Ronald A Kastelein

Sander Van Der Heul

Willem C Verboom

Nancy Jennings

Jan Van Der Veen

Dick De Haan

Reactions to sound by North Sea fish Kastelein et al. Page 1 of 24 MER Startle response of captive North Sea fish species to underwater tones between 0.1 and 64 kHz

Keywords: Acoustics, Atlantic herring, behaviour, Atlantic cod, common eel, horse mackerel, marine fish, pollack, pout, sea bass, thicklip mullet

World-wide, underwater background noise levels are increasing due to anthropogenic activities. Little is known about the effects of anthropogenic noise on marine fish, and information is needed to predict any negative effects. Behavioural startle response thresholds were determined for eight marine fish species, held in a large tank, to tones of 0.1-64 kHz. Response threshold levels varied per frequency within and between species. For sea bass, the 50% reaction threshold occurred for signals of 0.1-0.7 kHz, for thicklip mullet 0.4-0.7 kHz, for pout 0.1-0.25 kHz, for horse mackerel 0.1-2 kHz and for Atlantic herring 4 kHz. For cod, pollack and eel, no 50 % reaction thresholds were reached. Reaction threshold levels increased from ~100 dB (re 1 µPa, rms) at 0.1 kHz to ~ 160 dB at 0.7 kHz. The 50 % reaction thresholds did not run parallel to the hearing curves. This shows that fish species react very differently to sound, and that generalisations about the effects of sound on fish should be made with care. As well as on the spectrum and level of anthropogenic sounds, the reactions of fish probably depend on the context (e.g. location, temperature, physiological state, age, body size, and school size).

Reactions to sound by North Sea fish Kastelein et al.

Introduction

World-wide, underwater background noise levels are increasing due to anthropogenic activities (National Research Council, 2003;2005). Many marine organisms rely heavily on acoustics to survive. Fish, for instance, engage with their surroundings through sound, by using species-specific acoustic adaptations for hunting, territorial behaviour, mate attraction, spatial orientation, predator aversion, etc [START_REF] Popper | Sound detection mechanisms and capabilities of teleost fishes[END_REF]. Such ecologically important behaviours can be negatively influenced by anthropogenic noise. Little is known about the effects of anthropogenic noise on marine fish, and much information is needed to evaluate or predict any negative effects [START_REF] Popper | Anthropogenic sound: Effects on the behavior and physiology of fishes[END_REF].

The effect of a sound may depend on: 1) properties of the sound, such as frequency spectrum, source level (SL), duration, rise and fall times in level, and repetition rate, 2) background noise (masking), 3) sound level, duration and spectrum of the sound as received by the animal, 4) hearing properties of the species (sensitivity, directivity index and critical ratio), and 5) species-specific or individual variation in reaction to sound.

Limited information is available on the hearing sensitivity of only about 100 of the 27,000 marine fish species [START_REF] Popper | Sound detection mechanisms and capabilities of teleost fishes[END_REF]. Most audiograms of marine fish species indicate that their highest sensitivity to sounds falls within the 0.1 -2 kHz range. The fact that the bandwidth of hearing sensitivity is narrow could be due to mechanical limitations of the sense organs, which may include the swim bladder, or to physical constraints of the testing systems [START_REF] Ladich | Parallel evolution in fish hearing organs[END_REF]. However, some studies suggest that clupeid fish may also be able to detect ultrasound [START_REF] Mann | Ultrasound detection by clupeiforme fishes[END_REF] although Pacific herring cannot [START_REF] Mann | Herring do not detect ultrasound[END_REF]. Cod can detect ultrasound (3 ms 38 kHz pulses), but only at very high received levels (of > 194 dB re 1 Pa; [START_REF] Astrup | Detection of intense ultrasound by the cod Gadus morhua[END_REF][START_REF] Astrup | Discrimination between high and low repetition rates of ultrasonic pulses by the cod[END_REF].

The fact that a fish can detect a sound does not necessarily mean that it will react to that sound. In many species, a certain sound pressure level needs to be reached before the behaviour is affected, and some fish species do not show startle responses to sounds no matter how loud they are. Researchers have investigated the effects of specific sounds on the behaviour of marine fish species, and responses vary greatly [START_REF] Moulton | Annotated references concerning the effects of man-made sounds on the movements of fishes[END_REF][START_REF] Hawkins | Underwater sound and fish behavior[END_REF][START_REF] Myrberg | The effects of man-made noise on the behavior of marine animals[END_REF]Popper and Carlson, 1998;[START_REF] Luczkovich | Sounds of sex and death in the sea: bottlenose dolphin whistles suppress mating choruses of silver perch[END_REF]Kastelein et al., 2005).

The aim of the present study was to determine the behavioural reaction threshold levels of eight fish species from the North Sea to pure tones in the frequency range 0.1 to 64 kHz. Where possible, we compare our thresholds to hearing thresholds, and evaluate the relationship between sound detection and response. Though pure tones may not occur often in nature, an understanding of the responses of fish species to them is fundamental to understanding their responses to more complex sounds, which could be tested in applied follow-up studies.

Materials and methods

Study animals

Eight fish species that are found in the North Sea were selected for testing, based on their economic importance in fisheries, their availability, their ease of maintenance in captivity, and the temperatures at which they can be kept (the water temperature in our study tank was influenced by the environment). The animal welfare commission of the Netherlands Page 3 of 24 MER stipulated that the fish used must feed readily in captivity, so they had to come from aquaria or fish farms, though most were originally wild-caught. All came from the Netherlands. Six of the study species [sea bass (Dicentrarchus labrax), thicklip mullet (Chelon labrosus), pout (Trisopterus luscus), Atlantic cod (Gadus morhua), pollack (Pollachius pollachius) and horse mackerel (Trachurus trachurus)] were from Sea aquarium "Het Arsenaal", Flushing (Table 1). The fish had been wild-caught by hook and line or in a trap, so that no obvious damage had occurred to their swim bladders. The common eel (Anguilla anguilla) came from "Schot aquacultuur" fish farm, Bruinisse. The Atlantic herring (Clupea harengus) were borrowed from the Oceanium department of Blijdorp Zoo, Rotterdam. The fish were all adapted to captivity and were feeding voluntarily.

Except for the Atlantic herring, the animals were fed ad lib. on pieces of raw fish (food was given until the animals stopped eating) twice a week after the daily study sessions. The herring were fed Trouvit pellets (Nutreco Aquaculture) from a food dispenser throughout the day. The amount eaten was related to the water temperature. For one to two weeks before each species was tested, the fish were kept in white polyester 2.2 m diameter holding tanks with a water depth of 1 m. These tanks were very quiet, as they (and their water systems) had been designed specifically for acoustic research. In the holding tanks, most fish swam slowly or remained stationary most of the time. During the study the species were kept in a much larger tank (see below) in schools of 4-17 individuals. In this tank all species showed schooling behaviour, and the school size was mainly determined by the availability of the fish and the available space in the net enclosure.

Study area

The experiments were conducted in a large outdoor tank at SEAMARCO's research institute in Wilhelminadorp, The Netherlands. The rectangular tank (7.0 m long, 4.0 m wide; water depth 2.0 m) was made of plywood covered on both sides with fibreglass (Fig. 1). The tank was set into a 1 m deep hole in the ground, resting on a layer of rubber tiles, and the sides below ground level were covered with a layer of 3 cm thick Styrofoam. The pool walls and floor were blue (Ral colour 50/15).

To reduce predation by birds, algal growth, impact of noise from rain, glistening of the water surface, and to create a more even light pattern in the pool, a slanting roof (9 m x 6 m) made of Polyurethane between 2 sheets of metal was built above the pool (1.5 m above the water level on one side and 2.0 m on the other side). From October to December inclusive, artificial lighting was used during the first session of the day.

The water was pumped continuously from the nearby Oosterschelde (a lagoon of the North Sea). The turnover rate of water in the tank was once per day. The salinity was 30-33 ‰. To ensure the good water clarity needed to film the fish, the water was circulated via sand, UV light, and carbon filters. Water temperature was measured daily and remained well within the boundaries suitable for the fish species (Table 2). No influence of water temperature on the effects of the stimuli were observed in any of the species; a previous study [START_REF] Kastelein | Effects of acoustic alarms, designed to reduce small cetacean bycatch, on the behaviour of North Sea fish species in a large tank[END_REF] also showed that within the temperature range experienced in the present study, the fish reacted to sound independently of the temperature.

To make the environment inside the tank as quiet as possible, the filter unit had a low noise "whisper" pump. To reduce contact noise entering the pool, the pump and filter unit were placed on rubber tiles, and the filtration pump was connected to the tank with flexible tubes.

To ensure that during test sessions all fish could be filmed at any time with one or more (depending on the school length and width) of the three cameras, and to make a change in fish species easy for the fish and the researchers, the fish were kept in a net enclosure (4 m Page 4 of 24 MER long, 1.9 m wide and 2.5 m high) that was rigged over the width of the tank (Fig. 1). The net was made of white nylon (1.5 cm stretched mesh). By means of lead lines and four weights in the corners, the enclosure kept its rectangular shape. Two research cabins were placed 2 m apart on one side of the tank. One housed the sound generation equipment, three monitors, video recording equipment, and sound recording equipment. The other housed the sound calibration equipment.

Stimuli

The fish were subjected to pure tones at the following frequencies: 0.1, 0.125, 0.250, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 8, 16, 32, 45, and 64 kHz. These are mainly octave frequencies, but extra frequencies were tested below 1 kHz. The lowest frequency (0.1 kHz) was determined by the equipment available for this study. The stationary portion of all signals was 900 ms in duration. Rise and fall times (each 50 ms) preceded and followed the stationary portion, to prevent abrupt signal onset and offset transients. The tones were produced by a generator (Hewlett Packard, model 33120A), a signal shaper and attenuator (a modified audiometer, Midimate model 602; 5-dB steps), a power amplifier (HQ Power, model VPA2200BMN-2 x 200 W, rms), and one of the following three underwater transducers, depending on the frequency of the projected sounds: for signals of 0.1-0.250 kHz, an Ocean Engineering Enterprise transducer (model DRS-12; 30 cm diameter) and its impedance matching transformer; for signals of 0.4 -45 kHz, an Ocean Engineering Enterprise transducer (model DRS-8; 20 cm diameter) and its impedance matching transformer; for 64 kHz signals, an Airmar high frequency experimental transducer.

During a pre-test with each fish species, the signal levels for the main study were determined by increasing the sound pressure level of each frequency until a reaction, which was best described as a startle response, to the stimulus was observed. That sound pressure level was tested in the main study, as were levels 5 dB higher and lower. Some signal frequencies caused no reaction when produced at the highest sound pressure level that could be generated with the available equipment. In such cases, that maximum producible level was used during the main study.

During test sessions the audible stimuli and background noise were checked with a hydrophone (Labforce 1 BV, model 90.02.01), a charge amplifier (Brüel & Kjaer (B&K), 2635) and an amplified loudspeaker. For sounds above 16 kHz, the loudspeaker was replaced by a heterodyne frequency reducer (Stag Electronics, Batbox III). The outputs of the charge amplifier and frequency reducer were fed into the video recorders (via ground loop isolators), so that the behaviour of the fish at the exact times of stimulus presentation could later be analysed.

Sound measurements

Two types of sound measurement were carried out during the experiments: 1) determination of the background noise level in the pool, to check that the stimuli were not masked by background noise; 2) determination of the sound pressure levels (SPLs) at two locations in the net enclosure during sound emissions, to check the distribution of the stimulus sounds in the study area.

The equipment used to measure background noise and stimulus SPLs (up to 45 kHz) was the same and consisted of a broadband hydrophone (B&K 8101, 0-100 kHz), a voltage amplifier system (TNO TPD, 0-300 kHz) and a personal computer with spectral analysis software (Cool Edit Pro, Syntrillium Software Corp., USA; sample frequency 11-96 kHz, frequency range 0-48 kHz, df = 15-115 Hz). The whole system was calibrated with a Page 5 of 24 MER pistonphone (B&K 4223) and a white noise 'insert voltage signal' into the hydrophone preamplifier. Measurements were corrected for the frequency sensitivity of the hydrophone and the frequency response of the measurement equipment.

During the course of the study, background noise levels in the tank were determined several times. Levels were measured in the range 0.020-48 kHz as narrow-band Fast Fourier Transform (FFT) results, and were converted to Power Spectral Density (PSD) levels (1 Hz bandwidth), time-averaged over 32 s. The remote location of SEAMARCO's institute had been chosen to minimise mechanical and other sound sources, and so background noise levels were very low: generally below those caused by sea state 1 (spectrum level; very low wind conditions; [START_REF] Wentz | Acoustic ambient noise in the ocean: spectra and sources[END_REF]. Above wind force Beaufort 4, the background noise in the tank did rise, so no sessions were conducted under wind force conditions above Beaufort 4.

Stimulus sound levels were measured on five occasions, which were well distributed over the study period. Levels were determined in the area in which the fish usually swam (0.5 m above the bottom in the centre of the net enclosure), and at distances of 1.5 and 3.5 m from the sound sources (transducers). Two frequency ranges were applied to measure the sound distribution in the pool: 0.020-0.5 kHz (sample frequency 11.025 Hz) and 0.4-48 kHz (sample frequency 96 kHz). For each stimulus frequency, the spectra of three sound blocks (900 ms duration each) were determined (by FFT) and linearly averaged. Because for pure tones the pool was reverberant and there was a frequency cut-off below approximately 0.250 kHz, the propagation loss fluctuated considerably and deflected from the '20 log R' attenuation law. Additional SPL measurements throughout the net enclosure showed that the stimuli levels (depending on frequency) varied at most by ± 8 dB from the average received level (of the 1.5 m and 3.5 m recordings). This received level range was used to calculate the average 50 % reaction threshold as a range, rather than a line. Most stimuli showed no harmonics, and those that did, had harmonics at least 30 dB below the level of the fundamental frequency.

The 64 kHz signal was calibrated with a calibrated hydrophone (RESON, TC 4032, S/N 1704048), connected to an input module (RESON EC 6073) to transfer the signals to a computer and to power the hydrophone. A battery-powered amplifier (ETEC A1101) was used to condition the hydrophone signal and as a high-pass filter of 10 Hz. As the gain characteristics were flat to 1 MHz, a low-pass filter was used on the output of the amplifier to filter the HF noise above 150 kHz with 12 dB/octave. The output of the filter was connected via a coaxial input module (BNC 2110) to a 16-bit data acquisition card (National Instruments type PCI 6281M) on which the analogue signals were digitized with a sample rate of 512 kHz. For each data sample the SPL was computed, using the SPL/voltage relation of a pistonphone (G.R.A.S., model 42AC) reference source and a sound level meter (B&K 2239), to measure the SPL reduction with the hydrophone linked to the pistonphone. With this reference all system errors in the analogue/digital link could be eliminated, assuming a flat response curve of the hydrophone up to 100 kHz. The computer with the DAQ card was powered via a UPS (APC 1400) to maintain a floating earth circuit decoupled from the local earth system. The data monitoring/acquisition/analysis functions were conducted by using acoustic software modules developed in Labview 7.0 software (National Instruments) by IMARES. The spectrograms were computed in narrow-band FFT.

Observation equipment

The behaviour of the fish was recorded from above by three black-and-white underwater video cameras (Mariscope, model Micro, Kiel, Germany). The cameras were mounted in a row across the width of the pool (Fig. 1), with the lenses just below the water surface, so that about 80 % of the water volume in the net enclosure was in view. Just below Page 6 of 24 MER the water surface some areas were not in view, but these areas were never used by the fish, which swam near the bottom. The images of the three cameras matched without overlap.

Methodology

In each test a school of fish of only one species was used, in order to avoid the chance of the behaviour of one species influencing the behaviour of another. The 4 -17 fish of each species were placed in the tank at least a day before the first session with that species was conducted. This allowed the fish to habituate to the tank. The transducers and cameras were placed in the pool at the beginning of each working day and remained in the water during all sessions. As the pump in the pool was extremely quiet it was left on during the experiments. Each one-hour session consisted of ten 1-minute trials during which a sound was projected 30 s after the onset of the trial. The time between trials was 5 minutes. This inter-trial time was based on a pre-test in which the time between signals, at a particular level which caused a startle response when first projected, was reduced from 10 minutes to 1 minute. Often the fish stopped reacting to the individual stimuli when successive signals occurred with only one minute in between, but response was restored after two minutes. Therefore a conservative inter-trial time of five minutes was chosen for the main experiment.

Usually four sessions were conducted daily between 08.30 and 16.00 hrs. Per fish species, all frequency and level combinations, determined during the pre-test, were offered in random order during the approximately 15 day study period for that species. Each frequency and level combination was tested 12 times on each fish species (pollack 15 times). The study was conducted between October 2004 and December 2005.

Analysis

Two researchers collected and analysed the data. During the stimulus projection, one researcher (the operator), who could see the entire study area on three monitors in the research cabin, recorded (as a general impression) whether the fish reacted to the stimulus or not. After each session, the reaction of each fish in the school was recorded by the other researcher, from the three video recordings.

A reaction to a stimulus was characterised by a sudden change in swim speed, swim direction and/or body posture during the acoustic signal presentation. If 50 % or more of the fish in the school reacted to the stimulus, the trial was classified as a "reaction" trial. The two researchers alternated tasks between sessions, and when analysing the video recordings, were not aware of the other researcher's previous classification of the trials during the sessions. There was no reason for the researchers to be biased, as we were not expecting or predicting reactions or lack of reactions to any particular sound. The reactions of the fish were in fact so clear that no disagreement between the ratings of the two researchers occurred throughout the study.

Per signal frequency or level combination, the % of the 12 (15 for pollack) trials in which the fish reacted to the stimulus was calculated. Based on these percentages, psychometric curves were drawn (received level range versus % reaction). From these curves, the 50 % reaction threshold sound pressure level ranges were derived, and used to draw the upper and lower 50 % reaction threshold curves for each species.

Results

The fish that responded to the stimuli showed startle responses: they increased their swimming speed and often made tight turns. No startle response was ever seen during test Page 7 of 24 MER periods apart from during signal presentation. In almost all cases when a startle response was seen, the fish swam away from the sound source. The fish always resumed normal swimming behaviour within a few seconds of the end of the 900 ms acoustic stimulus presentation. For sea bass, 50 % reaction threshold ranges were reached for signals between 0.1 and 0.7 kHz (Fig. 2A). The sea bass did not react to the maximum received levels that could be produced for the higher frequency signals.

For thicklip mullet, 50 % reaction thresholds were reached for signals between 0.4 and 0.7 kHz (Fig. 2B). The fish did not react to the maximum received levels that could be produced for the other frequencies. However, the mullet reacted to one of the twelve 0.1 kHz signal trials and two of the 0.125 kHz signal trials, which suggests that the 50 % reaction threshold level for those frequencies was only a few dB above the maximum level that could be produced with the available equipment.

For pout, 50 % reaction thresholds were reached for signals between 0.1 and 0.250 kHz (Fig. 2C). The pout did not react to the maximum received levels that could be produced for the higher frequency signals.

For Atlantic cod and common eel, no 50 % reaction thresholds could be reached with the maximum levels for the frequencies that could be produced with the available equipment (Figs. 2D and2E).

For Pollack, no 50 % reaction thresholds could be reached with the maximum levels for the frequencies that could be produced with the available equipment (Fig. 2F). However, there was some reaction to the maximum levels that could be produced for signals of 0.1 kHz (reaction in 4 of the 15 trials), 0.125 kHz (4 trials), 0.250 kHz (2 trials) and 0.4 kHz (3 trials).

For horse mackerel, 50 % reaction thresholds were reached for signals between 0.1 and 2 kHz (Fig. 2G). The horse mackerel did not react to the maximum received levels that could be produced for the higher frequency signals.

Atlantic herring reacted to two frequencies. The 50 % reaction threshold was reached only for the 4 kHz signal (Fig. 2H). There was also some reaction to the 0.4 kHz signal (in 2 of the 12 trials). The herring did not react to the maximum received levels that could be produced for the other frequencies.

Discussion and conclusions

We judged that the researchers used consistent criteria for classing a trial as a response trial or a non-response trial, because their classifications were always identical, and the startle response was very obvious (not a subtle increase in swimming speed or swimming depth as was observed in a previous study; [START_REF] Kastelein | Effects of acoustic alarms, designed to reduce small cetacean bycatch, on the behaviour of North Sea fish species in a large tank[END_REF].

The size of their tank influences the general swimming behaviour of many fish species. Before the fish were put in the test tank, they were kept in much smaller circular tanks, in which they swam very slowly or not at all. In the net enclosure in the large test tank, the fish were much more active; they behaved in the same way as fish in the previous study in this tank, which had the entire tank available to them [START_REF] Kastelein | Effects of acoustic alarms, designed to reduce small cetacean bycatch, on the behaviour of North Sea fish species in a large tank[END_REF]. So, although the test tank was far from a natural environment, it was a much better study area than the smaller tanks used in several previous studies on reactions of fish to sound.

The study fish had been housed, for at least part of their lives, in tanks at aquaria and fish farms. However, those facilities had water filtration systems that were relatively quiet, so the study animals had probably not been exposed to higher sound levels than wild conspecifics. As the location of the study site was selected because of its remote location and quiet environment, the tank was designed specifically for acoustic research, and the area around the tank was strictly controlled (nobody was present within 100 m of the tank, except Page 8 of 24 MER the researchers who sat quietly), there was little background noise, and startle responses were not observed outside the signal presentations.

The reactions of the fish in the present study were probably dependent on the context in which the sounds were produced, and the fish probably responded differently than would wild fish. Even in the wild, animals behave differently depending on location, temperature, physiological state, age, body size, and school size. So, even if the present study had been conducted in the wild, the findings may not have been of universal value.

Comparison of response threshold levels with hearing threshold levels.

During recent years, underwater sound has been of growing interest to governments (e.g. National Research Council, 2003), as they have to set standards, for example, for acceptable sound levels for marine animals. However, for convenience, and because of a lack of knowledge, fixed levels above the basic hearing threshold of animals have often been used as criteria for acceptable sound levels (National Research Council, 2005;[START_REF] Southall | Marine Mammal Noise Exposure Criteria[END_REF]. Therefore, we compare the response threshold levels we found in the present study to hearing threshold levels, to see if this approach is valid for tones in the fish species tested, and to evaluate the relationship between detection of a sound and response to it.

The hearing sensitivity of only four of the fish species (sea bass, Atlantic cod, pollack, and Atlantic herring) has been tested, either physiologically or behaviourally [START_REF] Lovell | The hearing abilities of the bass, Dicentrarchus labrax[END_REF][START_REF] Offutt | Structures for the detection of acoustic stimuli in the Atlantic cod, Gadus Morhua[END_REF][START_REF] Buerkle | An audiogram of the Atlantic cod, Gadus morhua L[END_REF]Hawkings and Myrberg, 1983;[START_REF] Chapman | A field study of the hearing in cod, Gadus morhua L[END_REF]1969;[START_REF] Chapman | Field studies of hearing in teleost fish[END_REF][START_REF] Enger | Hearing in herring[END_REF]. Although the critical ratios are known only for cod (for 0.06, 0.160 and 0.380 kHz pure tones; [START_REF] Hawkins | Masked auditory thresholds in the cod, Gadus morhua L[END_REF], it is likely that the background noise level in the tank for these four species was sufficiently low not to mask the test stimuli (Figs. 2A, D, F, andH).

Atlantic herring is a hearing specialist. The members of the gadoid family Atlantic cod and pollack (and probably the close relative pout), and the eel, are considered to be hearing generalists with medium hearing ability. Sea bass is believed to be a hearing generalist with higher hearing thresholds. We found no information about hearing in thicklip mullet and horse mackerel.

In the sea bass, the 50 % reaction threshold levels were 0-30 dB above the hearing thresholds for the test frequencies (ABR audiogram method, Lovell, 2003; Fig. 2A). In the Atlantic cod, which has a gas-filled swim bladder, 50 % reaction threshold levels were not even reached when the test signals were 5-30 dB above the cod's hearing thresholds for those frequencies obtained by [START_REF] Buerkle | An audiogram of the Atlantic cod, Gadus morhua L[END_REF], who later stated that the hearing was masked by the ambient noise in the tank [START_REF] Buerkle | Relation of pure tone thresholds to background noise level in the Atlantic cod (Gadus morhua)[END_REF]; Fig. 2D). Reaction threshold levels were 15-50 dB above the more accepted hearing thresholds obtained for the same species in the ocean by [START_REF] Chapman | A field study of the hearing in cod, Gadus morhua L[END_REF], and 10-60 dB above those obtained in a physiological audiogram by [START_REF] Offutt | Structures for the detection of acoustic stimuli in the Atlantic cod, Gadus Morhua[END_REF]; Fig. 2D). Cod have directional hearing [START_REF] Schuijf | On the mechanism of directional hearing in cod (Gadus morhua L.)[END_REF][START_REF] Hawkins | Directional hearing in the median vertical plane by the cod[END_REF]. In the wild, directional swimming of cod in response to a sound source has also been observed (Løkkeborg and Soldal, 1993). Although Atlantic cod is considered to have relatively sensitive hearing, it is a species not easily frightened. When a novel object is introduced into a tank, cod investigate it, whereas many other species of fish flee and initially keep a great distance from it. Maybe this bold behaviour caused the animals in the present study not to react to the test signals, even at relatively high levels. On the other hand, avoidance of continuous low frequency pure tones between 0.025 and 0.125 kHz at sound pressure levels of 130 and 140 dB re 1µPa was observed in experiments with juvenile and adult cod (C. Mueller-Blenkle, pers. comm.). Cod is sensitive to infrasound down to 0.1 Hz [START_REF] Sand | Detection of infrasound by the Atlantic cod[END_REF][START_REF] Enger | Detection and reaction of fish to infrasound[END_REF], so the lack of response in the present study may be because cod is especially sensitive to frequencies lower than those we used.

Page 9 of 24 MER

In the pollack, 50 % reaction threshold levels were not reached when the test signals were 10-30 dB above the hearing thresholds for the test frequencies obtained by [START_REF] Chapman | The importance of sound in fish behaviour in relation to capture by trawls[END_REF], and 20-40 dB above the threshold reported by [START_REF] Chapman | Field studies of hearing in teleost fish[END_REF], although we did see some responses to the 0.1, 0.125, 0.250, and 0.4 kHz stimuli (Fig. 2F). This suggests that the 50 % reaction threshold level for those frequencies was only a few dB above the maximum level that could be produced with the available equipment.

Herring is considered a hearing specialist, as it has a swim bladder that is mechanically coupled to the inner ear, and air-filled vesicles (bullae) close to the inner ear [START_REF] Enger | Hearing in herring[END_REF]. At 4 kHz, the 50 % reaction threshold level was 30 dB above the estimated hearing threshold level for herring [START_REF] Enger | Hearing in herring[END_REF]; Fig. 2H). [START_REF] Blaxter | Startle response in herring: the effect of sound stimulus frequency, size of fish, and selective interference with the acoustico-lateralis system[END_REF] exposed herring to 0.07-0.2 kHz signals, found startle responses at received levels between 122-138 dB (re 1 Pa), and observed that the response depended on the size of the herring. Olson (1971) reports that herring showed a clear behavioural response to 0.1 kHz signals when the signal was 20-25 dB above the ambient noise level. Blaxter et al. (1981) found that 2-40 ms signals were sufficient for herring to detect stimuli and localize sound sources. Thus the 900 ms stimuli in the present study were sufficiently long to cause a startle response in herring. As in the present study, herring have been shown to react directionally to sound stimuli in previous studies [START_REF] Olsen | Directional responses in herring for sound and noise stimuli[END_REF][START_REF] Hering | Avoidance of acoustic stimuli by the herring[END_REF]Schwarz and Greer, 1984).

The present study shows that the difference between the hearing and reaction threshold levels varies per frequency, and within and between the fish species. This suggests that at sea, not only the masking effect of the background noise on a stimulus determines its effect on fish behaviour, but also the frequency spectrum and level of the stimulus. In addition, the present study clearly shows that fish species react very differently to sound, and that generalisations about the effects of sound on fish should be made with the utmost care.

Anthropogenic noise

In the present study only eight of the 224 fish species that occur in the North Sea [START_REF] Yang | The dominant fish fauna in the North Sea and its determination[END_REF] were tested. Because even within these eight species marked differences in reaction threshold received levels, and in the frequencies that elicited reactions, occurred, it would be useful to conduct similar tests on more fish species and stimuli of different frequency spectra, to be able to predict the reactions of marine fish of the North Sea to anthropogenic noise.

The fish species that responded to sounds in the present study would probably also react to sounds at frequencies lower than those we tested. The lowest frequency tested (0.1 kHz) was determined by the equipment available for this study. Except for herring, all the fish species that reacted did so to sounds below 2 kHz. Some of the reactions to low frequency stimuli in the present study may have been due to particle motion, which was not measured, but should be in future studies of this kind. In general, anthropogenic noise sources have wide spectra, but low frequency parts of the spectra travel further than high frequency parts, so that low frequencies (below 1 kHz) are dominant at distance from the source [START_REF] Richardson | Marine Page 12 of 24 MER Mammals and Noise[END_REF]. Therefore, the fish species tested in the present study could be influenced by anthropogenic activities if tonal signals were produced and if the acoustic received level was above the reaction threshold levels determined in the present study. The received level depends on, among other parameters, the source level of the sound, the water depth, and the propagation loss, which in turn depends on the distance between the sound source and the fish.

In the present study, only the reactions of the fish to pure tones were tested. It would be of interest to test the animals' reaction to sounds more similar to anthropogenic noise, to more complicated sounds, such as sweeps, and to the actual broad-band noise of, for instance, Page 10 of 24 MER wind turbines and shipping, both of which contain pure tones superimposed on a noise spectrum. As well as on the spectrum and level of anthropogenic sounds, the reactions of fish probably depend on the context (e.g. location, temperature, physiological state, age, body size, and school size).

Page 13 of 24 MER Tables:

Table 1. Mean standard body length of the study fish. N = number of individuals used in the test, SD = standard deviation. Because herring cannot be touched without damaging them, their body length was estimated. Fig. 2. The maximum received level range that could be produced in the for the test frequencies causing no reactions, and, for some species, the 50 % reaction SPL ranges (shaded areas represent ± 8 dB of average received level). A) Sea bass (0.1-0.7 kHz; school size: 17 fish), and the background noise range in the net enclosure, which applies to all species. Also shown is the auditory brainstem response (ABR) audiogram of sea bass. B) Thicklip mullet (0.4-0.7 kHz; school size: 11 fish). C) Pout (0.1-0.250 kHz; school size: 9 fish). D) Atlantic cod (school size: 5 fish). Also shown are three hearing thresholds of Atlantic cod. E) Common eel (school size: 10 fish). F) Pollack (school size: 4 fish). There was some reaction (< 50 %) to the maximum levels that could be produced for signals of 0.1 kHz, 0.125 kHz, 0.250 kHz and 0.4 kHz. Also shown are two hearing thresholds of pollack. G) Horse mackerel (0.1-2 kHz; school size: 13 fish). H) Atlantic herring (4 kHz; school size: 4 fish). Also shown is the hearing threshold of Atlantic herring.

Species

Page 16 of 24 MER

Fig. 1 .

 1 Fig.1. A schematic view of the study area, showing the tank, the net enclosure, the location of the three cameras, and the three transducers. The fish are shown to scale for the larger fish species.

  Figure 1

Table 2 .

 2 Water temperature during the test periods for each fish species. N = number of measurements, SD = standard deviation.

		N Standard body length (cm)
			Mean SD	Range
	Sea bass	17	23 2.4	18-26
	Thicklip mullet 11	17 5.3	8-24
	Pout	9	21 2.7	18-24
	Atlantic cod	5	44 1.7	42-46
	Common eel	10	46 6.5	35-57
	Pollack	4	24	2	22-26
	Horse mackerel 13	4 0.8	3-5
	Atlantic herring 4	27	-	25-30

Acknowledgements

We thank Rob Triesscheijn for all his help during various phases of this project, and Petra van der Marel, Janine Veenstra, Marieke Fennema, and Sonja de Wilde for part of the data collection and analysis. We thank Bert Meijering, director of Topsy Baits, for allowing us the use of his facilities, and Hein Hermans for his help with water quality. We thank Gerard Visser, Michaël Laterveer, and Peter van Putten (all of the Oceanium department of Blijdorp Zoo, Rotterdam), for lending us the Atlantic herring. We thank Christina Müller-Blenkle (Humboldt University, Berlin, Germany) for her comments on an earlier version of this manuscript. This project complied with the Dutch standards for animal experiments, and was funded by the Netherlands Ministry for Agriculture, Nature, and Food Quality (DKWprogram 418: North Sea and Coast). Supervision was done by IMARES, Texel, The Netherlands (via Han Lindeboom and Peter Reijnders).

Page 24 of 24 MER Figure 2h


Bayle-Sempere

M Vazquez-Luis

P Sanchez-Jerez

J Bayle-Sempere

M ; Sanchez-Jerez

Changes in amphipod (Crustacea) assemblages associated with shallow-water algal habitats invaded by Caulerpa racemosa var. cylindracea in the western Mediterranean Sea

Keywords: Amphipoda, Caulerpa racemosa var. cylindracea, detritus, invasive species, Mediterranean Sea, shallow rocky bottoms

Changes in amphipod (Crustacea) assemblages associated with shallow-water algal habitats invaded by var. in the western Mediterranean Sea

Introduction

The green alga Caulerpa racemosa (Forsskål) J. Agardh var. cylindracea (Sonder) Verlaque, Huisman et Boudouresque [START_REF] Verlaque | On the identity and origin of the Mediterranean invasive Caulerpa racemosa (Caulerpales, Chlorophyta)[END_REF] (hereafter C. racemosa) was first detected in Tunisia (Mediterranean Sea) in 1926. More recently, during the 1990s, C. racemosa spread steadily throughout the Mediterranean region (see [START_REF] Piazzi | Invasion of Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta) in the Mediterranean Sea: an assessment of the spread[END_REF]. Recent genetic analyses have shown that the C. racemosa population originated from the southwest coast of Western Australia [START_REF] Verlaque | On the identity and origin of the Mediterranean invasive Caulerpa racemosa (Caulerpales, Chlorophyta)[END_REF], and is not a Lessepsian migrant as thought originally. A remarkable spread of C. racemosa throughout the Mediterranean has been recorded during the last 15 years or so, during which the alga invaded different habitats located at water depths of 0 m to more than 60 m. The recorded rate of invasion has been much faster than that for other invasive species such as Caulerpa taxifolia [START_REF] Verlaque | On the identity and origin of the Mediterranean invasive Caulerpa racemosa (Caulerpales, Chlorophyta)[END_REF]. The rapid spread of C. racemosa can be explained by its very efficient reproductive strategies, both sexual and asexual (via propagules), which is not the case with Caulerpa taxifolia (Panayotidis and Žuljevi´c, 2001;Renoncourt and Meisenez, 2002).

Several studies have demonstrated that habitat heterogeneity and complexity play an important role in influencing the assemblage structure of epibenthic marine fauna [START_REF] Johnson | Variations in diversity within benthic marine communities[END_REF][START_REF] Stoner | Perception and choice of substratum by epifaunal amphipods associated with seagrasses[END_REF]Dean and Connell, 1987;[START_REF] Edgar | Patterns of colonization of mobile epifauna in Western Australian seagrass bed[END_REF][START_REF] Taylor | Mobile epifauna on subtidal brown seaweeds in northeastern New Zealand[END_REF]Cole, 1994, Ayala andMartín, 2003). The pattern of distribution and species richness and abundance of epifauna on rocky bottoms is affected by changes in the composition of associated macroalgae [START_REF] Connell | Community interactions on marine rocky intertidal shores[END_REF]. Therefore, changes in habitat structure of vegetated substrates resulting from the invasion of species such as C. racemosa could affect the associated fauna, including amphipods. Amphipods are one of the most important groups of invertebrates associated with vegetated habitats, and they play an important role as trophic resources for fish populations [START_REF] Sanchez-Jerez | Comparison of the epifauna spatial distribution in Posidonia oceanica, Cymodocea nododa and unvegetated bottoms: Importance of meadow edges[END_REF][START_REF] Stål | Food utilisation by coastal fish assemblages in rocky and soft bottoms on the Swedish west coast: Inference for identification of essential fish habitats[END_REF]. Amphipods respond to habitat alteration and can therefore be used as an indicator of environmental impacts on vegetated habitats [START_REF] Bellan-Santini | Relationship Between Populations of Amphipods and Pollution[END_REF][START_REF] Virnstein | Seagrass-associated invertebrate communities of the Southeastern U S A: a review[END_REF][START_REF] Conradi | The Amphipod Community as a Bioindicator in Algeciras Bay (Southern Iberian Peninsula) Based on a Spatio-Temporal Distribution[END_REF][START_REF] Sanchez-Jerez | Influence of the structure of Posidonia oceanica meadows modified by bottom trawling on crustacean assemblages: comparison of amphipods and decapods[END_REF].

Since the first records of spread of C. racemosa along Mediterranean coastal areas, several studies concerning the ecology of this species have been undertaken (see [START_REF] Ruitton | Seasonal changes of the introduced Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta) at th e northwest limit of its Mediterranean range[END_REF][START_REF] Cavas | Antiproliferative and newly attributed apoptotic activities from an invasive marine alga: Caulerpa racemosa var. cylindracea[END_REF]. However, information on changes on the associated fauna brought about by the invasive alga is lacking [START_REF] Argyrou | Expansion of the macroalga Caulerpa racemosa and changes in soft bottom macrofaunal assemblages in Moni Bay, Cyprus[END_REF][START_REF] Piazzi | The spread of Caulerpa racemosa var. cylindracea in in the Mediterranean Sea: an example of how biological invasions can influence beta diversity[END_REF]. The main aim of this study was to assess the effects of C. racemosa on amphipod assemblages associated with a shallow water rocky bottom habitat in the southwestern Mediterranean Sea, by comparing natural rocky algal assemblages and the same habitat type that has been invaded by C. racemosa. The hypothesis tested in our study was: invasion by C. racemosa should influence the habitat structure of rocky bottom algal assemblages and, consequently, the assemblage structure of associated epifauna such as amphipods may be affected, in terms of species richness and abundance.

Materials and Methods

Study area

Fieldwork was carried out along the Cape of Santa Pola (Alicante, south-east Spain; Fig. 1) on a shallow rocky platform. In Alicante, C. racemosa was first recorded in 2002 at a site located around ten kilometres north of our study area, where it colonised soft sediments and dead matte of Posidonia oceanica. Following two months, C. racemosa was detected on the rocky platform in our study area (Pena-Martin et al., 2003). The alien alga now occurs in extensive areas of ecologically important rocky bottoms, on sandy and muddy substrata, and on dead matte of Posidonia oceanica. It also occurs intermixed with Cymodocea nodosa in meadows of the seagrass within a wide depth range (0.2 -1.5 m) and with patchy distribution.

Sampling and experimental design

Shallow rocky habitat, located within the 0.2 m -0.5 m depth range, was sampled.

Several sampling sites, separated by hundreds of metres, with C. racemosa and without the alien alga, were randomly selected across the study area. All sites presented similar environmental conditions with respect to wave exposure and light [START_REF] Ferrandis-Ballester | Dulces bárbaros del este y del oeste (Análisis estadísticos de los vientos en la bahía de Alicante)[END_REF]. Three 'invaded' rocky habitat sites and another three 'noninvaded' rocky habitat sites were sampled in September 2004 (average water temperature = 27.5 ºC), which coincides with the period of maximum vegetative growth of the alga [START_REF] Piazzi | Développement et dynamique saisonnière d'un peuplement méditerranéen de l'algue tropicale Caulerpa racemosa (Forsskål) J. Agardh[END_REF]. Other sites were sampled again in March 2005 (water temperature = 13 ºC) which coincides with the period of minimum vegetative grow. At each site, three random replications were taken using a 20×20 cm quadrat by scraping the whole surface using a trowel [START_REF] Edgar | The influence of plant structure on the species richness, biomass and secondary production of macrofaunal assemblages associated with Western Australian seagrass beds[END_REF]. Samples were separated by tens of meters. A 300 m mesh bag was attached to the quadrat to avoid loss of the motile fauna. Samples were preserved in 4% solution of formaldehyde in seawater.

Each replicate was sieved in sea-water through a 500 m mesh, obtaining the fine fraction of detritus. In the laboratory, the amphipods were separated, identified, and counted. After that, algae were sorted as well as the bigger fragments of detritus.

Afterwards the macrophytes and the detritus were dried for 24 h at 80 ºC, and weighed.

The macrophytes were identified to the species level. Habitat structure was characterised using three attributes: species richness of macrophytes, biomass of each species (g) and quantity of detritus (g).

Data analysis

Two different statistical approaches were used to identify potential changes in the amphipod assemblages and habitat features, based on the null hypothesis of no change in amphipod assemblage composition between the two habitat types across different sampling times.

Multivariate analysis of assemblage structure

Non-parametric multidimensional scaling (MDS) was used as the ordination method for exploring changes in amphipod and macroalgal assemblages [START_REF] Clarke | Changes in Marine Communities: an approach to statistical analysis and interpretation[END_REF]. The similarity matrix, which was calculated using the Bray-Curtis index and double square transformation of data, was used to construct bivariate MDS plots.

Calculation of percentage similarities (SIMPER) was then applied to calculate the contribution of each species to the dissimilarity between sampling times and habitats. (PRIMER software;[START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF].

Univariate analysis

Algal biomass, C. racemosa biomass, species richness of seaweeds, biomass of detritus, total abundance of amphipods, species richness of amphipods and abundance of seven amphipod species (the most important by SIMPER) were analysed using ANOVA. To test whether the abundance of amphipods was similar across habitats and times we used an analysis of variance (ANOVA) which incorporated the following factors: 'Time of sampling', a fixed factor and orthogonal, with two treatments: September-04 and March-05; 'habitat', a fixed factor, with two treatments: macroalgal community with C. racemosa (invaded habitat) and without C. racemosa (non-invaded habitat), and 'Site', a random factor and nested with the both main factors, with three random sampling sites.

Prior to ANOVA, heterogeneity of variance was tested with Cochran's C-test. Data were x +1 transformed if variances were significantly different at p = 0.05, and log(x +1) transformed if variance was still heterogeneous. Where variance remained heterogeneous, untransformed data were analysed, as ANOVA is a robust statistical test and is relatively unaffected by heterogeneity of variances, particularly in balanced experiments [START_REF] Underwood | Experiments in Ecology: Their Logical Design and Interpretation Using Analysis of Variance[END_REF], however, in such cases special care was taken in the interpretation of results. Furthermore, in such cases, to reduce type I error, the level of significance was reduced to < 0.01. When ANOVA indicted a significant difference for a given factor, the source of difference was identified by applying the Student-Newman-Keul (SNK) test [START_REF] Underwood | Techniques of analysis of variance in experimental biology and ecology[END_REF][START_REF] Underwood | Experiments in Ecology: Their Logical Design and Interpretation Using Analysis of Variance[END_REF].

Results

Habitat features

A total of 24 taxa of macroalgae and one seagrass species were identified (Table 1).

Species richness showed significant seasonal differences (Table 2; Ti=P <0.05). During September, the invaded habitat showed a reduced number of macroalgal species: only five together with C. racemosa, compared with 11 species recorded from the noninvaded habitat. The most important species in terms of biomass in the non-invaded places were Jania rubens, Halopteris scoparia and Cystoseira brachicarpa, while in the invaded habitat C. racemosa, Jania rubens and Padina pavonica were most important (Table 1). In March, differences between invaded and non-invaded habitats were lower (14 where C. racemosa was present and 16 species where it was absent). The most important species in non-invaded sites during March were Corallina ellongata, Jania rubens and Dictiota fasciola; and C. racemosa, Cladophora sp. and Halopteris scoparia dominated at invaded sites (Table 1). The MDS two-dimensional representation of macrophyte biomass clearly showed segregation of sampling sites mainly related to sampling time and habitat (Fig. 2).

In the case of algal biomass, excluding C. racemosa, significant differences were noted for the invaded habitat with respect to the non-invaded habitat (Table 2; Fig. 3).

Total algal biomass recorded in September was similar between the two different habitats (347.67 ± 117.44 g dw m -2 for non-invaded habitat compared to 379.55 ± 64.63 g dw m -2 for invaded habitat; Table 1), however, C. racemosa contributed a high value (74.6%), and replacing other native species. Non-invaded sites showed the highest mean biomass value, which reached a maximum value of 826.46 ± 203.15 in September (Fig. 3). However, total biomass (326.52 ± 79.21 g dw m -2 ) at the invaded sites, without a recovery of the community in spite of the reduction of C. racemosa biomass.

Differences in accumulated detritus biomass was significant between habitats (P<0.05, Table 2). The highest values were recorded from invaded habitats during both sampling periods (472.48 g dw m -2 in September and 466.92 g dw m -2 in March). On the other hand, detritus biomass was relatively low at non-invaded habitats during September (5.87 ± 1.51 g dw m -2 ), and higher in March (242.22 ± 47.25 g dw m -2 ; Fig. 3).

Amphipod assemblage

A total of 31 amphipod taxa, belonging to 13 families, were recorded (Table 3).

Species richness was significantly different (Ti, P<0.05, Table 4; Fig. 4a) among sampling times but similar between habitats. In September, species richness recorded from the habitat dominated by C. racemosa was similar to that recorded from the noninvaded habitat (16 and 14 species respectively; Table 3), while for both habitats, values were higher in March (20 and 24 species, Table 3). Only ten species were present in both habitats and in both months: Amphilochus neapolitanus, Ampithoe ramondi, Microdeutopus spp., Caprella spp., Apocorophium acutum, Elasmopus spp., Lysianassa costae, Pereionotus testudo, Stenothoe monoculodes and Hyale schmidti. Seven species were found only in the non-invaded habitats: Amphilochus sp., Ampelisca serraticaudata, Ampithoe ferox, Lembos sp., Leptocheirus guttatus, Lysianassa sp. and Stenothoe tergestina; while there seemed to be a relationship between the invaded habitat and the following six amphipods: Ampelisca diadema, Corophium spp.

Corophium insidiosum, Gammarella fucicola, Melita palmata and Orchomene humilis.

Total abundance was similar between habitats in September, but there were significant differences between the two habitats in March, with a maximum value of 6497.22 ind/m 2 recorded from the non-invaded habitat (Ti x Ha P<0.01, Table 4; Fig. 4b).

The two-dimensional MDS plot for the amphipod assemblages showed segregation of sampling sites that was mainly related to the sampling period, while moderate segregation was noted by habitat type during March (Fig. 5). The main species responsible for dissimilarities in amphipod assemblages were Caprella spp., Ampithoe ramondi, Microdeutopus spp., Elasmopus spp., Apocorophium acutum, Hyale schmidti and Lysianassa costae, for which an average dissimilarity of 64.73 % was noted between habitats. Together, these species comprised 90 % of the total amphipod abundance.

The seven most important amphipod species showed different patterns of abundance values with respect to macroalgal assemblage. Abundance of Ampithoe ramondi and Hyale schmidti were significantly higher in non-invaded habitats (Ha, P<0.05, Table 4; Fig. 6a,b). On the other hand, Apocorophium acutum had a significantly higher abundance in the invaded habitat (Ha, P<0.05, Table 4; Fig. 6c).

Significant differences were recorded for Microdeutopus spp. in terms of sampling times (Ti, P<0.05, Table 4; Fig. 6d), while it was more abundant in September at invaded habitats but without significant differences.

With regard to differences between sampling times (Table 3), several species appeared only in September (Amphilochus sp., Melita palmata and Orchomene humilis); or in March (Ampelisca diadema, Ampelisca serraticaudata, Lembos sp., Leptocheirus guttatus, Corophium spp., Corophium insidiosum, Dexamine spiniventris, Gammarella fucicola, Melita hergensis, Lysianassa sp. and Stenothoe tergestina).

Dissimilarity between sampling times within non-invaded sites was not very high (48.09%); the most important species contributing to this dissimilarity were Caprella spp. and Hyale schmidti. Dissimilarity between sampling times within invaded sites was 51.71%, the most important species contributing to this dissimilarity were Caprella spp. and Lysianassa costae.

The abundance of two species (Caprella spp. and Elasmopus spp.) was not significantly different between the two habitat types, but there was a clear pattern of higher abundances in non-invaded habitat. The abundance of Caprella spp. varied significantly with respect to season, being more abundant in March, especially in the non-invaded habitat (Ti, P<0.05, Table 4; Fig. 6e). Elasmopus spp. seemed to be more abundant on native seaweeds, especially during September, but not statistical differences were noted (Table 4, Fig. 6f). Finally, the abundance of Lysianassa costae, was higher in non-invaded habitats, but this was true only in March (TixHa, P<0.01, Table 4, Fig. 6g); the pattern was opposite during September, but without any significant differences.

Discussion

Our results show that the presence and abundance of C. racemosa had a marked effect on the macroalgal assemblage structure of Mediterranean shallow rocky communities, affecting the species composition of vegetation and increasing the detritus stock. This causes important changes in the associated amphipod assemblage, in terms of both abundance and species composition. The effects of the invasive species on habitat structure were more important in September because the alga was dominant at that time. The seasonal variation in abundance values for C. racemosa recorded from our study area was similar to that described in other localities, i.e. with maximum development in the warm season, while biomass values were similar or even greater than those recorded from other Mediterranean localities [START_REF] Piazzi | Développement et dynamique saisonnière d'un peuplement méditerranéen de l'algue tropicale Caulerpa racemosa (Forsskål) J. Agardh[END_REF]. The peculiar growth of C. racemosa, with stolons that spread rapidly in all directions, and its colonisation pattern that results from sexual reproduction [START_REF] Panayotidis | Sexual reproduction of the invasive green alga Caulerpa racemosa var. occidentalis in the Mediterranean Sea[END_REF], together with dispersal of fragments detached from the plant (Ceccherelli et al., 2001;Ceccherelli and Piazzi, 2001) and propagules [START_REF] Renoncourt | Formation of propagules on a invasive strain of Caulerpa racemosa (Chlorophyta) in the Mediterranean Sea[END_REF] permit C. racemosa to colonize a habitat very efficiently. This results in strong modification of habitat structure, particularly that of macroalgal assemblages. On the SE coast of Spain and other locations (e.g. Nervi, Italy; [START_REF] Modena | Spreading of Caulerpa racemosa (Forsskål) J. Agardh (Bryopsidaceae, Chlorophyta) along the coasts of the Ligurian Sea[END_REF], colonisation by C. racemosa has been mostly recorded from rocky bottom habitats located in shallow waters (0.5 m), however, on a wider scale across the Mediterranean region (e.g.

France, Italy and Cyprus) more extensive spread of the alga have been recorded on soft bottom habitats in deeper waters (15-20 m) [START_REF] Argyrou | Expansion of the macroalga Caulerpa racemosa and changes in soft bottom macrofaunal assemblages in Moni Bay, Cyprus[END_REF][START_REF] Verlaque | Blitzkrieg in a marine invasion: Caulerpa racemosa var. cylindracea (Bryopsidales, Chlorophyta) reaches the Canary Islands (northest Atlantic)[END_REF], and on dead matte of Posidonia oceanica. Therefore, one would expect that the spread of C. racemosa will extend to both rocky and soft bottom habitats, as well as to degraded P. oceanica meadows.

With respect to changes in vegetation resulting from the presence of C. racemosa, an important reduction in species richness was recorded in September. On the other hand, contrary to results from other studies [START_REF] Piazzi | Comparative study of the growth of two co-occurring introduced green algae Caulerpa taxifolia and Caulerpa racemosa along the Tuscan coast (Italy, western Mediterranean)[END_REF], species richness was similar between invaded and non-invaded habitats in March (the colder month), which implies that some native species colonized the habitat irrespective of the different species composition and biomass of benthic vegetation. Some authors have shown that several years are necessary for the development of dense and continuous C. racemosa stands [START_REF] Ruitton | Seasonal changes of the introduced Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta) at th e northwest limit of its Mediterranean range[END_REF]; possibly during the forthcoming years the effects will persist also during the coldest period at the study area, reducing the possibility of algae colonization. However, the total biomass of C. racemosa recorded from our study (259 g dw m -2 ) is comparable or higher than that recorded from studies carried out on French and Italian coasts, and is also higher than values obtained for C. taxifolia and C. prolifera meadows at some localities (see a review in [START_REF] Capiomont | Phenology of a deep-water population of Caulerpa racemosa var. cylindracea in the northwestern Mediterranean Sea. Short communication[END_REF].

One of the remarkable impacts of C. racemosa is the accumulation of detritus, which persists throughout the year. In spite of the important seasonal variability of C. racemosa biomass, detritus accumulation persist in winter. The mesh generated by the stolons of this species ensure that detritus is retained, while the multilayered structure formed by the thallus traps the sediment [START_REF] Piazzi | Comparative study of the growth of two co-occurring introduced green algae Caulerpa taxifolia and Caulerpa racemosa along the Tuscan coast (Italy, western Mediterranean)[END_REF]. Detritus values recorded during both seasons were 2 to 6 times higher than those recorded from native seaweeds. This accumulation of detritus causes import changes in the structure of the sediment and in the granulometric composition [START_REF] Walker | Seagrass, sediment and infauna: a comparison of Posidonia australis, Posidonia sinuosa and Amphibolis antarctica in Princess Royal Harbour, south-western Australia. I. Seagrass biomass, productivity and contribution to sediments[END_REF], leading to anoxic conditions in which toxic substances can be possibly accumulated. On the other hand, detritus plays a very important role as a trophic resource for marine invertebrates, being one of the main trophic pathways of the marine ecosystem (Valiella, 1995) and is one of the most important features of habitat structure in vegetated habitats [START_REF] Allesina | The consequences of the aggregation of detritus pools in ecological networks[END_REF].

Therefore, any modification of the detritus compartment can affect the overall trophic web, because it could lead to a change in the faunal assemblage structure resulting from modification of trophic guilds due to different trophic requirements.

With respect to the epifauna, various studies have demonstrated that modification of habitat complexity affects crustacean assemblages (Hicks, 1977[START_REF] Hicks | Habitat structure, disturbance, and equilibrium in crustacean communities[END_REF][START_REF] Stoner | Perception and choice of substratum by epifaunal amphipods associated with seagrasses[END_REF][START_REF] Virnstein | Seagrass-associated invertebrate communities of the Southeastern U S A: a review[END_REF][START_REF] Virnstein | Motile epifauna of marine macrophytes in the Indian River Lagoon, Florida. I. Comparisons among three species of seagrasses from adjacent beds[END_REF][START_REF] Edgar | The ecology of South-East Tasmanian phytal animal communities. IV. Factors affecting the distribution of ampithoid amphipods among algae[END_REF][START_REF] Edgar | Patterns of colonization of mobile epifauna in Western Australian seagrass bed[END_REF][START_REF] Sanchez-Jerez | Comparison of the epifauna spatial distribution in Posidonia oceanica, Cymodocea nododa and unvegetated bottoms: Importance of meadow edges[END_REF][START_REF] Ayala | Relaciones entre la comunidad de anfípodos y las macroalgas a las que están asociados, en una plataforma rocosa del litoral central de Venezuela[END_REF]. In our study, the amphipod assemblages were affected by changes in habitat structure due to colonisation by C. racemosa. The total abundance of amphipods was greater on native seaweeds, mainly in March. However, the species richness of the amphipod assemblage did not decrease because of the spread of C. racemosa but because of an indirect effect; namely the increase in detritus biomass in invaded habitat resulted in an increase in the abundance of detritivores species (e.g. Apocorophium acutum), and increase in the abundance of herbivorous species (e.g. Ampithoe ramondi and Hyale schmidti), hence resulting in a large overall modification of the assemblage species composition.

The main modification in amphipod assemblage structure was manifested as an increase in abundance of Apocorophium acutum in habitat invaded by C. racemosa, which was four times higher than that of other Corophiidae species in eutrophic habitats [START_REF] Guerra-García | Oxygen levels versus chemical pollutants: do they have similar influence on macrofaunal assemblages? A case study in a harbour with two opposing entrances[END_REF]. Species belonging to the genus Apocorophium are tube-dwelling amphipods that inhabit small U-shaped tubes and are selective deposit feeders [START_REF] Ciarelli | Reproducibility of spikedsediment bioassays using the marine benthic amphipod, Corophium volutator[END_REF], and feed on bacteria, algae, and diatoms adsorbed onto the surface of sediment particles [START_REF] Meadows | The behaviour of Corophium volutator[END_REF]. Several species of this genus are continuously exposed to toxicants in the sediment, which they ingest while feeding [START_REF] Bat | Sediment toxicity testing: a bioassay approach using the amphipod Corophium volutator and the polychaete Arenicola marina[END_REF], hence they have been used for sediment toxicity tests. The accumulation of detritus in habitats invaded by C. racemosa seems to favour colonization by Corophium spp., resulting in a dramatic increase in abundance and establishment of a large population in an altered habitat. Microdeutopus spp. also showed a small increase in abundance in habitat invaded by C. racemosa, but the effect was only recorded in September. This genus is generally associated with Posidonia oceanica and fine sand habitats [START_REF] Ruffo | The Amphipoda of the Mediterranean. Part 1, Gammaridea[END_REF], but in experiments carried out recently by [START_REF] Roberts | Habitat configuration affects colonisation of epifauna in marine alga bed[END_REF], the family Aoridae were noted to prefer bare rock with a thin cover of sediment than an algal substratum. Furthermore, other taxonomic groups, such as the polychaetes, are favoured by accumulation of detritus amongst the C. racemosa stolons, as shown by [START_REF] Argyrou | Expansion of the macroalga Caulerpa racemosa and changes in soft bottom macrofaunal assemblages in Moni Bay, Cyprus[END_REF] from their study carried out in Cyprus.

On the other hand, the abundance of some species, such as Ampithoe ramondi, Hyale schmidti or Caprella spp., was reduced in habitat invaded by C. racemosa, since these species have a preference for native seaweeds [START_REF] Kocatas | Note préliminaire sur les amphipodes recueillis dans les horizons supérieurs de l'étage infralitoral rocheux du Golfe d'Izmir (Turquie)[END_REF][START_REF] Russo | Epifauna living on sublittoral seaweeds around Cyprus[END_REF]Guerra-Garcia and García-Gómez, 2001;[START_REF] Roberts | Habitat configuration affects colonisation of epifauna in marine alga bed[END_REF]. Other studies have also shown displacement of Ampithoe ramondi resulting from the presence of other Caulerpa species, e.g. Caulerpa prolifera [START_REF] Sánchez-Moyano | Effect of the vegetative cycle of Caulerpa prolifera on the spatio-temporal variation of invertebrate macrofauna[END_REF]. The displaced species have a close relationship with seaweeds, as has been demonstrated by [START_REF] Viejo | Mobile epifauna inhabiting the invasive Sargassum muticum and two local seaweeds in northern Spain[END_REF]. In the case of other species, such as Lysianassa costae, the effects of C. racemosa seem to be seasonal, inhabiting habitats that have a large algal biomass, irrespective of the species composition.

Comparing the results of the present study with ones on the effects of C. taxifolia on amphipod assemblages, the species richness recorded from the latter appears to be lower [START_REF] Bellan-Santini | Faune d' invertébrés du peuplement à Caulerpa taxifolia. Données préliminaires pour les côtes de provence (Méditerranée nord-occidentale[END_REF]. Furthermore, the effects of C. taxifolia on the abundance of amphipods also seem to be more critical [START_REF] Bellan-Santini | Faune d' invertébrés du peuplement à Caulerpa taxifolia. Données préliminaires pour les côtes de provence (Méditerranée nord-occidentale[END_REF]. Such differences could be due to several factors, such as the species composition of Caulerpa beds. Stands of C. taxifolia are monospecific, while in our case; the C. racemosa grew intermixed with other macroalgal species during both sampling periods. Consequently, during the initial stages of invasion of a host habitat by an invasive seaweed, an increment in habitat complexity will occur if the initial cover is low, hence a new and additional habitat for the local epifauna becomes available [START_REF] Viejo | Mobile epifauna inhabiting the invasive Sargassum muticum and two local seaweeds in northern Spain[END_REF]. However, if growth of the invasive species continues, leading to a monospecific and dense meadow, it could lead to impoverishment of the amphipod assemblage as has been noted for C. taxifolia elsewhere.

Several studies have demonstrated that consequences of C. racemosa in host systems are very important, particularly since it appears that this introduced species most invasive in the Mediterranean Sea [START_REF] Verlaque | On the identity and origin of the Mediterranean invasive Caulerpa racemosa (Caulerpales, Chlorophyta)[END_REF]. The presence of C. racemosa in the southeastern coast of Spain is an incipient problem. It was recorded from Alicante in 2002 and, in less than three years, important changes in the structure of shallow rocky habitat assemblages occurred, although to date the distribution of C. racemosa has been rather patchy, hence its effects are not very widespread locally.

Several years may be necessary for development of a dense and continuous C. racemosa meadow [START_REF] Ruitton | Seasonal changes of the introduced Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta) at th e northwest limit of its Mediterranean range[END_REF]; therefore in the near future, one can expect dramatic changes in rocky bottom habitats, leading to a direct impact on marine benthic communities along the western Mediterranean coast. It is therefore very important to increase our research effort to establish the ecological implications of the spread of C. racemosa in the Mediterranean Sea, as this will provide the necessary information and tools to develop new mitigation and eradication programs.

Acknowledgements

This work was supported by Instituto Alicantino de Cultura Juan Gil Albert, Excma. Diputación de Alicante. We would like to thank Judith Klein and Marc Verlaque, Centro Oceanológico de Marsella (Université de la Mediterranée, Parc Scientifique et Technologique de Luminy) for identify the variety of C. racemosa present along the coast of Alicante. We also thank several colleagues for their assistance in the field. We are very grateful to Joseph A Borg to improve the English version and for his comments.

Table 1 Mean biomass (g dw m -2 ) values ± SE for the different species, together with total biomass, total species richness and detritus weight for the two sampling periods and habitats.

SEPTEMBER -04 MARCH -05 Species

Non-invaded habitat

Invaded habitat

Non-invaded habitat

Invaded habitat

Chlorophyta Acetabularia acetabulum (Linnaeus) P.

Fig. 1 .

 1 Fig. 1. Map showing the location of Alicante on the southeastern coast of Spain, and the study area.

Fig. 2 .

 2 Fig. 2. Two-dimensional MDS plot for algal biomass; S: September-04; M: March-05; I: Invaded habitat; NI: Non-invaded habitat, for each of the six sampling sites.

Fig. 3 .

 3 Fig. 3. Mean seaweed biomass (± SE) recorded from the two different sampling periods and habitats. Seaweed = biomass of seaweeds (without C. racemosa); C. racemosa = biomass of C. racemosa; and Detritus = biomass of detritus.

Fig. 4 .

 4 Fig. 4. (a) Mean number of amphipod species (individuals m -2 ± SE) and (b) total abundance (individuals m -2 ± SE), recorded from the two different sampling periods and habitats.

Fig. 5 .

 5 Fig. 5. Two-dimensional MDS plot amphipod species abundance. S = September-04; M = March-05; I = Invaded habitat; NI = Non-invaded habitat for each of the six sampling sites.

Fig. 6 .

 6 Fig. 6. Mean abundance (number of individuals ±SE) of the seven most abundant amphipods: Ampithoe ramondi (a); Hyale schmidti (b); Apocorophium acutum (c); Microdeutopus spp. (d); Caprella spp. (e); Elasmopus spp. (f) and Lysianassa costae (g)

Table 2

 2 Result of ANOVA (three-factor) for the different habitat attributes: macrophyte species richness, macrophyte biomass, macrophyte biomass without C. racemosa, and detritus. MS = mean square; P = level of probability; df = degrees of freedom; Si= site; ns = non-significant; * significant at p < 0.05

	Source of variation df	Species richness	Macrophyte biomass	Macrophyte biomass without C. racemosa	Detritus	F versus
			MS	P	MS	P	MS	P	MS	P
	Sampling time=Ti	1	53.78 0.0158*	6.59 0.2154	6.05	0.1066	191.75 0.3812 Si(TixHa)
	Habitat=Ha	1	2.78 0.5077	5.88 0.2395	10.04 0.0472* 1720.49 0.0241* Si(TixHa)
	TixHa	1	11.11 0.2029	12.34 0.1030	0.27	0.7110	210.67 0.3599 Si(TixHa)
	Si (TixHa)	8	5.78 0.0043*	3.64 0.0821	1.83 0.0002*	223.34 0.0001*	Res
	Residual	24	1.47		1.77		0.29		31.75
	Cochran's C-test		C=0.3585 ns	C=0.3917 ns	C=0.2616 ns	C=0.3368 ns
	Transformation		None		Sqrt(X+1)	Ln(X+1)	None

Table 3

 3 Abundance of amphipod species (number of individuals per m 2 ± SE) recorded from the different habitats for each of the two different sampling periods, together with total abundance and species richness for the two sampling periods and habitats.

	SEPTEMBER -04	MARCH -05

Table 4

 4 Results of ANOVA (three-factor) for amphipod species richness, total abundance, and abundance of the seven most abundant species. MS = mean square; P = level of probability; df = degrees of freedom; Si= site, ns = non-significant; * significant at p < 0.05

	Source of variation df	Species richness MS P	Total abundance MS P	Ampithoe ramondi MS P	F versus
	Sampling time=Ti	1	78.03	0.0158*	6889.00 0.6864	13.28	0.0348*	Si(TixHa)
	Habitat=Ha	1	42.25	0.0551	84487.11 0.1807	12.04	0.0420*	Si(TixHa)
	TixHa	1	56.25	0.0321*	54444.44 0.0033*	2.47	0.3054	Si(TixHa)
	Si (TixHa)	8	8.39	0.0481*	39278.50 0.0033 *	2.06	0.0028*	Res
	Residual	24	3.53		9534.44		0.48
	Cochran's C-test		C=0.1654 ns	C=0.2210 ns	C=0.2488 ns
	Transformation		None	None	Ln(X+1)
	Source of variation df	Hyale schmidtii	Apocorophium acutum	Microdeutopus spp.	F versus
			MS	P	MS	P	MS	P
	Sampling time=Ti	1	14.69	0.0104*	230.02	0.0980	23.49	0.0008*	Si(TixHa)
	Habitat=Ha	1	16.63	0.0076*	890.02 0.0062*	0.82	0.3590	Si(TixHa)
	TixHa	1	6.96	0.0511	132.25	0.1934	0.0003	0.9853	Si(TixHa)
	Si (TixHa)	8	1.32	0.0508	65.58	0.7064	0.87	0.0943	Res
	Residual	24	0.56		96.77		0.44
	Cochran's C-test		C=0.3841 ns	C=0.3720 ns	C=0.2565 ns
	Transformation		Ln(X+1)	None	None
	Source of variation df	Caprella spp. MS P	Elasmopus spp. MS P	Lysianassa costae MS P	F versus
	Sampling time=Ti	1	85069.44 0.0166*	15.39	0.1746	16.00	0.5058	Si(TixHa)
	Habitat=Ha	1	24753.77 0.1420	8.57	0.2986	53.77	0.2374	Si(TixHa)
	TixHa	1	20640.11 0.1753	0.21	0.8648	386.77	0.0090*	Si(TixHa)
	Si (TixHa)	8	9331.33 0.0359*	6.93	0.0033*	32.97	0.5053	Res
	Residual	24	3650.83		1.68		35.19
	Cochran's C-test		C=0.3862 ns	C=0.2690 ns	C=0.3228 ns
	Transformation		None	Sqrt(X+1)	None


Maria Cristina Fossi
email: fossi@unisi.it

Letizia Marsili

Silvia Casini

Daniela Bucalossi

First detection of CYP1A1 and CYP2B induction in Mediterranean cetacean skin biopsies and cultured fibroblasts by western blot analysis

Keywords: Mediterranean cetaceans, Integument biopsy, Fibroblast cell cultures, CYP1A1, CYP2B, Western blot, OCs, PBDEs

The need to develop powerful tools to detect exposure and effects of POPs and emerging contaminants in Mediterranean cetaceans led us to develop a suite of sensitive nonlethal biomarkers in integument biopsies of free-ranging animals. In order to propose induction of CYP1A1 and CYP2B, detected by western blot analysis, as biomarkers of exposure to OCs, PAHs and PBDEs, a three-phase experimental protocol (in vitro experiments, calibration experiments and field applications) was followed using fibroblast cell cultures and biopsies of Mediterranean Stenella coeruleoalba and Tursiops truncatus. This methodology was confirmed to be sensitive and stable in comparison to previous methods used to detect CYP1A1 in biopsies, enabling analysis of several inducible proteins in nonlethal samples and analysis of material from stranded animals.

In the last 20 years, there has been growing concern about the potential risk to Mediterranean cetaceans of high bioaccumulation of organochlorines (OCs) (Fossi et al., 1992;Fossi et al., 2003a) and emerging contaminants, such as polybrominated diphenyl ethers (PBDEs) (Alaee et al., 2003;Fossi et al., 2006). While levels of organochlorine compounds are decreasing, concentrations of PBDEs seem to be increasing in the Mediterranean environment.

Several international institutions (IWC, ACCOBAMS) have encouraged research and development of sensitive non-lethal biomarkers in biopsies of free-ranging animals, to define the toxicological status of cetaceans. The objective of our lab was to develop a "multi-trial biomarker tool" applied to cetacean integument biopsies to detect "toxicological stress syndromes". We used a multidisciplinary approach, merging genomic and proteomic studies (Western blot, RT-PCR, cultured fibroblasts) with analysis of POPs, PAHs and PBDEs, and genetic population studies. Here, we summarize the main results of part of this project, focusing on detection of CYP1A1 and CYP2B induction in cetacean biopsies and cultured fibroblasts by Western blot (WB) analysis, as biomarkers of exposure to OCs, PAHs and PBDEs.

In order to validate this methodology, a three phase experimental protocol was followed. In the first phase (in vitro experiments), we used WB to detect CYP1A1 and CYP2B induction caused by exposure of cultured fibroblasts of Stenella coeruleoalba and Tursiops truncatus to PBDEs and OCs. In the second phase (calibration experiments), we validated WB analysis of CYP1A1 and CYP2B as a semi-quantitative detection tool using calibration curves of CYP450 obtained from liver and skin of stranded specimens of Tursiops truncatus. Finally in the third phase (field applications), we applied WB analysis of CYP1A1-

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CYP2B as a semi-quantitative tool to explore differences in exposure to OCs, PAHs and PBDEs of three sub-populations of Stenella coeruleoalba.

Sub-samples of skin and liver of stranded Tursiops truncatus (n=2) were obtained 4-8 hours after death. Integument biopsies (Fossi et al, 2003a) were obtained from free-ranging Stenella coeruleoalba (n=60) from the Mediterranean Whale Sanctuary, Ionian Sea and Strait of Gibraltar.

Gender was determined genetically according to Berube and Palsboll (1996).

Fibroblasts were cultured from biopsies according to Marsili et al. (2000) and were subject to two different experimental protocols for 48 h, using two classes of CYP-inducers (Fossi et al., 2006). Firstly, a mixture of Arochlor 1260, pp'DDT and pp'DDE in DMSO were used at three concentrations: 1, 5 and 25 µg/ml, plus DMSO (0.05%) as control. Secondly, a mixture containing 27 PBDEs in nonane were used at three concentrations: 0.1, 0.05 and 0.01 µg/ml, plus nonane (0.01 µg/ml) as control.

Homogeneous sub-samples of biopsies (including epidermis, dermis and blubber layers; Angell et al., 2004) were homogenized in aryl-hydrocarbon-receptor (AhR) buffer (Wilson et al., 2007) in a Tissue Lyser (Qiagen). The homogenate was centrifuged twice and the supernatant (S9) was analyzed for total proteins and then by WB.

For WB analysis, S9 tissue homogenates (in duplicate) and fibroblast extracts were separated by SDS-PAGE (10% polyacrylamide gels) and blotted onto nitrocellulose; the membranes were saturated with blocking solution for 1h. Primary polyclonal antibodies were from Oxford Biochemical Research. Goat anti rabbit CYP1A1 and anti CYP2B4, diluted 1:5000 and 1:1000, respectively, in TTBS-1% gelatin, were incubated with cetacean proteins overnight. Incubation with anti-goat HRP-labelled secondary antibody (1:3000) was performed (1h) and detected according to the BioRad Immun-Star-HRP-Chemiluminescent-Kit booklet.

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In order to validate WB analysis of CYP1A1 and CYP2B as a semi-quantitative detection tool in biopsies, a series of calibration curves of CYP450 from liver (ranging from 1.2 to 4.8 pmol of CYP450) and skin (from 0.095 to 2.400 pmol) of stranded Tursiops truncatus were analysed in triplicate. A triplicate skin standard (SS) was analyzed within the curves and maintained as internal standard in subsequent WBs. Semi-quantitative analysis was performed for each WB with Quantity-One software (BioRad) using Adjusted Volume (Intensity *mm 2 ) as quantitative parameter. The data was correlated with increasing pmol of CYP450 and two Excel Macros (one for CYP1A1 and one for CYP2B) were produced incorporating the normalizing factor of SS for quantification of unknown biopsy samples. The calibration curves were validated for semi-quantitative analysis of CYP1A1 and CYP2B in three different populations of striped dolphin.

In the first phase (in vitro experiments), we applied the WB technique to detect CYP1A1 and CYP2B induction produced by PBDEs and OCs in fibroblasts of the two species of dolphins (Fossi et al., 2006). Increasing induction was detected in both species with increasing concentrations of contaminants. In female striped dolphin cells, CYP2B increased 3-, 15-and 16-fold with respect to control, with increasing concentrations of OCs. The highest trend of induction (CYP2B) was found in Tursiops truncatus cells treated with PBDEs. In the second phase (calibration experiments), we validated WB analysis of CYP1A1 (Fig. 1A) and CYP2B (Fig. 1B) as a semi-quantitative detection tool with the calibration curves of CYP450 obtained from liver and skin of stranded specimens of Tursiops truncatus.

A linear correlation was found between relative pmol CYP1A1 and Adjusted Volume (Int. *mm 2 ) (R=0.99) and between relative pmol CYP2B and Adjusted Volume (R=0.99). The two Excel Macros (CYP1A1 and CYP2B), incorporating the normalizing factor of SS (Skin Standard) (Fig 1A and1B) will be used for quantification of unknown biopsy samples (third phase). In the third phase (field applications), we applied the WB analysis of CYP1A1 and

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CYP2B as semi-quantitative detection tools, in biopsy samples, to explore different exposure to planar (polycyclic aromatic hydrocarbons and planar halogenated aromatic hydrocarbons) (Angell et al., 2004;Godard et al., 2004;Wilson et al., 2007) and globular (globular halogenated aromatic hydrocarbons) lipophilic contaminants, respectively, in three subpopulations of Mediterranean Stenella coeruleoalba. Here the calibration curves were validated (as pilot experiment) for semi-quantitative analysies of CYP1A1 (Fig. 2) and CYP2B in male sub-samples collected in the three areas. Future studies will concern correlations between CYP1A1/CYP2B and blubber levels of contaminants.

In conclusion this methodology applied to skin biopsies and stranded cetaceans was confirmed to be sensitive and stable in comparison to previous methods used to detect CYP1A1 in biopsies (enzymatic (Fossi et al., 1992;Fossi et al., 2003a) and immunohistochemistry techniques), enabling analysis of several inducible proteins in nonlethal samples and material from stranded animals. Future development of more sensitive techniques (RT-PCR and proteomics) with chemical data (OCs, PBDEs, PAHs) will provide more complete information about cetacean toxicological stress syndromes.

Figure Captions

Fig. 1 .

 1 Fig. 1. Western blot analysis (WB) of CYP1A1 (A) and CYP2B (B) calibration curves of

Fig. 2 .

 2 Fig. 2. Western blot (WB) analysis of CYP1A1 in male specimens of three sub-populations

  Figure 1a

Acknowledgements

We thank Dr. Mori, Dr. Porcelloni, Dr. Caliani, Dr. Spinsanti, Dr. Jimenez, Dr. Gaspari, Dr. Panigada and Dr. De Stephanies for technical support in the sampling and laboratory activities. This project was supported by the Italian Ministry for Environment.


Maria Gonzalez-Rey

Angela Serafim

Rui Company

Tânia Gomes

Maria João Bebianno

Detoxification mechanisms in shrimp: comparative approach between hydrothermal vent fields and estuarine environments

Keywords: Metallothionein, Lipid Peroxidation, Metals, Hydrothermal vents, Shrimp

Hydrothermal vents are extreme deep-sea habitats that, due to their singular features, still intrigue scientific communities. Swift growth rates and profuse biomass of biological communities can be observed, despite of their inherently unstable physicalchemical and toxic conditions, indicating that organisms inhabiting this environment must be well adapted to these inhospitable conditions. The caridean shrimp, Chorocaris chacei, Mirocaris fortunata and Rimicaris exoculata, together with bathymodiolid mussels, dominate the vent fauna along the Mid-Atlantic Ridge (MAR). Crustacean species are widely used as biological indicators of environmental alterations, since they play a key ecological role as planktivorous grazers, epibenthic scavengers or as prey species. The biological consequences of the hydrothermal metal-rich environment in shrimp species are still largely unknown. Therefore, the aim of this study was the determination of the metal levels (Ag, Cd, Cu, Fe, Mn and Zn), metallothioneins (MT)

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and lipid peroxidation (LPO) in shrimp species collected in Rainbow, Lucky Strike and Menez-Gwen vent sites, in order to evaluate their different adaptation strategies toward metals when compared with two common coastal shrimp species (Palaemon elegans and Palaemonetes varians) from a fairly unpolluted estuarine system in south Portugal (Ria Formosa). Results show significant differences in metal concentrations, MT levels and lipid peroxidation between vent and coastal shrimp and also between shrimp species from the same site. This indicates that biochemical responses in both vent and coastal shrimp are affected not only by the environmental characteristics but also by inter-specific differences. Nevertheless, these responses apparently grant a successful adaptation for the survival in a metal-extreme environment.

Metals can be extremely toxic to organisms when present at high levels.

Nonetheless, metal-enriched hydrothermal vent fields: Rainbow (RB) (36°13'N, 33°54'W, 2300m), Lucky-Strike (LS) (37°17'N, 32°16'W, 1700 m) and Menez-Gwen (MGw) (37°51'N, 31°31´W, 850 m) manage, despite of their geochemical and physical distinct settings (Douville et al., 2002), to simultaneously comprise high metal concentrations with prolific biological communities. Trying to understand this apparent contradiction, metal concentrations (Ag, Cd, Cu, Fe, Mn and Zn), metallothionein (MT) and lipid peroxidation (LPO) levels were measured and compared between three vent shrimp species: Rimicaris exoculata and Mirocaris fortunata collected from RB and Chorocaris chacei from both LS and MGw with two common coastal species from a fairly unpolluted estuarine system Ria Formosa (RFor) (37º03´N; 07º47´W) in South Portugal (Bebianno, 1995).

Hydrothermal vent species were collected using the remote operated vehicle VICTOR6000 during SEAHMA I cruise. Coastal shrimp were collected with a subsuperficial tow using a shrimp net (~40 cm diameter). Both vent and coastal species were immediately frozen (-80ºC) and stored until analysis. Before dissection, shrimp were thoroughly cleaned with 0.02M TRIS-HCl buffer (pH 8.6), assuring minimal surface/particle contamination. Exoskeletons were separated from soft tissues; gills and hepatopancreas were not considered for analysis. MT analyses were performed in the soft tissues according to Bebianno and Langston (1989). Metal concentrations were determined in the soft tissues and exoskeleton by AAS. Lipid peroxidation was determined according to Erdelmeier et al. (1998). Total protein concentrations in supernatants were determined according to Lowry method (Lowry, 1951). Nonparametric test of Kruskall-Wallis (p<0.05) was used to compare statistical differences among the variables and Tukey's test was used when significant differences between averages were observed.

The most accumulated metals were Fe, Zn, Mn and Cu while Ag and Cd were the less accumulated in all shrimp species (Fig. 1). The concentration of metals accumulated in different tissues of crustaceans vary widely between metals and between taxa [START_REF] Rainbow | Metal metabolism in aquatic environment[END_REF] and is related its speciation, function of tissues and other biotic variables (Pourang et al., 2004). In hydrothermal species, this tendency reflects the metal concentrations in the hydrothermal fluids. Metal concentrations for MAR hydrothermal vents ranged from: Ag (4.3-17 nM), Cd (9-12 nM), Cu (2 µM), Fe (2-18 µM), Mn (59-68 µM) and Zn (2 µM) in Menez-Gwen; Ag (4.7-25 nM), Cd (18-79 nM), Cu (2 -30 µM), Fe (70-920 µM), Mn (77-450 µM) and Zn (2 -40 µM) in Lucky Strike and Ag (47 nM), Cd (130 nM), Cu (140 µM), Fe (24000 µM), Mn (2250 µM) and Zn (160 µM) in Rainbow (Douville et al., 2002). In the Ria Formosa system metal concentrations ranged from: Cd (0.9 -4.5 nM), Cu (0.02 -0.05 µM), Fe (8 -52 µM), Mn (2.5 -6.3 µM) and Zn (0.02 -0.03 µM) (Caetano et al., 1997). Since Fe, Zn and Cu are essential elements, high levels of these metals in shrimp tissues are attributed to their essentiality (Pourang et al., 2004). Accordingly, the low levels of Cd (a nonessential metal) in the tissues reflect the lower metal concentrations in the seawater where shrimp were collected (Géret et al., 2002a).

The measured metal concentrations vary with site, species and type of tissue, i.e. for hydrothermal shrimp, Fe is the most prominent metal, whilst for coastal shrimp is Mn. In M. fortunata from RB and C. chacei from LS the exoskeleton had significantly higher metal concentrations than the soft tissues (Fig. 1). Exoskeleton contains a significant proportion of metals, which is reduced prior to moulting. Moulting is an important aspect of crustacean physiology that influences metal concentrations and its

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distribution between soft tissues and exoskeleton (Pourang et al., 2004). However, in C. chacei from MGw all metals except Mn occur in higher concentrations in the soft tissues when compared to the exoskeleton (Fig. 1). Since Mn can substitute calcium in calcium carbonate, this metal is predominately found in calcified parts. The carapace of decapods acts as a depository in which Mn can be accumulated (Pourang et al., 2004).

In coastal metals occur in higher concentrations in soft tissues compared to exoskeleton. Metals are taken up by crustaceans in a form initially available to bind with metabolites in the receiving cell, with the potential to be transported elsewhere in the body via the haemolymph (Marsden and Rainbow 2004). Hence, significant differences in metal content between tissues reflect different bioavailability, function and efficiency in accumulating metals. [START_REF] Rainbow | Metal metabolism in aquatic environment[END_REF] concluded that Zn body concentrations in decapods are regulated to approximately those required for metabolic demand with relatively little Zn stored in detoxified form. However this seems not to be the case especially in hydrothermal vent species.

Like metal levels, MT concentrations also varied between species. MT levels in C. chacei from LS were four-fold higher than those for the same species from MGw (Table 1) and significantly higher than other vent or coastal shrimp species. C. chacei collected from MGw may reflect a more recent exposure to metals, contrarily to what occurred in shrimp from LS. Therefore, it seems that metals were not yet remobilized from the soft tissues to the exoskeleton, in order to be detoxified by MT. This protein does not respond immediately to the metal exposure as observed in laboratory studies (Géret et al., 2002b) and this may explain the significant differences observed between the levels of metals and MT in C. chacei from these two different hydrothermal vents.

However, LPO levels in C. chacei were significantly lower than those of R. exoculata and M. fortunata from RB and similar to the coastal shrimp (Table 1).

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Moreover, positive correlations exist between MT levels and Ag (P. elegans -MT = 2.14 + 0.71 Ag, r = 0.997), Zn (P. varians -MT = 1.03 + 1.32 Zn, r = 0.998) and Cd (C. chacei from LS -MT = -43.33 + 112.9 Cd, r = 0.989). Since, no crustacean regulates the body concentration of non-essential metals [START_REF] Rainbow | Metal metabolism in aquatic environment[END_REF]; MT may have a role in metal detoxification in the coastal species. Although no significant differences in MT levels between the two shrimp species were found, M. fortunata had four-fold higher LPO levels than P. varians (p<0.05). Considering that RB is the vent site with higher metal concentrations, basal levels of LPO in native shrimp may be naturally elevated.

Therefore, LPO levels point out that shrimp from RB are exposed to more significant stressful environmental conditions when compared to their coastal counterparts.

Nevertheless, a negative correlation between antioxidant enzymes and MT levels was detected in vent species (Gonzalez-Rey et al., 2007). Hence, MT (particularly for C. chacei from LS) may have an antioxidant role in ROS detoxification inside the cells, mainly in the elimination of hydroxyl radical, as described by Chubatsu and Meneghini (1993). Finally, the results suggest that biochemical responses in vent and coastal shrimp are not exclusively affected by environmental characteristics (i.e. microhabitats) but also by interspecific differences (i.e. feeding habits and moulting cycles), and only a complex combination of metal detoxification systems grant a successful adaptation to survival in metal-rich environments.

Figure Caption

Fig. 1. Non-essential (Ag and Cd) and essential metal concentrations (Cu, Fe, Mn and Zn) ( g. g -1 dry weight) in soft and exoskeleton tissues of shrimp species. R. exoculata

(1), M. fortunata C. chacei (3,4) from Mid-Atlantic hydrothermal vent fields and P. elegans (5) and P. varians (6) from Ria Formosa coastal area (Portugal). The data represent average and ± standard deviation (SD) for n=24. Values followed by the same lower case letter represent a non statistical difference between soft tissues for each metal and values followed by the same upper case letter represent a non statistical difference between exoskeleton tissues for each metal ( > 0.05). * Significant differences between soft tissues and exoskeleton for each species within every metal ( < 0.05).

Table 1.

Metallothionein concentrations (MT) and lipid peroxidation levels (LPO) in soft tissues of five shrimp species: R. exoculata, M. fortunata and C. chacei from Mid-Atlantic hydrothermal vent fields and P. elegans and P. varians from Ria Formosa coastal area (Portugal). The data represent average and ± standard deviation (SD) for n= 24. Values followed by the same letter are not statistically different ( > 0.05)