the existing tools to study these processes are not appropriate. Using a novel microscopic technique-digital holographic microscopy (DHM)-we studied the mucus release of L. pertusa under various experimental conditions. DHM technology permits µm-scale observations and allows the visualization of transparent mucoid substances in real time without staining. Fragments of L. pertusa were first maintained in flow-through chambers without stressors and imaged with DHM, then exposed to various stressors (suspended particles, particulate food and air exposure) and re-imaged. Under non-stressed conditions no release of mucus was observed, whilst mucus strings and sheaths were produced in response to suspended particles (activated charcoal and drill cuttings sediment) i.e. in a stressed condition. Mucus strings and so-called 'string balls' were also observed in response to exposure to particulate food (brine shrimp Artemia salina). Upon air-exposure, mucus production was clearly visible once the fragments were returned to the flow chamber. Distinct optical properties such as optical path length difference (OPD) were measured with DHM in response to the various stimuli suggesting that different mucus types are produced by L. pertusa. Mucus produced to reject particles is similar in refractive index to the surrounding seawater, suggesting that the energy content of this mucus is low. In contrast, mucus produced in response to either food particle addition or air exposure had a higher refractive index, suggesting a higher metabolic investment in the production of these mucoid substances. This paper shows for the first time the potential of DHM technology for the detection, characterization and quantification of mucus production through OPD measurements in L. pertusa.

Short- and long-term responses and recovery of mussels Mytilus edulis exposed to heavy fuel oil no. 6 and styrene.

Biomarkers have the potential to be used to assess the impact of anthropogenic discharges in marine waters. We have used a suite of biomarkers spanning from enzymatic to histopathological alterations and general stress responses to assess the short- and long-term impact on mussels Mytilus edulis of heavy fuel oil no. 6 and styrene. Mussels were exposed for 5 months, with a refilling of the exposure system, to a water soluble fraction of heavy fuel and, then, kept for a month in clean water for recovery. In a second experiment, mussels were exposed to styrene for 19 days and maintained in clean water for up to 4 months. Chemical body tissue levels reflected the weathering processes of these compounds. Acyl-CoA oxidase activity was induced in oil-exposed mussels after refilling, whereas styrene inhibited it after 19 days of exposure and after 2 weeks in clean water. Gamete development and alkali-labile phosphate levels suggest that neither oil nor styrene behaved as endocrine disruptors. Neutral red retention time was lower in treated groups than in controls. Lysosomal membrane stability was significantly reduced in exposed groups and recovered after withdrawal of oil but not after removal of styrene. Neither oil nor styrene exposure affected the condition index except for the reduction seen in mussels exposed to oil for 1 month. Biomarker response index discriminated exposed mussels, which showed higher values, and returned to control levels after recovery. Results obtained from these pilot experiments can help to identify relevant monitoring tools to assess the impact of oil and chemicals in marine spill scenarios.