Underwater high frequency noise: Biological responses in sea urchin Arbacia lixula (Linnaeus, 1758).

Marine life is extremely sensitive to the effects of environmental noise due to its reliance on underwater sounds for basic life functions, such as searching for food and mating. However, the effects on invertebrate species are not yet fully understood. The aim of this study was to determine the biochemical responses of Arbacia lixula exposed to high-frequency noise. Protein concentration, enzyme activity (esterase, phosphatase and peroxidase) and cytotoxicity in coelomic fluid were compared in individuals exposed for three hours to consecutive linear sweeps of 100 to 200 kHz lasting 1 s, and control specimens. Sound pressure levels ranged between 145 and 160 dB re 1µPa. Coelomic fluid was extracted and the gene and protein expression of HSP70 with RT-PCR was evaluated on coelomocytes. A significant change was found in enzyme activity and in the expression of the HSP70 gene and protein compared to the control. These results suggested that high-frequency stimuli elicit a noise-induced physiological stress response in A. lixula, confirming the vulnerability of this species to acoustic exposure. Furthermore, these findings provide the first evidence that cell-free coelomic fluid can be used as a signal to evaluate noise exposure in marine invertebrates.

Effects of acoustic stimulation on biochemical parameters in the digestive gland of Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819).

Underwater sounds generated by anthropogenic activity can cause behavior changes, temporary loss of hearing, damage to parts of the body, or death in a number of marine organisms and can also affect healing and survival. In this study, the authors examined the effects of high-frequency acoustic stimulations on a number of biochemical parameters in the Mediterranean mussel, Mytilus galloprovincialis. During the experiment, animals were placed in a test tank and exposed to acoustic signals [a linear sweep ranging from 100 to 200 kHz and lasting 1 s, with a sound pressure level range of between 145 and 160 dBrms (re 1µParms)] for 3 h. Total haemocyte count was assessed and glucose levels, cytotoxic activity and enzyme activity (alkaline phosphatase, esterase and peroxidase) in the digestive gland were measured. For the first time, this study suggests that high-frequency noise pollution has a negative impact on biochemical parameters in the digestive gland.

The effect of low frequency noise on the behaviour of juvenile Sparus aurata.

Anthropogenic activities are causing increased noise levels in the marine environment. To date, few studies have been undertaken to investigate the effects of different noise frequencies on the behaviour of juvenile fish. In this study, the behavioural changes of juvenile gilthead seabream (Sparus aurata) are evaluated when exposed to white noise filtered in third-octave bands centred at 63, 125, 500, and 1000 Hz (sound pressure level, 140-150 dB re 1 µΡa) for 7 h. The group dispersion, motility, and swimming height of the fish were analysed before and during the acoustic emission. Dispersion of the fish was found to reduce immediately upon application of low frequency sound (63 and 125 Hz) with a return to control condition after 2 h (indicative of habituation), whereas at 1 kHz, dispersion increased after 2 h without any habituation. The motility decreased significantly at 63 Hz throughout the 7 h of sound exposure. The swimming height decreased significantly for all frequencies other than 125 Hz. The results of this study highlight significant variations in the behavioural responses of juvenile fish that could have consequences on their fitness and survival.

Underwater acoustic communication during the mating behaviour of the semi-terrestrial crab Neohelice granulata.

In semi-terrestrial crabs, the production of sounds has been recognized to be related to courtship communication dynamics. The present study aimed to assess if the crab Neohelice granulata (Varunidae) was able to emit acoustic signals and if they played a role in the crab's behaviour. We also assessed the locomotor behaviours to examine these parameters in different mating contexts of crabs. The study was divided into two different experimental conditions: 'solitary experiment' (consisting of three combination layouts with male, unreceptive and receptive females alone) and 'group experiment' (consisting of mixed combinations layouts of males, unreceptive, and receptive females). Synchronized acoustic and video monitoring systems were used to record the acoustic signals and locomotor behaviours of alone and grouped specimens. The greatest values of locomotor behavioural parameters were observed in layouts with receptive females alone and with 2 males plus 1 receptive female, probably related to courtship behaviour. N. granulata produced two distinct signals, a multi-pulse rasp signal (highest numbers were recorded in layouts with male alone and with 2 males plus 1 receptive female) and a single rasp signal. These results may suggest that males use the multi-rasp signal to advertise their presence to other males or to attract receptive females.

Sound production mechanism in the semiterrestrial crab Neohelice granulata (Brachyura, Varunidae).

Very few studies of sound production in the Brachyura have simultaneously identified the type of individuals (e.g., sex) producing acoustic signals, the structures involved in making sound and the social context. The emission and type of sound signals in Neohelice granulata were previously characterized, but the sex and the body structures involved in the sound production mechanism were not determined. In the present study, experiments conducted in the laboratory demonstrated that acoustic signals were produced by males through an up-down movement of the cheliped by rubbing the merus against the pterygostomial area of the carapace. The micromorphology of the merus showed that it has a ridge of tubercles which may act as a plectrum, while the pterygostomial area bears tubercles and might function as the pars stridens. Acoustic signals were displayed more frequently in the presence of receptive females. Agonistic encounters among males also occurred more often in the presence of receptive females. The authors propose that Neohelice granulata males use their chelipeds to produce sound signals in a mating context, probably to attract the receptive female and/or to repel other males when a receptive female is present. Thus, the display might have a reproductive function influencing mate choice.

Evolutionary conserved mechanisms pervade structure and transcriptional modulation of allograft inflammatory factor-1 from sea anemone Anemonia viridis.

Gene family encoding allograft inflammatory factor-1 (AIF-1) is well conserved among organisms; however, there is limited knowledge in lower organisms. In this study, the first AIF-1 homologue from cnidarians was identified and characterised in the sea anemone Anemonia viridis. The full-length cDNA of AvAIF-1 was of 913 bp with a 5' -untranslated region (UTR) of 148 bp, a 3'-UTR of 315 and an open reading frame (ORF) of 450 bp encoding a polypeptide with149 amino acid residues and predicted molecular weight of about 17 kDa. The predicted protein possesses evolutionary conserved EF hand Ca2+ binding motifs, post-transcriptional modification sites and a 3D structure which can be superimposed with human members of AIF-1 family. The AvAIF-1 transcript was constitutively expressed in all tested tissues of unchallenged sea anemone, suggesting that AvAIF-1 could serve as a general protective factor under normal physiological conditions. Moreover, we profiled the transcriptional activation of AvAIF-1 after challenges with different abiotic/biotic stresses showing induction by warming conditions, heavy metals exposure and immune stimulation. Thus, mechanisms associated to inflammation and immune challenges up-regulated AvAIF-1 mRNA levels. Our results suggest its involvement in the inflammatory processes and immune response of A. viridis.

Underwater vocal complexity of Arctic seal Erignathus barbatus in Kongsfjorden (Svalbard).

In this study the description of underwater vocal repertoire of bearded seal in Svalbard (Norway) was extended. Two autonomous passive acoustic recorders were deployed for one year (August 2014-July 2015) in the inner and outer parts of the Kongsfjorden, and 1728 h were recorded and 17 220 vocalizations were found. Nine different vocalization classes were identified and characterized using ten acoustic parameters. The calls showed heterogeneous spectral features, but share the descending trend of frequency modulation. The different classes emerged were discriminated primarily by bandwidth and duration, and then by minimum frequency, central frequency, and maximum frequency in this order. This study represents a step forward to improve the understanding of the acoustic behaviour and the social function of these calls, and identified long passive acoustic monitoring as an effective method to assess vocal complexity and the ecology of marine species producing sounds.

Are mussels able to distinguish underwater sounds? Assessment of the reactions of Mytilus galloprovincialis after exposure to lab-generated acoustic signals.

This study examined the effects of lab-generated acoustic signals on the behaviour and biochemistry of Mediterranean mussels (Mytilus galloprovincialis). The experiment was carried out in a tank equipped with a video-recording system using six groups of five mussels exposed to five acoustic treatments (each treatment was replicated three times) for 30min. The acoustic signals, with a maximum sound pressure level of 150dB rms re 1µPa, differed in frequency range as follows: low (0.1-5kHz), mid-low (5-10kHz), mid (10-20kHz), mid-high (20-40kHz) and high (40-60kHz). The exposure to sweeps did not produce any significant changes in the mussels' behaviour. Conversely, the specimens exposed to the low frequency band treatment showed significantly higher values of the following biochemical stress parameters measured in their plasma and tissues: glucose, total proteins, total haemocyte number (THC), heat shock protein 70 (Hsp70) expression, and Acetylcholinesterase (AChE) activity. The responses observed in the mussels exposed to low frequency sweeps enable us to suppose a biological and ecological role for this sound, which contains the main frequencies produced by both shipping traffic and the acoustic emissions of fish.

Vessel noise pollution as a human threat to fish: assessment of the stress response in gilthead sea bream (Sparus aurata, Linnaeus 1758).

This study examined the effects of boat noise pollution on the stress indices of gilthead sea bream (Sparus aurata, Linnaeus 1758). To assess the stress response in these fish, biometric values and plasma parameters such as ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, total protein, cholesterol, triglycerides and osmolarity were analysed. After acclimatization of the animals, the experiment was carried out in a tank fitted with underwater speakers where the fish were exposed to sound treatments (in duplicate) consisting of: 10 days of no sound (control treatment; the animals were only exposed to the experimental tank's background noise) and 10 days of noise derived from original recordings of motor boats, including recreational boats, hydrofoil, fishing boat and ferry boat (vessel noise treatment). The exposure to noise produced significant variations in almost all the plasma parameters assessed, but no differences were observed in weights and fork lengths. A PERMANOVA analysis highlighted significantly increased values (p < 0.05) of ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, cholesterol, triglycerides and osmolarity in the fish exposed to vessel noise for 10 days. This study clearly highlights that anthropogenic noise negatively affects fish, and they are valuable targets for detailed investigations into the effects of this global pollutant. Finally, these experimental studies could represent part of the science that is able to improve the quality of the policies related to management plans for maritime spaces (Marine Strategy Framework Directive 56/2008 CE) that are aimed at stemming this pollutant phenomenon.

Synthesis of water suitable as the MEPC.174(58) G8 influent water for testing ballast water management systems.

Here, we describe the methodologies adopted to ensure that natural seawater, used as "influent water" for the land test, complies with the requirement that should be fulfilled to show the efficacy of the new ballast water treatment system (BWTS). The new BWTS was located on the coast of SW Sicily (Italy), and the sampled seawater showed that bacteria and plankton were two orders of magnitude lower than requested. Integrated approaches for preparation of massive cultures of bacteria (Alcanivorax borkumensis and Marinobacter hydrocarbonoclasticus), algae (Tetraselmis suecica), rotifers (Brachionus plicatilis), and crustaceans (Artemia salina) suitable to ensure that 200 m(3) of water fulfilled the international guidelines of MEPC.174(58)G8 are here described. These methodologies allowed us to prepare the "influent water" in good agreement with guidelines and without specific problems arising from natural conditions (seasons, weather, etc.) which significantly affect the concentrations of organisms at sea. This approach also offered the chance to reliably run land tests once every two weeks.

Combining whistle acoustic parameters to discriminate Mediterranean odontocetes during passive acoustic monitoring.

Acoustic observation can complement visual observation to more effectively monitor occurrence and distribution of marine mammals. For effective acoustic censuses, calibration methods must be determined by joint visual and acoustic studies. Research is still needed in the field of acoustic species identification, particularly for smaller odontocetes. From 1994 to 2012, whistles of four odontocete species were recorded in different areas of the Mediterranean Sea to determine how reliably these vocalizations can be classified to species. Recordings were attributed to species by simultaneous visual observation. The results of this study highlight that the frequency parameters, which are linked to physical features of animals, show lower variability than modulation parameters, which are likely to be more dependent on complex eco-ethological contexts. For all the studied species, minimum and maximum frequencies were linearly correlated with body size. DFA and Classification Tree Analysis (CART) show that these parameters were the most important for classifying species; however, both statistical methods highlighted the need for combining them with the number of contour minima and contour maxima for correct classification. Generally, DFA and CART results reflected both phylogenetic distance (especially for common and striped dolphins) and the size of the species.

Partaking of Archaea to biogeochemical cycling in oxygen-deficient zones of meromictic saline Lake Faro (Messina, Italy).

We used a combination of molecular and microbiological approaches to determine the activity, abundance and diversity of archaeal populations inhabiting meromictic saline Lake Faro (Messina, Italy). Analysis of archaeal 16S rRNA, amoA, accA and hbd genes and transcripts revealed that sub- and anoxic layers of Lake Faro are primarily inhabited by the organisms related to the clusters of Marine Group I.1a of Thaumarchaeota frequently recovered from oxygen-depleted marine ecosystems. These organisms dominated the metabolically active archaea down to the bottom of the lake, indicating their adaptation to recurrent changes in the levels of water column hypoxia. The upper microaerobic layer of Lake Faro redoxcline has the maximal rates of dark primary production much lower than those of other previously studied pelagic redoxclines, but comparable to the values of meso- and bathypelagic areas of Mediterranean Sea. Application of bacterial inhibitors, especially azide, significantly declined the CO2 fixation rates in the low interface and monimolimnion, whereas archaea-specific inhibitor had effect only in upper part of the redoxcline. Based on these findings, we hypothesize that dark bicarbonate fixation in suboxic zone of Lake Faro results mainly from archaeal activity which is affected by the predicted lack in oxygen in lower layers.

Physiological and agonistic behavioural response of Procambarus clarkii to an acoustic stimulus.

This study examined the effects of an acoustic stimulus on the haemolymph and agonistic behaviour of the red swamp crayfish, Procambarus clarkii. The experiment was conducted in a tank equipped with a video recording system using six groups (three control and three test groups) of five adult crayfish (30 specimens in total). After 1 h of habituation, the behaviour of the crayfish was monitored for 2 h. During the second hour, the animals in the test groups were exposed to a linear sweep (frequency range 0.1-25 kHz; peak amplitude 148 dB(rms) re. 1 µPa at 12 kHz) acoustic stimulus for 30 min. Exposure to the noise produced significant variations in haemato-immunological parameters as well as a reduction in agonistic behaviour.

Evidence of accelerated evolution and ectodermal-specific expression of presumptive BDS toxin cDNAs from Anemonia viridis.

Anemonia viridis is a widespread and extensively studied Mediterranean species of sea anemone from which a large number of polypeptide toxins, such as blood depressing substances (BDS) peptides, have been isolated. The first members of this class, BDS-1 and BDS-2, are polypeptides belonging to the ß-defensin fold family and were initially described for their antihypertensive and antiviral activities. BDS-1 and BDS-2 are 43 amino acid peptides characterised by three disulfide bonds that act as neurotoxins affecting Kv3.1, Kv3.2 and Kv3.4 channel gating kinetics. In addition, BDS-1 inactivates the Nav1.7 and Nav1.3 channels. The development of a large dataset of A. viridis expressed sequence tags (ESTs) and the identification of 13 putative BDS-like cDNA sequences has attracted interest, especially as scientific and diagnostic tools. A comparison of BDS cDNA sequences showed that the untranslated regions are more conserved than the protein-coding regions. Moreover, the KA/KS ratios calculated for all pairwise comparisons showed values greater than 1, suggesting mechanisms of accelerated evolution. The structures of the BDS homologs were predicted by molecular modelling. All toxins possess similar 3D structures that consist of a triple-stranded antiparallel ß-sheet and an additional small antiparallel ß-sheet located downstream of the cleavage/maturation site; however, the orientation of the triple-stranded ß-sheet appears to differ among the toxins. To characterise the spatial expression profile of the putative BDS cDNA sequences, tissue-specific cDNA libraries, enriched for BDS transcripts, were constructed. In addition, the proper amplification of ectodermal or endodermal markers ensured the tissue specificity of each library. Sequencing randomly selected clones from each library revealed ectodermal-specific expression of ten BDS transcripts, while transcripts of BDS-8, BDS-13, BDS-14 and BDS-15 failed to be retrieved, likely due to under-representation in our cDNA libraries. The calculation of the relative abundance of BDS transcripts in the cDNA libraries revealed that BDS-1, BDS-3, BDS-4, BDS-5 and BDS-6 are the most represented transcripts.

The underwater acoustic activities of the red swamp crayfish Procambarus clarkii.

This study describes the underwater acoustic behavior of the red swamp crayfish Procambarus clarkii. The study was conducted both in a tank and in the natural environment. The tank was equipped with video and acoustic recording systems. Observations were conducted to identify the underwater acoustic signals produced and their association with behavioral events and the movement status of the animals. In a lake in a natural reserve, a remote acoustic recording station was used to study the circadian underwater acoustic activity of the crayfish and to assess the acoustic features of the signals. The red swamp crayfish produces irregular trains of wide-band pulses (duration 0.4 ms, SPL(PK) 128 dB re 1 µPa, peak frequency 28 kHz, bandwidth(RMS) 20 kHz). The production of signals is positively related to intraspecific interactions (encounter/approach, fighting and successive Tail Flips). In the natural environment, acoustic activity is almost absent during the day, increases abruptly at sunset and continues until dawn. This study reveals the previously unknown underwater acoustic signals of Procambarus clarkii and the potential of passive acoustic methods to monitor the presence, the abundance and the behavioral activities of this invasive species.

Distribution of Cd and As in organs and tissues of four marine mammal species stranded along the Italian coasts.

Concentrations of Cd and As were determined in organs and tissues (muscle, heart, kidney, lung and liver) of four cetacean species (Stenella coeruleoalba, Truncatus truncatus, Grampus griseus and Ziphius cavirostris) stranded along the Italian coasts during the period 2000-2009. Significant differences were found between Cd concentrations in the different analysed tissues. Particularly, the kidney shows the highest concentrations of Cd in all analysed specimens, followed by the liver. No systematic or statistically significant difference in As concentrations was found in the analysed tissues. Considerable differences of Cd and As values in samples from different geographical areas (Sicily Channel, Adriatic Sea and Tyrrhenian Sea) were found, with the highest values measured in samples from the Sicily Channel. This basically suggests that anthropogenic and natural sources significantly affect Cd and As content in cetaceans.

Unveiling microbial life in new deep-sea hypersaline Lake Thetis. Part I: Prokaryotes and environmental settings.

In September 2008, an expedition of the RV Urania was devoted to exploration of the genomic richness of deep hypersaline anoxic lakes (DHALs) located in the Western part of the Mediterranean Ridge. Approximately 40 nautical miles SE from Urania Lake, the presence of anoxic hypersaline lake, which we named Thetis, was confirmed by swath bathymetry profiling and through immediate sampling casts. The brine surface of the Thetis Lake is located at a depth of 3258 m with a thickness of ≈ 157 m. Brine composition was found to be thalassohaline, saturated by NaCl with a total salinity of 348‰, which is one of highest value reported for DHALs. Similarly to other Mediterranean DHALs, seawater-brine interface of Thetis represents a steep pycno- and chemocline with gradients of salinity, electron donors and acceptors and posseses a remarkable stratification of prokaryotic communities, observed to be more metabolically active in the upper interface where redox gradient was sharper. [(14) C]-bicarbonate fixation analysis revealed that microbial communities are sustained by sulfur-oxidizing chemolithoautotrophic primary producers that thrive within upper interface. Besides microaerophilic autotrophy, heterotrophic sulfate reduction, methanogenesis and anaerobic methane oxidation are likely the predominant processes driving the ecosystem of Thetis Lake.

The key role played by the Augusta basin (southern Italy) in the mercury contamination of the Mediterranean Sea.

The Augusta basin, located in SE Sicily (southern Italy), is a semi-enclosed marine area, labelled as a highly contaminated site. The release of mercury into the harbour seawater and its dispersion to the blue water, make the Augusta basin a potential source of anthropogenic pollution for the Mediterranean Sea. A mass balance was implemented to calculate the HgT budget in the Augusta basin. Results suggest that an average of ∼0.073 kmol of HgT is released, by diffusion, on a yearly basis, from sediments to the seawater, with a consequent output of 0.162 kmol y(-1) to coastal and offshore waters; this makes the Augusta area an important contributor of mercury to the Mediterranean Sea. Owing to the geographical location of the Augusta basin, its outflowing shelf-waters are immediately intercepted by the surface Atlantic Ionian Stream (AIS) and mixed with the main gyres of the eastern Mediterranean Sea, thus representing a risk for the large-scale marine system.

Growth-related trophic changes of Thunnus thynnus as evidenced by stable nitrogen isotopic values in the first dorsal spine.

The bluefin tuna, Thunnus thynnus, is a highly migratory and long-living fish at the top of the pelagic food web. As top predator, it plays a key role in the stability of marine food webs by exerting top-down control on its prey. The diet composition of bluefin tuna varies in relation to its growth, seasons and migratory patterns, making it difficult to evaluate spatial and temporal effects. This latter aspect is further complicated to be determined during the first months of life, when T. thynnus specimens have a rapid growth rate leading to changes in the trophic status. In this study, the potential collagen-related effects on δ15N and δ13C values were evaluated on the whole spine of adult tuna specimens collected in the central Mediterranean Sea. Obtained results showed non-significant differences between extracted and non-extracted collagen samples for δ15N in whole spine, allowing adopting the isotopic analysis both for annuli in the spine section of adults and for younger specimens, whose spine size does not permit the collagen extraction. Specifically, isotopic analysis of whole spine of the young of the year specimens, showed a rapid change in δ15N values with length, following an exponential model. For older specimens, δ15N values were higher and varied around a plateau, likely due to a higher specificity in the choice of prey and/or to change in the geographical location. Such variability was also mirrored in annuli of spines sections of adult tunas. As far as δ13C values are concerned, a strong collagen-related effect was evidenced, likely highlighting the influence of lipids. Consequently, δ13C analysis may be used only on adult specimens where collagen extraction is possible. This research also showed how isotopic analysis of both whole sample and sequence of annuli in the cross-section of dorsal spine might produce isotopic profiles useful to detect specific trophic dynamics along the bluefin tuna growth.

Condition of pteropod shells near a volcanic CO2 vent region.

Natural gradients of pH in the ocean are useful analogues for studying the projected impacts of Ocean Acidification (OA) on marine ecosystems. Here we document the in situ impact of submarine CO2 volcanic emissions (CO2 vents) on live shelled-pteropods (planktonic gastropods) species Creseis conica in the Gulf of Naples (Tyrrhenian Sea, Mediterranean). Since the currents inside the Gulf will likely drive those pelagic calcifying organisms into and out of the CO2 vent zones, we assume that pteropods will be occasionally exposed to the vents during their life cycle. Shell degradation and biomass were investigated in the stations located within and nearby the CO2 vent emission in relation to the variability of sea water carbonate chemistry. A relative decrease in shell biomass (22%), increase in incidence of shell fractures (38%) and extent of dissolution were observed in Creseis conica collected in the Gulf of Naples compared to those from the Northern Tyrrhenian Sea (control stations). These results suggest that discontinuous but recurrent exposure to highly variable carbonate chemistry could consistently affect the characteristic of the pteropod shells.