ARTICLE

Shipping noise affecting immune responses of European spinylobster (Palinurus elephas)Monica Celi, Francesco Filiciotto, Mirella Vazzana, Vincenzo Arizza, Vincenzo Maccarrone,Maria Ceraulo, Salvatore Mazzola, and Giuseppa Buscaino

Abstract: Marine crustaceans are influenced by numerous environmental factors. Environmental stress from pollutants seemsto affect their metabolism, growth, moulting, survival, and immune defence. Recently, it has become clear that there is animpact of the global increase in sea noise levels due to shipping traffic on crustacean welfare. Considering the ecological andcommercial importance of the European spiny lobster (Palinurus elephas (Fabricius, 1787)) in most parts of the Mediterraneancoastal area, in the present study we investigated whether shipping noise pollution contribute to changing the immuneparameters of stress in the European spiny lobster. The animals were exposed to a mix of noises produced by different typologiesof the boat played back in a tank, and the values of cellular and humeral parameters were evaluated. Total haemocyte count(THC), haemolymphatic protein concentration, phenoloxidase (PO) activity in cell-free haemolymph, and heat shock protein 27(Hsp27) expression in haemocyte lysate were considered potential biomarkers of stress. THC and PO activity decreased signifi-cantly, whereas total protein and Hsp27 expression increased significantly. Overall, the results demonstrate that the stressfulconditions investigated in this study affect both cellular and biochemical parameters in the European spiny lobster.

Key words: European spiny lobster, Palinurus elephas, noise pollution, immune response, PO activity, Hsp27.

Résumé : Les crustacés marins sont sous l’influence de nombreux facteurs environnementaux. Le stress environnemental provenantdes polluants semble avoir un impact sur le métabolisme, le développement, la mue, la capacité de survie et les défensesimmunitaires. Il a récemment été découvert que l’influence croissante du niveau sonore marin, causé par le trafic maritime,avait un impact non négligeable sur la santé des crustacés. Compte tenu de l’importance économique et écologique de lalangouste européenne (Palinurus elephas (Fabricius, 1787)) tout au long de la côte Méditerranéenne, nous nous sommes demandédans cette étude si la pollution sonore liée au trafic maritime jouait un rôle sur les paramètres du stress immunitaire de lalangouste européenne. Pour le vérifier, ces animaux ont été placés dans des aquariums et ont été exposés a un échantillon debruits produits par différents bateaux. Pour cela, des données sur les paramètres cellulaires et humorales ont été recensés. Lesparamètres suivants tels que : le compte total d’haemocyte (THC), la concentration en protéines haemolymphatiques, l’activitéde la phenoloxidase (PO) sur les cellules libres de l’haemolymphe, l’expression de la protéine de choc thermique 27 (Hsp27) surl’haemocyte lysate, ont été considérés comme de potentiels indicateurs de stress. Le THC et l’activité du PO présentent des baissessignificatives tandis que le taux de protéines et l’expression du Hsp27 ont augmentés de manières significatives. Dansl’ensemble, les résultats de cette étude ont montré que les paramètres de stress qui ont été testés dans ce cas ont un impact, a lafois sur les paramètres cellulaires et biochimiques de la langouste européenne.

Mots-clés : langouste européenne, Palinurus elephas, la pollution sonore, la réponse immunitaire, l’activité du PO, Hsp27.

IntroductionThe underwater noise generated by human activities in the

marine environment includes commercial shipping, offshoreconstruction, sonar exploration, seismic surveys, and underwa-ter explosions. These sounds have increased in both spatial andtemporal diffusion over the past 50 years, producing increasingbackground sea noise pollution (Ross 2005; Hildebrand 2009). Thischange in the underwater acoustic scene may pose a threat tomarine habitats and animals (Myrberg 1990; McIntyre 1995;Popper et al. 2003; Slabbekoorn et al. 2010; Popper and Hawkins2011). In consideration of recently growing awareness of this issue,

underwater noise has been listed in the European Union’s MarineStrategy Framework Directive 2008/56/EC as one of the descrip-tors for achieving good environmental status (GES).

Recently, many studies have shown the impact of anthro-pogenic acoustic pollution on fish and marine mammals (Au et al.1974; Myrberg 1990; Thomas et al 1990; Engås et al. 1996; Scholikand Yan 2001; Sun et al. 2001; Amoser and Ladich 2003; NRC 2003;Romano et al. 2004; Popper et al. 2005; Sandström et al. 2005;Codarin et al. 2009; Buscaino et al. 2010; Filiciotto et al. 2013).

Compared with sea mammals and fish, very few noise studieshave evaluated the impact of acoustic pollution on crustaceans

Received 10 August 2014. Accepted 12 November 2014.

M. Celi, F. Filiciotto, V. Maccarrone, S. Mazzola, and G. Buscaino. Istituto per l’Ambiente Marino Costiero U.O. di Capo Granitola – ConsiglioNazionale delle Ricerche, Via del Faro no. 3, 91021 Granitola, TP, Italy.M. Vazzana. Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Viale delle Scienze; Edificio 16, Universita degli Studi diPalermo, Italy.V. Arizza. Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Viale delle Scienze; Edificio 16, Universita degli Studi diPalermo, Italy; IEMEST – Istituto Euromediterraneo di Scienza e Tecnologia, Via E. Amari, 123 – 90100 Palermo, Italy.M. Ceraulo. Istituto per l’Ambiente Marino Costiero U.O. di Capo Granitola – Consiglio Nazionale delle Ricerche, Via del Faro no. 3, 91021 Granitola, TP,Italy; Department of Basic Sciences and Foundations – University of Urbino – Campus Scientifico ‘Enrico Mattei’ – Urbino, Italy.Corresponding author: Mirella Vazzana (e-mail: mirella.vazzana@unipa.it).

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Can. J. Zool. 93: 113–121 (2015) dx.doi.org/10.1139/cjz-2014-0219 Published at www.nrcresearchpress.com/cjz on 28 November 2014.

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(Christian 2003; Andriguetto-Filho et al. 2005; Celi et al. 2013;Wale et al. 2013), despite their high diversity, abundance, andeconomic importance.

Various haematological and immunological parameters such astotal haemocyte count (THC), haemolymph protein concentration(PC), phenoloxidase (PO) activity, and heat shock proteins (Hsps)expression have been considered to be potential health or diseasemarkers in crustaceans and can be used to assess stress responses(Hose et al. 1984; Persson et al. 1987; Bachère et al. 1995; Le Moullacet al. 1998; Jussila et al. 1997; 2001; Rodríguez and Le Moullac 2000;Le Moullac and Haffner 2000). Influences of stress on immuneresponses are diverse and usually depend on the nature of thestressor, the immune parameter studied, and the physiologicalstatus of the animal (Lacoste et al. 2002). Thus, under stressfulconditions, immune competence may be compromised.

In crustaceans, changes in the numbers of circulating haemo-cytes and their proportion appear to be associated with changes inenvironmental factors. Elevated haemocyte counts have been re-ported in crustaceans exposed to physical disturbance and air(Hauton et al. 1997; Jussila et al. 1997, 1999, 2001; Gomez-Jimenezet al. 2000).

Recently there have been reports in the literature that underacoustic stress, THC is persistently lowered, indicating that thesepractices may alter, at least temporarily, the number of circulat-ing haemocytes (Celi et al. 2013; Filiciotto et al. 2014).

Another potential indicator of environmental stress is the in-duction and (or) elevated expression of stress proteins, also known asHsps. Recently, we showed that noise pollution upregulate Hsp70protein expression in the red swamp crayfish (Procambarus clarkii(Girard, 1852)) and European spiny lobster (Palinurus elephas(Fabricius, 1787)) (Celi et al. 2013; Filiciotto et al. 2014). According totheir molecular size, they are usually classified into the followingfamilies: Hsp100s, Hsp90s, Hsp70s, Hsp60s, Hsp40s, and smallheat shock proteins (smHsps). The smHsp family is the most struc-turally divergent, ranging in size from 17 to 26 kDa in plants, from20 to 27 kDa in vertebrates, from 20 to 24 kDa in Drosophila, from24 to 43 kDa in yeast, and from 16 to 21 kDa in bacteria (Ingolia andCraig 1982; Caspers et al. 1995; Wotton et al. 1996; Verghese et al.2012).

These proteins are usually encoded by multigene families thatare expressed in many cell types, are often upregulated by biolog-ical stresses, and are under developmental control (Caspers et al.1995; Arrigo 2005). Although their exact roles in vivo are un-known, they are likely to serve a protective function under stressconditions (Landry et al. 1989; Yeh et al. 1994) and may be involvedin modulating cytoskeleton assembly and dynamics (Lavoie et al.1993; Nicholl and Quinlan 1994). While little is known aboutsmHsps in crustacean, Huang et al. (2008) identified a smHsp inthe giant tiger prawn (Penaeus monodon Fabricius, 1798) with amolecular mass of 21 kDa that was highly expressed in almost allorgans such as the epithelium, gill, stomach, midgut, lymphoidorgan, hepatopancreas, nervous tissue, and heart, but less ex-pressed in haemolymph, suggesting that gene regulation of Hsp21was seriously affected by WSSV (white spot syndrome virus).

The crustacean immune system also includes other proteincomponents involved in non-self-recognition, clotting, and foreign-agent elimination (Rosas et al. 2004). Alterations in haemolymphprotein concentration have been associated with various environ-mental stressors (Baden et al. 1990; Bjerregaard 1991; Chen et al.1994; Sánchez et al. 2001). Haemolymph protein concentrationhas been considered a means of determining the physical condi-tion or vitality of live lobsters (Stewart and Li 1969; Bolton et al.2009; Celi et al. 2013).

Among the plasma proteins in crustaceans, a very importantrole is played by prophenoloxidase (proPO) located in the plasmaand haemocytes (Hernández-López et al. 2003; Perdomo-Moraleset al. 2007; Masuda et al. 2012). For a long time, it has been recog-nized that the prophenoloxidase-activating system (proPO system)

has an important role as a non-self-recognition system in arthro-pods, and participates in the innate immune response throughmelanization, cytotoxic reactions, cell adhesion encapsulation,and phagocytosis (Cerenius and Söderhäll 2004; Jiravanichpaisalet al. 2006). The proPO system is composed of, among other pro-teins, the enzyme proPO, which is the zymogen of phenoloxidase(PO; EC 1.14.18.1). The conversion of proPO into PO occurs throughproteolytic attack by a serine proteinase named prophenoloxidase-activating enzyme (ppA) (Söderhäll and Cerenius 1998; Cereniusand Söderhäll 2004; Jiravanichpaisal et al. 2006). Changes in POactivity have been related to health and environmental conditions(Sung et al. 1998).

In this study, we investigated whether shipping noise pollutioncontributes to changing immune parameter indices of stress inEuropean spiny lobster. The lobsters were exposed to a mix ofnoises produced by different typologies of boat played back in atank; the assessed haemolymphatic parameters included PO ac-tivity, total proteins, THC, and heat shock protein 27 (Hsp27).

Materials and methods

Study speciesThe European spiny lobster is a common crustacean species

along the Mediterranean and northeastern Atlantic coasts (Hunter1999). This species, which is primarily active at night for feedingand reproduction (Goñi and Latrouite 2005), represents one of themajor targets of Mediterranean artisanal fisheries and, althoughcatches are now reduced and sporadic, this fishery has a longhistory (Goñi and Latrouite 2005; Groeneveld et al. 2006).

Animal maintenanceThe European spiny lobsters used in this study were captured

in May 2013 at a depth of 20–25 m near Mazara del Vallo (south-west Sicily, Italy) by fishermen using a commercial trammel net(54 mm inner-panel mesh size, 1200 m length). After capture, thelobsters were transferred to an indoor circular polyvinyl chloride(PVC) tank (2.35 m diameter and 1.5 m depth) at the Institute forthe Marine and Coastal Environment of the National ResearchCouncil (CNR–IAMC) of Capo Granitola (southwest Sicily, Italy) fora month-long acclimation period. Before the beginning of theacclimation phase, males were marked using a nontoxic water-resistant paint (Markservice, Milan, Italy) that was spread on thecentral portion of the lobsters’ carapace to distinguish them fromthe females. The lobsters were fed frozen molluscs, shrimps, andfish ad libitum.

The holding tank was equipped with an independent flow-through seawater system from a common source (25 ± 3.7 L/min;mean ± SD). Salinity was 36.4 ± 0.81 ppt (mean ± SD) and thetemperature was 18.61 ± 0.39 °C (mean ± SD). All animals werekept under natural photoperiods during the entire study period.

Experimental setupTwenty lobsters (10 males and 10 females) 298.57 ± 52.8 g in

mass and 7.81 ± 0.5 cm in carapace length (mean ± SD) were testedafter the acclimation period. In particular, we used 10 specimens(5 females and 5 males) for test condition and 10 specimens forcontrol (5 females and 5 males). Animals were individually andrandomly collected from the holding tank, assigned to the trials,and were used in only one experiment to meet the assumption ofexperimental independence. For the experimental procedure, weused only lobsters that had not recently molted. The lobsters werereleased into the centre of an experimental tank that was identi-cal in size, shape, flow-through seawater system equipment, andwater composition with the holding tank (Fig. 1). No shelter waspresent in the experimental tank. The lobsters were deprived offood for 5 days before the start of the experimental trials.

The experiment was performed during the month of July 2013.After a 1 h habituation period, the trials consisted of 30 min ofsilence before 30 min of sound exposure only for the treated

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animals (boat noise condition). Specimens assigned to the controlgroup were not exposed to any stimuli but only the low-level noiseof the experimental tank environment (control condition).

The sound exposure consisted of the playback of a randomsequence of boat noises, including recreational boats, hydrofoils,fishing boats, and ferryboats, with the aim to reproduce the noisefrom a marine area with high anthropogenic acoustic pollution.Control lobsters were exposed only to the low-level noise of theexperimental tank’s background noise.

At the end of the experimental phase, both lobsters exposed toboat noise and control animals were captured with a net andplaced on crushed ice for 15 min to induce torpor or “cold anaes-thesia” to allow sampling of the haemolymph. The samples werecollected from the experimental animals, and the lobsters werethen transferred into a postexperimental tank and released afterrecovery.

The protocols for animal husbandry and experimentation werereviewed and approved in accordance with Directive 86/609/EECby the Council of the European Communities regarding the treat-ment of animals used for scientific purposes.

Acoustic stimuli recording and played backTo obtain acoustic recordings of noises from boats for use in the

experimental tank, we used a calibrated hydrophone (model 8104;Bruel & Kjer, Naerum, Denmark) with a sensitivity of –205.6 dB re1 V/�Pa ± 4.0 dB in the 0.1 Hz to 80 kHz frequency band. Thehydrophone was used with a preamplifier (VP1000; Reson A/S,Slangerup, Denmark) with a 1 MHz bandwidth single-ended volt-age that had a high-pass filter set at 10 Hz and a 32 dB gain. Theequipment was connected to a digital acquisition card (USGH416HB;Avisoft Bioacoustics, Berlin, Germany; set with no gain) managedby Avisoft Recorder USGH software (Avisoft Bioacoustics). The sig-nals were acquired at 300 000 samples/s at 16 bits and were anal-ysed by the Avisoft-SASLab Pro program (Avisoft Bioacoustics).The format of the file was *.wav.

The marine traffic noise condition was obtained by recording inan area near a harbour, characterized by the alternating passagesof different types of boats (Fig. 2A). The noise in the experimentaltank was recorded to characterize the baseline noise in the studyenvironment (Fig. 2A). In Fig. 2B, the power spectrum of boatstimuli and sea background noise were reported. During the en-tire experimental period, the seawater-recirculating flow was de-ployed directly beyond the tank water’s surface to prevent anybubbles and no air pumps were used.

To play back the acoustic stimulus of different boats inside theexperimental tank, a playlist with the acquired wave file was cre-ated. The playlist was played back using the “loop mode” functionof the Avisoft-SASLab recorder software (Avisoft Bioacoustics)through the stereo output of the computer connected to a poweramplifier (type 2713; Bruel & Kjer). The amplifier was, in turn,

connected to the underwater loudspeaker (model UW30; Lubell,Columbus, Ohio, USA; transmit response: 125 ± 5 dB re 1 �Pa/V re1 m in the frequency range 100–1000 Hz; 120 ± 10 dB re 1 �Pa/V re1 m in the frequency range 1–20 kHz). The loudspeaker remainedin the tank during the control trial.

Immunological analysis

Haemolymph collectionHaemolymph was withdrawn from the ventral sinus located at

the first abdominal segment into a 2.5 mL sterile syringe contain-ing an equal volume of precooled anticoagulant solution (0.45 mol/LNaCl, 30 mmol/L sodium citrate, 26 mmol/L citric acid, 10 mmol/LEDTA). Haemocytes were examined by light microscopy to per-form THC using a Neubauer haemocytometer chamber and, afterthe cell counts, the diluted haemolymph was centrifuged at 500gfor 5 min at 4 °C and the haemocyte pellet was washed once withcacodylate buffer (CAC buffer: 10 mmol/L sodium cacodilate,0.45 mol/L sodium chloride, 20 mmol/L calcium chloride, pH 7).The resulting haemocyte pellet was then used for the PO enzymeactivity assay and biochemical analysis.

Haemocyte lysate supernatant (HLS)HLS was prepared by lysing cells in 500 �L of RIPA buffer,

pH 7.5, supplemented with a cocktail of protease inhibitors usinga glass piston homogenizer on ice. Lysates were centrifuged at15 000 g for 30 min at 4 °C. The supernatants were collected anddialyzed against 50 mmol/L Tris-HCl (pH 7.5).

Haemolymph protein concentrationThe total protein concentration in plasma and HLS was deter-

mined using a Quibit® 2.0 fluorometer (Invitrogen, Life Technol-ogies, Inc., Burlington, Ontario, Canada). The data were quantifiedwith standards.

Assay for PO activityPrior to the enzyme activity assay, the supernatant proteins in

each sample were adjusted to the same concentration of 500 �g/mL.Phenoloxidase activity was measured spectrophotometrically inplasma by recording the formation of dopachrome from L-3,4-dihydroxyphenylalanine (L-DOPA) using the microplate method(Hernández-López et al. 1996), with slight modifications. Briefly,100 �L of plasma was incubated with either 50 �L of L-DOPA, 3 mg/mLin CAC buffer followed by 50 �L of CAC buffer added 5 min later. Theoptical density at 490 nm was measured using a microplate reader(GloMax®-Multi Detection System; Promega Corporation, Madison,Wisconsin, USA). The control solution, which consisted of 150 �L ofCAC buffer and 50 �L of L-DOPA, was used for the background POactivity. Where indicated, the reaction velocity was defined as theincrement of absorbance per minute and was obtained from theslope of the linear region of the plot of absorbance versus time (min).

Zymographic assay of PO activityThe plasma samples were analysed by sodium dodecyl sulfate –

polyacrylamide gel electrophoresis (SDS–PAGE) as described byLaemmli (1970) in 6% polyacrylamide gels. Phenoloxidase activityafter electrophoresis was demonstrated using L-DOPA (3 mmol/Lin CAC buffer and 0.1% SDS) as substrate. Half of the gel wasstained with Coomassie Brilliant Blue R250 for identification oftotal proteins after the gels were photographed.

CytochemistryFor chemical and histochemical analyses, 100 �L of haemocyte

suspension were fixed on a side with 1% glutaraldehyde in filteredsea water plus 1% sucrose for 10 min. The May–Grunwald–Giemsastain was used to identify the different cell types according toFiliciotto et al. (2014). To identify the cell type that presents POactivity, smears after washing in CAC buffer were incubated with

Fig. 1. Schematic view of the experimental arena.

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100 �L L-DOPA solution (CAC buffer with 0.1% SDS) and observedafter 20 min using a light microscope.

Hsp protein expressionThe equivalent of 25 �g of serum protein and 25 �g of total

tissue lysate were separated on 10% SDS–PAGE under reducingconditions (Laemmli 1970). SDS – polyacrylamide minigels weretransferred to nitrocellulose membranes using a semidry transferapparatus (Bio-Rad Laboratories, Hercules, California, USA) andblocked with 5% bovine serum albumin (BSA) in TBS-T (20 mmol/L

Trizma base, pH 7.5, 300 mmol/L NaCl, 0.1% (v/v) Tween-20 with0.02% sodium azide) for 1 h at room temperature. The mem-branes were incubated over night at 4 °C with a mouse mono-clonal anti-Hsp27 antibody (Sigma-Aldrich, St. Louis, Missouri,USA; 1:800 dilution), washed with TBS-T (three times for 5 mineach), and incubated with an alkaline phosphatase-conjugatedgoat anti-mouse IgG (1:7500 for 1 h at room temperature). Afterwashing with TBS-T (three times for 5 min each), the membraneswere incubated with the 5-bromo-4-chloro-3-indolyl phosphate –nitro blue tetrazolium (BCIP–NBT) liquid substrate system. AlphaImager software was used for densitometric analysis of the immu-noblotted bands.

Fig. 2. (A) Spectrogram of the experimental tank background noise and of different boat noise stimuli: frequency (kHz) versus time (s).(A.1) Experimental tank environment; (A.2) recreational boat 1; (A.3) recreational boat 2; (A.4) recreational boat 3; (A.5) recreational boat4; (A.6) recreational boat 5; (A.7) hydrofoil; (A.8) fishing boat; (A.9) ferry boat. The intensity is reflected by the colour scale (dB re 1 �Pa, root meansquare, 1024-sample FlatDown window, sampling frequency 96 kHz). For interpretation of the colour scale refer to the colour version on the Web.(B) Mean power spectrum of the boat noise stimuli (top, solid black line) with the standard deviation (broken lines) and of the experimental tankbackground noise (bottom, solid grey line). Sound pressure level expressed in dB re 1 �Pa (wave files were undersampled to 96 kHz, fast Fouriertransform (FFT) size 1024, bandwidth 121.9 Hz, Hamming window) versus frequency expressed in Hertz.

Table 1. Summary of two-way ANOVA results for phenoloxidase (PO),protein concentration (PC), total haemocyte count (THC), and heatshock protein 27 (Hsp27) data.

Value FEffectdf

Errordf p

Intercept 0.004 847.739 4 13 <0.05Acoustic exposure 0.028 112.160 4 13 <0.05Sex 0.860 0.525 4 13 0.72Acoustic exposure × sex 0.654 1.717 4 13 0.20

Note: Two factors (fixed and orthogonal) on PO, PC, THC, and Hsp27 param-eters: acoustic exposure (two levels: boat noise condition, control) and sex (twolevels: male, female).

Table 2. Total haemocyte count (THC), protein concentration (PC),and phenoloxidase (PO) values (mean ± SD) in European spiny lobsters(Palinurus elephas) under control and boat acoustic conditions.

Control Boat

THC (1 × 106 cell/mL) 12±1 7±0.9PC (�g/�L) 3.98±0.6 9.04±1.9PO activity (optical density at 490 nm) 0.789±0.37 0.126±0.038

Note: There were significant differences between control lobsters and lob-sters exposed to boat noise (p < 0.001).

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Statistical analysisThe Kolmogorov–Smirnov test was used to check the normal

distribution of the haemolymphatic parameter data. Because nor-mal distributions in all haemolymphatic and biochemical datawere observed, parametric tests were used to compare differentvalues among the trials.

A two-way ANOVA was applied with two factors (fixed and or-thogonal) on each set of data: acoustic exposure (two levels: boatnoise condition, control) and sex (two levels: male, female).

The Newman–Keuls’ multiple comparisons test was used for allpost hoc comparisons.

P values of <0.05 were considered statistically significant. Allstatistical analyses were performed using the STATISTICA version7.0 (StatSoft, Inc., Tulsa, Oklahoma, USA) software package.

ResultsThe sex factor did not show significant differences among bio-

chemical parameters (Table 1).Instead, significant differences in all biochemical parameters

measured were detected between lobsters exposed to the boatcondition and control lobsters (Table 1). In particular, the numberof circulating haemocytes (THC) in the control European spinylobsters was approximately 12 × 106 cells/mL. After shipping noisepollution, the THC in lobsters decreased significantly, reachingvalues of 7 × 106 cells/L (Table 2).

Otherwise, significantly higher values of PC were found in ani-mals under the boat acoustic condition relative to the controllobsters (Table 2).

The PO activity in plasma decreased significantly in lobstersexposed to acoustic stimuli from boats relative to the controllobsters (Table 2).

Electrophoretic analysis by SDS–PAGE of plasma showed a pat-tern of PO activity bands with L-DOPA staining (Figs. 3A, �) and adifferent protein pattern in the same samples with CoomassieBrilliant Blue R250 staining (Fig. 3A, �). In the plasma, a well-defined band corresponding to around 160 kDa was produced. Atthe same concentration (20 �g per well), the densitometric anal-ysis of PO protein (Fig. 3B) revealed a significant decrease in ex-pression in the plasma from lobsters exposed to noise. In agreementwith Filiciotto et al. (2014), we identified three cellular types beforeand after staining (Figs. 4A, 4B). As shown in Figs. 4C and 4D, thehaemocytes of lobsters contained dark black granules located withingranulocytes.

The expression of Hsp27 was analysed by the Western blotmethod. Figure 5A shows the anti-rabbit Hsp27 mAb cross-reacted

with a 27 kDa band in circulating haemocytes from tested lob-sters. Densitometric analysis of Hsp27 protein levels revealed asignificant increase in expression in haemocytes collected fromlobsters exposed to noise (Fig. 5B). Hsp27 expression peaked(three-fold higher than in the untreated samples) after the periodof acoustic stimulation.

DiscussionMarine crustaceans are under the influence of numerous envi-

ronmental factors. Environmental stress from pollutants seems toaffect their metabolism, growth, moulting, survival (Staples andHeales 1991; Chen et al. 1995), and immune defence (Rodríguezand Le Moullac 2000).

Very few studies have considered the disturbances of boats oncrustaceans (Davis and Dodrill 1989; Blonder et al. 1992; Parsonsand Eggleston 2005; Wale et al. 2013), and none of these evalua-tions took into account the effects of boat noises. Considering theecological and commercial importance of the European spiny lob-ster in most parts of the Mediterranean coastal area, it is surpris-ing that until now the impact of the global increase in sea noiselevels from shipping traffic on this crustacean species has notbeen assessed. Only recently, Filiciotto et al. (2014) showed for thefirst time that boat noise pollution affects the behavioural activ-ity, as well as the cellular and biochemical parameter indices ofstress, of the European spiny lobster. To continue and completethe findings of the above-described experimental work, this studyaimed to gain more knowledge about the immune response ofEuropean spiny lobsters exposed to boat noises.

Among the examined haemato-immunological parameters inthis study, THC was considered. We showed that THC decreasedby about 38% in individuals subjected to acoustic stress relative tocontrol individuals. A decrease in THC is frequently reported inmarine crustaceans exposed to certain stress conditions (Lorenzonet al. 1999). In the blue shrimp (Penaeus stylirostris Stimpson, 1874 =Litopenaeus stylirostris (Stimpson, 1874)), a significant decrease inTHC was observed in shrimps exposed to hypoxia (Le Moullac et al.1998). Under acoustic stress, we previously reported THC was con-sistently lower, indicating that these practices may alter, at leasttemporarily, the number of circulating haemocytes (Celi et al.2013).

Changes in the levels of other plasma components have beendescribed in shrimp under several conditions. Haemolymph pro-tein concentration is considered a means of determining the phys-ical condition or vitality of live lobsters (Stewart and Li 1969;Bolton et al. 2009). In accordance with Filiciotto et al. (2014), the

Fig. 3. (A) Representative sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) analysis in 6% polyacrylamide. Each of foursamples under nondenaturing conditions from the same population of European spiny lobster (Palinurus elephas) (lines 1–2: plasma fromcontrol lobsters; lines 3–4: plasma from tested lobsters) was loaded twice on the same gel. After electrophoresis, the gel was cut in half. Theleft half was stained with Coomassie Brilliant Blue R250 (�) and the right half with L-DOPA solution, which is a standard substrate ofphenoloxidase (PO) (�). (B) Densitometric analysis of PO protein. There were significant differences between control (ctrl) lobsters and lobstersexposed boat noise (p < 0.01).

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present research showed that the levels of protein in the haemo-lymph increase significantly, by about two-fold, in individualssubjected to acoustic stress relative to control individuals.

The PO activity in haemolymph was measured by recording theformation of dopachrome from L-DOPA spectrophotometrically(Hernández-López et al. 1996; Le Moullac et al. 1997; Anggraeniand Owens 2000). Changes in PO activity have been related tohealth and environmental conditions (Sung et al. 1998). A declinein PO activity has been reported following cooling and also afterexposure to higher temperatures (Vargas-Albores et al. 1998;Cheng et al. 2005), while Ridgway et al. (2006) reported a decline inPO activity in the Norway lobster (Nephrops norvegicus (L., 1758))following prolonged aerial exposure; this is likely associated with

a decline in haemocytes that induces the PO pathway. We ob-served that acoustic stress was able to produce a decrease in POactivity in the plasma, probably due a decline in granulocytes, inagreement with a study on red swamp crayfish and Europeanspiny lobster that showed a decrease in the granular cell type afterexposure to an acoustic stimulus (Celi et al. 2013; Filiciotto et al.2014).

Hernández-López et al. (2003) reported that the plasma of thespiny lobster contains high PO activity, which is derived fromproPO itself. Similarly, the PO activity in crustacean plasma mayderive from proPO released from haemocytes (Jaenicke and Decker2003).

Fig. 4. The cytological location of the phenoloxidase (PO) activating system detected by cytochemical analysis and observed with a bright-fieldmicroscope: (A) untreated haemocytes from the European spiny lobster (Palinurus elephas) as control; (B) haemocytes incubated with L-DOPA asa substrate; (C) haemocytes stained with May–Grünwald–Giemsa; (D) granular cells positive for PO.

Fig. 5. Effect of the acoustic stimuli on expression levels of the heat shock protein 27 (Hsp27) in the European spiny lobster (Palinurus elephas).(A) Representative Western blot of Hsp27 levels in control (ctrl) animals and after noise exposure (boat). (B) Integrated density value (% I.D.V.)of the Hsp27 protein bands. Data are means ± SE (N = 5 female and 5 male for control and N = 5 female and 5 male test specimens). There weresignificant differences between ctrl and boat conditions (p < 0.01).

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In response to a variety of stress sources, living organisms ex-press particular sets of proteins such as smHsps. Mehlen et al.(1996) have shown the expression of smHsp from different spe-cies, such as mammalian Hsp27 and DHsp27 from the fruit flygenus Drosophila. In the giant tiger prawn, the Hsp21 gene wasidentified and categorized as a member of the smHsp family, andthe EST databases established from virus-infected shrimp (Huanget al. 2008) were annotated. In this study, we identified Hsp27 forthe first time in a crustacean and demonstrated that this smHspincreases after acoustic treatment. Hsp27 is a ubiquitous proteinand it is known to play multiple protective roles in cells exposedto a host of environmental insults (Tsvetkova et al. 2002; Salinthoneet al. 2007; Fu 2014).

Recently, we showed that noise pollution upregulates Hsp70protein expression in the same crustacean species (Filiciotto et al.2014); both Hsp27 and Hsp70 could be involved in cytoprotectiveand antiapoptotic activities after noise exposure.

Several studies have demonstrated that measurement ofhaematological parameters, such as THC, PC, PO activity, andHsps protein expression, are useful in evaluating stress-inducedimmunosuppression in marine crustaceans (Jussila et al. 1997;Lorenzon et al. 2011; Celi et al. 2013). Celi et al. (2013) observed thatred swamp crayfish showed altered aggressive behavioural pat-terns and changes in components of the haemato-immunologicalsystem such as serum glucose concentration, PC, agglutinatingactivity, and THC, differential haemocyte counts (DHC), andHsp70 expression when exposed to an acoustic stimulus at a fre-quency band of 0.1–25 kHz, clearly reflecting a stress condition.

In the present study, the effects of sea noise pollution on bothcellular and biochemical parameters in the haemolymph fromEuropean spiny lobsters were evaluated, demonstrating that theimpact of anthropogenic acoustic pollution induces alterations intheir immune response, often immunosuppressive in terms ofhaemocyte number, PO activity, total PC, and Hsps protein expres-sion, increasing the risk of infections by opportunistic and (or)pathogenic microorganisms.

Although many fundamental questions remain (i.e., the physi-ological responses to noise exposure of this marine species in anopen field where the acoustical field is closer to the natural envi-ronment), we argue that the insights from our study provide im-portant implications for underwater noise impact assessments.Indeed, we believe that haemolymphatic variations are criticalwhen assessing or predicting the severity of anthropogenic noiseimpact on crustaceans and may have applied value for devisingmitigating measures.

AcknowledgementsThis work has been funded by the Flagship Project RITMARE –

The Italian Research for the Sea coordinated by the Italian Na-tional Research Council and funded by the Italian Ministry ofEducation, University, and Research within the National ResearchProgram 2011–2013, and by the Project “L’industrializzazione dellaproduzione di Grandi Pelagici in Sicilia attraverso tecniche di ac-quacoltura responsabile” funded by Sicilian Regional Authority –Framework Convention between the Department for Fisheriesand the IAMC–CNR2.

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