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Physiological and agonistic behavioural response of Procambarus clarkii to an 1

acoustic stimulus 2

3

Monica Celi1, Francesco Filiciotto2, Daniela Parrinello1, Giuseppa Buscaino2,*, Alessandra 4

Damiano1, Angela Cuttitta2, Stefania D’Angelo3, Salvatore Mazzola2, Mirella Vazzana1 5

6 1Laboratory of Marine Immunobiology, Department of Environmental Biology and Biodiversity, Division of Animal 7

Biology and Anthropology, University of Palermo, Via Archirafi 18, Palermo, Italy 8 2Istituto per l’Ambiente Marino Costiero UOS Capo Granitola — National Research Council, Via del Faro No. 3 - 9

TG, 91021, Campobello di Mazara (TP), Italy 10 3 WWF Italia, via Lo Zano n. 29, 91026 Mazara del Vallo (TP), Italy 11

12

*To whom correspondence should be addressed: giuseppa.buscaino@cnr.it 13

14

Abstract 15

This study examined the effects of an acoustic stimulus on the haemolymph and agonistic 16

behaviour of the red swamp crayfish Procambarus clarkii. The experiment was conducted in a 17

tank equipped with a video recording system using 6 groups (3 control and 3 test groups) of five 18

adult crayfish (30 specimens in total). After one hour of habituation, the behaviour of the crayfish 19

was monitored for two hours. During the second hour, the animals in the test groups were exposed 20

to a linear sweep (frequency range 0.1-25 kHz; peak amplitude 148 dBrms re 1 µPa at 12 kHz) 21

acoustic stimulus for 30 minutes. Exposure to the noise produced significant variations in 22

haemato-immunological parameters as well as a reduction in agonistic behaviour. 23

24

INTRODUCTION 25

More than 500 recognised species of crayfish are distributed in aquatic habitats of all substrata 26

types across all continents except Antarctica and Africa (Taylor, 2002). Shelters range from 27

natural assemblages of rocks to constructed burrows in mud or sand. The red swamp crayfish 28

Procambarus clarkii (Girard, 1852) is an invasive freshwater species that originated in the south-29

central United States and currently shows a cosmopolitan distribution. This species has been 30

imported to Italy for farming purposes since 1987. Escaped crayfish have invaded natural habitats 31

and become stabilised in many ponds, lakes, and streams across Italy in recent years (Gherardi et 32

al., 1999). Although this crayfish is an aquatic species, it is highly resistant to air exposure and is 33

able to survive for several days outside the water (McMahon and Stuart, 1999). Several eco-34

ethological features of P. clarkii explain its rapid spread in the wild. The species’ biological cycle 35

reflects the hydrogeological cycle and water temperature changes in the invaded areas (Gutierrez-36

http://jeb.biologists.org/lookup/doi/10.1242/jeb.078865Access the most recent version at J Exp Biol Advance Online Articles. First posted online on 1 November 2012 as doi:10.1242/jeb.078865

Copyright (C) 2012. Published by The Company of Biologists Ltd

http://jeb.biologists.org/lookup/doi/10.1242/jeb.078865Access the most recent version at First posted online on 1 November 2012 as 10.1242/jeb.078865

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Yurrita et al., 1999). This crayfish is also highly resistant to environmental stress, including 37

extreme temperatures (Gherardi and Holdich, 1999; Paglianti and Gherardi, 2004), the absence of 38

water, salinity, low oxygen concentrations, and the presence of pollutants (Gherardi et al., 2002). 39

The success of P. clarkii as an invader is further supported by its generalist feeding habits as well 40

as its competitive superiority over native species due to its larger and big dimensions and high 41

grip claws and highly aggressive behaviour (Gherardi and Cioni, 2004). Individuals of both sexes 42

and assorted sizes usually live together, and social activity is particularly notable from spring to 43

autumn. During this period, adult individuals commonly leave their burrows after sunset and move 44

around the populated area. Crayfish exhibit agonistic behaviour when competing for habitats, 45

shelters, mates, and food (Bergman and Moore, 2003). The primary result of such agonistic 46

interactions is the establishment of a dominance relationship that can alter each individual’s access 47

to resources. Aggressive encounters between individuals (agonistic behaviour) are very common 48

(Bergman and Moore, 2003; Buscaino et al, 2012). The crayfish touch each other and assume 49

stereotyped postures aimed at threatening the opponent (Graham and Herberholz, 2009). Crayfish 50

have been used as a behavioural model system to study aggression (Dingle, 1983; Hyatt, 1983) 51

because of their very efficient (big dimensions and high grip) chelipeds (Garvey and Stein, 1993; 52

Schroeder and Huber, 2001) and the ritualised nature of their agonistic fights (Bruski and 53

Dunham, 1987). In particular, due to the high frequency of the agonistic behaviour in 54

Procambarus clarkii (Bergman and Moore, 2003; Buscaino et al, 2012), the observation of the 55

agonistic event and the motility (as a factor that could further the agonistic encounters) could 56

evidence alteration in the baseline behaviour due to external factor, such as an acoustic stimulus. 57

Moreover, the red swamp crayfish that is characterized by a high resistance to environmental 58

stress, could serve as a good model to examine the impacts of acoustic stimuli on behavioural 59

dynamics and the physiological parameters that reflect stress conditions. In addition, because P. 60

clarkii emits acoustic signals in both air and underwater (Favaro et al., 2011; Buscaino et al., 61

2012), it is possible that in this species sound (pressure variation and/or particle movements) can 62

play an ecological role (e.g. predator or conspecific movements perception) so that make these 63

animals sensible to an acoustic stimulus. Otherwise, some studies have evaluated the effects of 64

very high sound pressure levels stimuli (air guns used for seismic surveys) on marine crustacean 65

behaviour and biochemical parameters such as haemocytes, serum proteins, and enzymes without 66

significant effects (Christian et al., 2003; Andriguetto-Filho et al., 2005). While, Payne et al. 67

(2007) found that lobster exposed to very high as well as low sound level had experienced no 68

effect on delayed mortality or damage to mechanosensory system associated with animal 69

equilibrium and posture. However sub-lethal effects were observed with respect to feeding and 70

serum biochemistry with effect sometimes being observed weeks to months after exposure. 71

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Crustaceans might experience pain and stress in ways that are analogous to those all vertebrates 72

(Elwood et al., 2009). Potentially painful stimuli applied to vertebrates typically produce 73

vegetative modifications (behavioural changes as avoidance reaction), changes in blood flow, 74

respiratory patterns, biochemical and endocrine changes (Elwood et al., 2009). There has, 75

however, been limited examination of similar responses in crustaceans. 76

The behavioural observations in combination with physiological assessment, could give a 77

complete understanding of an external stimulus impact on an organism/population/species. In 78

particular, this combination could assume a significant relevance in crustaceans where the 79

behavioural patterns in response to stress condition are not yet well known. For example, 80

behavioural, physiological and biochemical adaptations have been identified in cave crayfish such 81

as, a decrease in locomotion and oxygen consumption, as well as a decrease in metabolic rates 82

after exposure to environmental stress (Caine, 1978). One possible avenue for evaluation of the 83

impact of an external stimulus is through the cardiac and respiratory systems. It is well known that 84

autonomic control of the respiratory and cardiovascular systems can regulate oxygen availability 85

and nutrients to specific target tissues needed for an impending behavioural response. On this 86

regard, Schapker et al., (2002) showed that crayfish rapidly alter heart rate (HR) and ventilatory 87

rate (VR) with changes in the environment and that HR and VR indicators were far more sensitive 88

than behavioural data alone. Moreover, in crayfish Bierbower (2010) used the tail flip response in 89

combination to HR and VR as bioindexes of the whole animal status to CO2 exposure as 90

environmental stressor. Specifically, the author observed a repellent/avoidance behaviour that 91

could be the result of avoiding the paralytic action resulting with CO2 exposure and a decrease 92

until cessation of HR and VR in correlation with CO2 increasing levels. In fish, Buscaino et al. 93

(2010) showed the relationship between behaviour and haematological parameters in relation to a 94

noise exposure. In particular, this short-term noise experiment showed an increase in motility and 95

glucidic metabolism of sea bream and sea bass. Hyperglycemia is a typical response of many 96

aquatic animals exposed to an external stress stimuli. In particular, in crustaceans increased 97

circulating crustacean Hyperglycemic Hormone (cHH) titres and hyperglycemia are reported to 98

occur following exposure to several environmental stressors (Durand et al., 2000; Lorenzon et al., 99

2002). Moreover, environmental stress seems to be an important factor for determining reduction 100

of immunocompetence with increasing prevalence of disease in crustaceans (Sinderman, 1979). 101

Several immune mechanism in Crustacea have been described and they derived essentially from 102

haemolymph cells (Destomieux et al., 1997). 103

Haemolymph cells play a central role in the immune mechanisms in Crustacea (Soderhall 104

and Smith, 1983; Hose and Martin, 1989; Hose et al., 1992; Smith and Chisholm, 1992; Clare and 105

Lumb, 1994; Destoumieux et al., 1997). Three cell types, hyaline, semigranular and granular cells, 106

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are commonly recognised in crustaceans (Bauchau, 1981; Tsing et al., 1989; Hose et al., 1990) 107

and are involved in coagulation, phagocytosis and the production of melanin by the 108

prophenoloxidase (proPO) system. Haemocytes are activated by microorganisms (Vargas-109

Albores, 1995; Vargas-Albores, et al. 1997) and are involved to eliminate foreign particles (Hose 110

and Martin, 1989; Bachère et al., 1995). The immune responses include the release of 111

peroxinectin, from the blood cells. This protein is involved in cell adhesion, degranulation, 112

opsonic, and peroxidase activity (Johansson et al., 1995). Others proteins, involved in defence 113

mechanisms are the stress proteins, also known as heat shock proteins (Hsps), are a highly 114

conserved class of proteins that show elevated expression during periods of stress in organisms as 115

phylogenetically divergent as bacteria and humans. Hsp70 is present at low levels in many cells 116

but is highly induced by stress, regardless of the stage of the cell cycle (Hang and Fox, 1996). In 117

decapod crustacean larvae, the elevation in Hsp70 expression was prolonged depending on the day 118

of pesticide exposure. This effect was directly related to the observed increase in mortality 119

(Snyder and Mulder, 2001). Liberge and Barthélémy (2007) showed that heat stress induced the 120

expression of Hsp70 and superoxide dismutase in the shell glands (structures involved in 121

reproduction) and, more particularly, during the formation of the diapause egg envelope in 122

Hemidiaptomus roubaui (Copepoda, Crustacea). The modulation of certain immunological and/or 123

physiological parameters in response to stressful conditions may serve as an important indicator of 124

health status (Perazzolo et al., 2002). 125

In this context, our understanding of crustacean immune mechanisms and the signals that 126

trigger haemolymph cells (Jiravanichpaisal et al., 2006) together with behavioural observation 127

could provide to monitor the effects of stress factors. In this study, we measured changes in 128

agonistic behaviour and haemolymph parameters in red swamp crayfish (Procambarus clarkii) 129

exposed to 30 minutes of an acoustic stimulus using the motility, the n. of tail flip, the n. of fights 130

and the analyses of total and differential haemocyte counts (THC and DHC, respectively), 131

glycaemic serum levels, total serum protein concentration (PC) and Hsp70 protein expression 132

levels. 133

134

MATERIALS AND METHODS 135

Collection and housing of animals 136

Thirty adult red swamp crayfish (Procambarus clarkii) (17 males and 13 females) weighing 26.1 137

± 9.3 g (mean ± SD) and measuring 9.4 ± 1.0 cm in total length and 4.7 ± 0.6 cm in carapace 138

length were used for this study. The crayfish were captured at the Preola and Gorghi Tondi 139

Natural Reserve (NW Sicily) and acclimated for one month at the Capo Granitola/CNR laboratory 140

(SW Sicily) in 2 shaded, outdoor PVC circular tanks (3.0 m in diameter, 1.0 m in depth) supplied 141

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with a thin layer of sand (1 cm deep). The temperature and salinity levels were monitored using a 142

multiparametric probe (556 MPS, YSI incorporated, USA) and kept constant at 24.02 ± 0.38°C 143

(mean ± SD) and 0.9 ± 0.01 ppt (mean ± SD), respectively, with a constant flow of water at a rate 144

of 25 ± 3.7 l min–1 (mean ± SD). The animals were fed pellets and frozen fish ad libitum. The P. 145

clarkii specimens were deprived of food for 2 days before the start of the experimental trials. All 146

animals were kept under natural photoperiods. 147

148

Rationale and experimental procedures 149

The crayfish were randomly collected from the holding tanks in groups of 5 individuals, assigned 150

to the control or test group and used in one experiment only. In total, 6 experimental trials (3 151

controls and 3 tests) were performed in two experimental tanks (control and test tanks) that lacked 152

shelter. 153

The animals, 5 control specimens and 5 test specimens, were simultaneously released into 154

the control and test tanks, respectively (Fig. 1). After a 1-h habituation period, we monitored and 155

video-recording the behaviour of the crayfish for two hours (1h = pre-experimental phase; 30 156

min= during-experimental phase; 30 min= post-experimental phase). In the during-experimental 157

phase individuals in the test groups were exposed to an acoustic stimulus for 30 min. Members of 158

the control group did not receive any stimuli. At the end of the post-experimental phase, both 159

control and stimulated animals were captured with a net and placed on crushed ice for 30 min to 160

induce torpor or “cold anaesthesia” to make sampling of the haemolymph. The samples were 161

immediately collected from five control and five experimental animals, and the crayfish then were 162

transferred into a small tank and released after recovery. This experimental procedure was 163

repeated three times. 164

165

Acoustic stimulus 166

Although the ability of the red swamp crayfish to perceive acoustic signals is unknown, this 167

species is able to generate wide-band pulse in air (Favaro et al. 2011) and in water (Buscaino et 168

al., 2012). Based on the idea that animals that produce acoustic signals may be able to perceive 169

said signals (e.g., for conspecific movements perception or communication), we decided to use a 170

stimulus with frequencies contained in both of the signals (air and aquatic environment) produced 171

by P. clarkii. The acoustic stimulus was therefore set to emit at a frequency band of 0.1-25 kHz. 172

Moreover, in the natural environmental, this band frequency is mainly produced by vessel traffic 173

(Sarà et al., 2007). 174

A 10-second linear sweep with a peak amplitude of 148 dB re 1 µPa rms at 12 kHz was 175

used to cover the selected frequency band (see Fig. 2). The linear sweep was repeated for 30 min 176

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without pause. The signals were generated by a waveform generator (Model 33220A, Agilent 177

Technologies, Santa Clara, CA, United States) connected to an underwater moving coil 178

loudspeaker (Model UW30, Lubell, Columbus, Ohio, USA) with a 100 Hz – 10 kHz-rated 179

frequency response. 180

The acoustic stimulus was recorded using a calibrated hydrophone (model 8104, Bruel & 181

Kjer, Nærum, Denmark) with a sensitivity of -205.6 dB re 1V/μPa ± 4.0 dB in the 0.1 Hz – 80 182

kHz frequency band. The hydrophone was connected to a digital acquisition card (USGH416HB, 183

Avisoft Bioacoustics, Berlin, Germany; set with a 40-dB gain) managed by dedicated Avisoft 184

Recorder USGH software (Avisoft Bioacoustics, Berlin, Germany). 185

The signals were acquired at 300 kilosamples per second at 16 bits and analysed by the 186

Avisoft-SASLab Pro software (Avisoft Bioacoustics, Berlin, Germany). The digital acquisition 187

card was calibrated with pure tone sine waves at different frequencies (1 and 20 kHz) and 188

different intensities (peak-to-peak 0.1 and 0.5 V) produced by a signal generator (AGILENT 189

33220, United States) using the SASLab Pro software. 190

191

Video monitoring system and analysis 192

To avoid disturbing the animals, we placed the equipment required for video monitoring and 193

recording in a laboratory located 5 m away from the tank. The video monitoring was carried out 194

using a low light cameras (Model CCD colour camera 1090/205, Urmet Domus SPA) placed 195

above the centre of the tanks for an overall view of the experimental space (Fig. 1). The signals 196

from the cameras were digitised and stored using a DAQ card (Model DV-RT4 Real Time, D-197

Vision) managed by custom-written software (Model DSE, D-Vision). 198

The video data were analysed in continuous mode. We identified the agonistic behavioural 199

events reported in other decapods (Buscaino et al., 2011a) and other Procambarus species 200

(Bergman and Moore, 2003; Buscaino et al., 2012): fight and tail flip. Moreover, we considered 201

“encounter” when a specimen approached another one without any threat display (Bergman and 202

Moore, 2003; Buscaino et al., 2012). 203

The fight was considered the approach between two or more specimens that continued in 204

series of agonistic activities including: a) the contact with chelae and progressing to pushing with 205

closed chelae, b) opened chelae used to grab an opponent until c) the most intense interaction in 206

which an individual appears to attempt to injure or injiure an opponent by grasping at chelae, legs, 207

or antennae (Bergman and Moore, 2003). The achievement of one or more of these behavioural 208

stages in continuous progression was considered as a single fight event. 209

The tail flip is a typical avoidance-behaviour event consisting in a rapid abdominal flexion 210

resulting in a new position away from the opponent and in crustaceans is highly associated with 211

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sound production (Buscaino et al., 2011a, b; Buscaino et al., 2012). 212

The total number of events (encounter, fight and tail flip) was counted each 6 minutes. 213

Moreover, because the number of encounters/fights could be influenced by the motility, at 214

the end of 6 minutes interval the number of specimen in movements (walking) or stopped (resting) 215

was counted. 216

The observers that analysed the video did not know if them were referred to the pre, during 217

or post experimental phase, neither control or acoustic treatment. 218

These events are behavioural indices that are useful for assessing intraspecific interactions 219

in decapods (Bergman and Moore, 2003). Variations in these events represent alterations of the 220

baseline activities. 221

222

Haemolymph analysis 223

Haemolymph sampling 224

One millilitre of haemolymph was drawn from the ventral sinus between the first and second 225

abdominal segments using a 2-ml syringe fitted with a 23-gauge needle. To delay or prevent 226

coagulation, the syringe was filled with an equal volume of anticoagulant. After the cellular 227

counts the samples were centrifuged at 800 g for 10 min at 4°C to obtain plasma serum and pellets 228

which were stored at -20°C for further use. 229

230

Total (THC) and differential (DHC) haemocyte counts 231

The total number of haemocytes per mm3 (THC) was determined using a Neubauer 232

haemocytometer chamber. Haemocytes were classified according to Lanz et al. (1993) using the 233

presence or absence of cytoplasmic granules as simple criteria. To perform the differential 234

haemocyte count (DHC, %), a small drop of haemolymph was smeared on a slide, fixed in 235

absolute methanol for 6 min, stained with diluted May-Grünwald-Giemsa (3 min in 10-fold 236

diluted May-Grünwald and 10 min in 10-fold diluted Giemsa), dehydrated with absolute ethanol 237

(1 min) and xilene (6 min) and then mounted in permount. Cells were counted in random areas on 238

each slide, and the relative proportions of various classes were computed (Mahmood and Yousaf, 239

1985). A total of 200 cells were counted on each slide. DHCs were calculated using the following 240

equation: 241

DHC (%) = number of different haemocyte cell typestotal haemocyte cells counted

x 100DHC (%) = number of different haemocyte cell typestotal haemocyte cells counted

x 100 242

243

Scanning electron microscopy (SEM) 244

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Haemolymph samples mixed with anticoagulant were dropped directly onto a coverslip pretreated 245

with 0.1% poly-l-lysine. The adherent monolayer was fixed in cacodylate buffer (0.1 M, pH 7.3) 246

containing 2.5% glutaraldehyde, post-fixed in osmium tetroxide (1%), dehydrated in a graded 247

alcohol series and dried at the critical point. The samples were mounted on stubs, gold-coated in a 248

sputter coater and observed by SEM (LEO 420). 249

250

Glucose, osmolarity, and protein assessment 251

Glucose levels were measured with the Accutrend GC kit (Boehringer Mannheim, Germany). 252

Osmolarity was estimated with an osmometer (Roebling, MESSTECHNIK, Berlin, Germany). 253

The total protein concentration of the crayfish haemolymph serum plasma was estimated using the 254

Bradford method (1976). Bovine serum albumin was used like protein standard. 255

256

Haemagglutination assay 257

The haemagglutinating activity (HA) of two-fold diluted samples was assayed in a 96-well 258

microtitre U plate containing a 1% rabbit red blood cells (RRBC) or sheep red blood cells (SRBC) 259

suspension in PBS (PBS-E: 6 mM KH2PO4, 0.11 mM Na2HPO4, 30 mM NaCl, pH 7.4). 260

Erythrocytes were supplied by the Istituto Zooprofilattico della Sicilia (Palermo, Italy) and 261

maintained in sterile Alsever’s solution (27 mM sodium citrate, 115 mM D-glucose, 18 mM 262

EDTA, and 336 mM NaCl in distilled water, pH 7.2). Tris-buffered saline (TBS; see below) 263

enriched with 1% RRBC and SRBC with 0.1% (w/v) gelatin was used as the reaction medium. 264

Twenty-five microlitres of plasma were mixed with an equal volume of RRBC or SRBC 265

suspension and incubated at 37°C for 1 h. Divalent cation requirements were estimated by adding 266

CaCl2 or MgCl2 to the reaction medium, up to a final concentration of 5-10 mM. The titre of the 267

haemagglutinating activity (HT) was expressed as the reciprocal of the highest dilution showing a 268

positive score for agglutination. 269

TBS was used in place of plasma for the negative controls. Each assay was performed in 270

duplicate using serum samples from different specimen preparations. The HA titre was expressed 271

as the average of the recorded values. 272

273

Haemocyte homogenate supernatant preparation (THS) 274

Cells were crushed on ice for 1 h in 1 ml of lysis buffer (RIPA: 0.5% sodium deoxycholate 275

(minimum 97%); 1% NP40; 0.1% SDS with PBS-T (1 M Na2HPO4, 1 M NaH2PO4, 1.5 M NaCl, 276

and 0.1% Tween-20, pH 7.5, supplemented with a cocktail of protease inhibitors: 2 µg/µl antipain, 277

leupeptin and bestatin, 1 µg/µl aprotinin and pepstatin, 1 mM benzamidine, and 0.1 mM AEBSF). 278

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The samples were then centrifuged at 15000 g for 30 min at 4°C. The supernatants were 279

collected and dialysed against 50 mM Trizma base (Tris [hydroxymethyl] aminomethane), pH 7.5, 280

and the protein contents were estimated. 281

282

283

SDS-PAGE and Western blot 284

The equivalent of 25 µg of total lysates for each sample was separated on 7.5% SDS-PAGE under 285

reducing conditions according to the Laemmli method (1970). SDS-polyacrylamide minigels were 286

transferred to nitrocellulose membranes using a semidry transfer apparatus (BioRad) and blocked 287

with 5% bovine serum albumin (BSA) in TBS-T (20 mM Trizma base, pH 7.5, 300 mM NaCl, 288

0.1% (v/v) Tween-20 with 0.02% sodium azide) for 1 h at room temperature (r.t.). According to 289

Celi et al. (2012), the membrane was incubated over night at 4°C with the primary antibody 290

(monoclonal anti-heat shock protein 70 antibody produced in mouse, Sigma Aldrich; 1:800 291

dilution), washed with TBS-T (three times for 5 min each), and incubated with alkaline 292

phosphatase-conjugated goat anti-mouse IgG (1:7500 for 1 h at r.t.). After washing with TBS-T 293

(three times for 5 min each), the membranes were incubated with the 5-bromo-4-chloro-3-indolyl 294

phosphate/nitro blue tetrazolium liquid substrate system (BCIP/NBT). The Alpha Imager software 295

was used for densitometric analysis of the immunoblotted bands. 296

Five specimens from each experimental group (control and test groups, 30 samples in 297

total) were examined, and each test was repeated in triplicate. 298

299

Statistical analysis 300

Because the behavioural data were not normally distributed, the Mann-Whitney U test was used to 301

compare the “encounters”, “fight” and “tail flip” events between control and test groups as well as 302

among pre-, during- and post-experimental phases. An unpaired t-test was used to determine 303

significant differences in plasma glucose, total protein, THC, DHC and Hsp70 expression levels. 304

305

RESULTS 306

Behavioural events 307

A total of 836 behavioural events were recorded during the 6 experimental trials, of which 379 308

were encounters, 380 were fights and 77 were tail flips. No significant differences in behavioural 309

responses were observed between control and test groups in the pre- or post-experimental phases 310

(Fig. 3). Conversely, in during-experimental phase, significant differences in the numbers of 311

encounters, fights and tail flip events were observed between the control and test groups (P < 312

0.05; Fig. 3). In particular, during the acoustic stimulus, the test crayfish exhibited a lower number 313

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of encounters, fights and tail flips as compared with the control animals. No significant 314

differences were found in the motility in during-experimental phase between control and test 315

group, as well as in the ratio of the motility during/pre/post experimental phase for the control and 316

test groups (P > 0.05). 317

318

319

Microscopic observations of circulating haemocytes 320

The haemocyte monolayers were comprised of flattened and well-spread cells and contained three 321

morphologically distinct cell types that could be differentiated by the presence and size of 322

granules. Three types of circulating haemocytes were identified by light and SEM. Granulocytes 323

are ovoid or fusiform and contain large acidophilic granules (Fig. 4a) that give a rough texture to 324

the cell surface (Fig. 4b). Semigranulocytes can be fusiform and generally display a smooth shape 325

(Fig. 4d). These cells contain small cytoplasmic granules that are typically eosinophilic (Fig. 4c). 326

Hyaline cells appear ovoid or fusiform in shape and are characterised by the absence of 327

cytoplasmic granules (Fig. 4e) and a smooth surface (Fig. 4f). 328

329

THC and DHC 330

The number of circulating haemocytes (total haemocyte count, THC) was approximately 4.7x106 331

± 0.4x105 cells ml-1. Hyalinocytes (H) represented approximately 20 ± 2.4% of the total 332

circulating haemocytes, whereas semigranulocytes accounted for 22.5 ± 3.3%, and granulocytes 333

for 57.5 ± 2.2%. 334

Acoustic stimuli significantly affected both THC and DHC. Following the 30-min acoustic 335

stimulus, the THC of the stressed crayfish decreased by approximately 50% relative to the initial 336

count (P < 0.001) (Table1). A different pattern was observed for the DHC. In tested crayfish, a 337

significant increase in hyaline cell number (from 20% to 58%, P < 0.001) was accompanied by 338

significant decreases in the relative proportions of granular and semigranular cells (P < 0.001 and 339

P < 0.01, respectively) relative to the values determined for the control group (Table 1). 340

341

Serological parameters 342

Glucose levels were significantly higher (575 ± 34 mg/dl, P < 0.01) in the test group than the 343

control group (Table 2). However, no differences in osmolarity or total protein content were 344

observed between groups (Table 2). 345

346

Haemagglutination titre 347

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The serum samples from the control crayfish agglutinated both RRBC and SRBC. The highest 348

titre was found with sheep SRBC (7.2 ± 1.66), whereas the RRBC yielded a score of 6.3 ± 2.1. 349

The HA of the serum from stressed specimens was decreased by approximately 50% (P < 0.01) 350

(Table 2). 351

352

Hsp70 protein expression after treatment 353

As shown in Fig. 5A, the anti-mouse Hsp70 mAb cross-reacted with a 70-kDa band in circulating 354

haemocytes from both the control and test groups. A densitometric analysis of Hsp70 protein 355

levels (Fig. 5B) revealed a significant increase in expression in the haemocytes collected from 356

stressed animals. Hsp70 expression peaked (3-fold higher than the untreated samples) after the 357

period of acoustic stimuli. 358

359

360

DISCUSSION 361

This study showed that an acoustic stimulus can reduce the agonistic behaviour of the crayfish 362

Procambarus clarkii, as demonstrated by the significantly reduced numbers of both fights and tail 363

flip events. According to Bergman & Moore (2003), P. clarkii crayfish engage in agonistic 364

interactions with high frequency to establish dominance relationships that regulate access to 365

resources. Similarly, Capelli and Hamilton (1984) have shown that in a laboratory environment, 366

food and shelter affect the agonistic behaviour of the crayfish Orconectes rusticus. In particular, 367

aggressive activity decreases with the increased availability of both shelter and food. To avoid 368

these effects, we observed crayfish held in tanks without shelter and deprived of food for two days 369

before the experimental trials. 370

In the during-experimental phase, although the motility of specimens of test group was lower, we 371

did not observe a significant reduction in comparison to the specimens of the control group. 372

Otherwise, a significant lower number of encounters of test group were observed in the during-373

experimental phase. Similarly to this result, the acoustic stimulus induced a decrease in the natural 374

aggressive activity also (number of fights and tail flip events) of the crayfish. Accordingly, when 375

the acoustic stimulus was interrupted (post-experimental phase), an increase in aggressive 376

agonistic behaviour was observed. 377

Our results indicate that P. clarkii could perceive all or part of the acoustic stimuli used in 378

this study (0.1-25 kHz bandwidth) within the wider bandwidth of their underwater acoustic 379

emissions (Buscaino et al., 2012). However, no data on the anatomical-functional structures with 380

which they detect acoustic energy (such as variation in pressure) are currently available. The 381

sensitivity of aquatic decapods to particle displacement and hydrodynamic stimulation is poor 382

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compared to other aquatic organism like fishes (Breithaupt and Tautz, 1990; Goodall et al., 1990; 383

Popper et al., 2001). From our study is not possible to discern the quantitative and qualitative 384

receptors involved to the perception of the variation of pressure or particle water movement or if 385

the sound produced physical effects on entire animal body without stimulating a specific receptor. 386

Lobster and crayfish primarily respond to hydrodynamic stimulation (behavioural study) than 387

pressure (Goodall et al., 1990; Popper et al., 2001). However, these studies focused on acoustic 388

frequencies lower (20-180 Hz) than our study (0.1-25 kHz). 389

In crustaceans, mechanoreceptors are located in cuticular extensions of the exoskeleton 390

and are called sensilla (Ali, 1987). Decapod mechanoreceptors include setae (hair-like cells), 391

chordotonal organs, and internal statocysts (Popper et al., 2001). In macruran decapods (crayfish), 392

the hairs are sensitive to vibration (Breithaupt and Tautz, 1990) and can respond to frequencies up 393

to 100 Hz. In P. clarkii, the antennules (lateral plus medial flagella) possess both chemosensory 394

setae and mechanosensory setae. The latter respond to hydrodynamic stimuli up to 100 Hz 395

(Breithaupt and Tautz, 1990). In particular, the medial flagellum functions as a hydrodynamic 396

receptor (Horner et al., 2008; Monteclaro et al., 2010). In our study, the animals didn’t show any 397

preference in the choice of the tank’s side to take up (near or far from the underwater loudspeaker) 398

in the during-experimental phase. Otherwise, is possible that the reverberation/reflection effects of 399

the sound inside the tank make animals unable to detect the sound source, inhibiting them to 400

perform the avoidance behaviour. Moreover, the agonistic behaviours were reduced during the 401

stimulation probably in consequence of the impact of an external stress condition that may inhibit 402

aggressive state as a result of a preservation instinct. A suggestion could be that crayfishes give 403

the priority to the external stimuli (considered as a stress source and confirmed by the increase of 404

glycaemic and Hsp70 levels and decrease of THC) respect other baseline agonistic behaviours. 405

In marine shrimps and crabs (Christian et al., 2003; Andriguetto-Filho et al., 2005), 406

exposure to stronger acoustic stimuli (air guns) produced no obvious effects on behaviour or 407

biochemical parameters (serum proteins, serum enzymes, calcium, and haemocyte types). 408

Otherwise, Payne et al. (2007) observed sub-lethal effect as well as feeding rate on lobsters 409

exposed to air gun. The discrepant findings of these prior studies and the present investigation 410

suggest that the effects of acoustic stimuli are perceived differently under different environmental 411

conditions (e.g. tank or natural environment, distance from the acoustic source) and acoustic 412

typologies (e.g. source level, frequency, duration) or even between different crustacean species. In 413

fish, Santulli et al. (1999) reported that exposure to air gun blasts affected biochemical parameters 414

(cortisol, glucose, lactate, AMP, ADP, ATP, and cAMP) in sea bass, and Buscaino et al. (2010) 415

showed that the exposure of sea bass and gilthead sea bream to a 0.1-1 kHz linear sweep (150 416

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dBrms re 1 µPa) caused a significant increase in motility that influenced haematological 417

parameters. 418

In aquatic crustaceans, various types of stress, including hypoxia (Le Moullac et al., 1997, 419

1998), low salinity (Perazzolo et al., 2002), viral infection and administration of an 420

immunostimulant (Hennig et al., 1998; Sritunyalucksana et al., 1999), affect haematological 421

parameters. In aquatic animals, hyperglycaemia is a typical stress response to harmful physical 422

and chemical environmental changes, including hypoxia and exposure to air during commercial 423

transport (Spicer et al., 1990; Zou et al., 1996; Kuo and Yang, 1999; Morris and Oliver 1999; 424

Durand et al., 2000; Speed et al., 2001). Hyperglycaemia has been associated with increased 425

circulating crustacean hyperglycaemic hormone (CHH) titres (Lorenzon et al., 1997, 2002; 426

Durand et al., 2000; Santos et al., 2001) and has been used as an index to assess CHH activity and 427

environmental stress (Webster, 1996; Bergmann et al., 2001; Toullec et al., 2002). Accordingly, in 428

P. clarkii, acoustic stress led to a significant increase (P < 0.01) in haemolymph glucose levels. 429

However, exposure of P. clarkii to an acoustic stimulus no significant effects on internal 430

osmoregulatory capacity and on total serum protein concentration (PC). In aquatic crustaceans and 431

particularly in decapods, the organs of the branchial chambers are the primary source of the 432

osmotic and ionic regulation (Péqueux, 1995). Exposure to environmental stressors and 433

pathological agents on osmoregulation usually, in crutacean, results in a decrease of its Na+ and 434

Cl- regulation. The partial or complete loss of osmoregulatory and ionoregulatory capacity is 435

generally linked to distruptions of the osmotic and ionic regulations. Studies on crustacean 436

responses to various environmental stressors revealed that the effect of stress upon osmotic and 437

ionic metabolism was time and dose-dependent (Charmantier et al., 1989; Charmantier and Soyez, 438

1994; Lignot et al., 2000). 439

Changes in the protein composition of haemolymph has been used like a stress indicator to 440

monitor shrimp health status and exposure to environmental stress (Chen et al., 1994; Chen and 441

Cheng, 1995) seem to depend from certain physiological and environmental variables (Bursey and 442

Lane, 1971; Chen and Cheng, 1993; Chen et al., 1994; Chen and Cheng, 1995), sex and animal 443

size (Chen and Cheng, 1993). 444

Naturally occurring agglutinins, including those with erythrocyte targets 445

(haemagglutinins), are involved in innate immunity in invertebrates (reviewed in Marques and 446

Barracco, 2000). In our study, acoustic stress induced a significant decrease in the agglutinating 447

titre of P. clarkii serum, as assayed with sheep and rabbit erythrocytes. Further analyses using 448

sugar inhibition might elucidate whether the serum haemagglutinins are lectins (Sharon, 2007). 449

In crustaceans, haemocytes are involved in organismal homeostasis and manage several 450

immune functions, including coagulation, phagocytosis, degranulation, opsonisation and 451

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production of melanin by the prophenoloxidase (proPO) system (Vargas-Albores, 1995; Vargas-452

Albores et al., 1997). According to Lanz et al. (1993), P. clarkii granular and semigranular 453

haemocytes may participate in the proPO system as well as phagocytic or cytotoxic functions. 454

THCs and DHCs have been used to assess crustacean health and the effects of stressful 455

conditions (Jussila et al., 1997). Decreases in the THC under stressful conditions have been 456

reported for several marine crustacean species (Smith et al., 1995; Hennig et al., 1998; Le Moullac 457

et al., 1998; Sánchez et al., 2001). Similarly, under acoustic stimuli, the THCs of our P. clarkii 458

specimens were reduced, suggesting the possibility of immune depletion as well as an increased 459

risk of infection. Moreover, acoustic stimuli also altered the DHC (relative proportions of HC, 460

SGC and SGC in the THC). Although the response of the DHC to different stressors is not well 461

understood, it has been used as a stress indicator in crustaceans (Jussila et al., 1997; Johansson et 462

al., 2000). Acoustic stimuli resulted in an increase in the relative number of hyaline cells and 463

decreases in semigranulocytes and granulocytes. These results are similar to those reported in the 464

literature (Jussila et al., 1997; Fotedar et al., 2001, 2006), where the proportions of granulocytes 465

and semigranulocytes were lower in moribund lobsters than healthy individuals (Bauchau, 1981; 466

Sequeira et al., 1995). 467

Stress proteins, also known as heat shock proteins (Hsps), are a highly conserved class of 468

proteins that show elevated transcription during periods of stress in organisms as phylogenetically 469

divergent as bacteria and humans. These proteins have been shown to play numerous important 470

roles in maintaining organismal health, e.g., in the host responses to environmental pollutants and 471

food toxins as well as the development of inflammation. In shrimp, Hsps are involved in the 472

specific and non-specific immune responses to bacterial and viral infections (Roberts et al., 2010). 473

In particular, Hsp70 acts to repair damage to proteins following acute stress and thus plays a key 474

role in cytoprotection (Feder and Hofmann, 1999). 475

In crustaceans, Hsp70 expression serves as a good bioindicator of stressful conditions, 476

including pesticide exposure and heat stress (Snyder and Mulder, 2001; Chang, 2005; Liberge and 477

Barthélémy, 2007). Little is known about the effects of noise on Hsp expression. Wu et al. (2001) 478

showed that Hsp70 expression increased after exposure to a stressful noise in humans, and 479

Hokestra et al. (1998) reported that the expression of Hsp70 (but not Hsp30, Hsp60, or Hsp90) is 480

increased in birds after exposure to a loud noise. We have also recently detected increased Hsp70 481

expression in fish Chromis chromis after exposure to sounds similar to those resulting from 482

human activities (Celi et al., unpublished). In the present study, we show for the first time that 483

acoustic stimuli induce Hsp70 overexpression in P. clarkii haemocytes as expression of a stress 484

status. 485

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In conclusion, exposure to acoustic stimuli altered certain aggressive behavioural patterns 486

and components of the haemato-immunological system of P. clarkii. Among the assessed 487

haemato-immunological parameters, the serum glucose concentration, PC, agglutinating activity, 488

THC, DHC and Hsp70 expression are the most promising parameters reflecting stress status in 489

crayfish. The haemato-immunological responses to the stressful conditions occur in conjunction 490

with behavioural changes. 491

In most natural aquatic environments, the soundscape has been permanently altered due to 492

anthropogenic activities (e.g., traffic vessels, wind turbines, electroacoustic instruments for 493

exploration and navigation), and the impact on aquatic organisms should be investigated over 494

brief, medium and long-term exposure periods (Payne et al., 2007). 495

However, in according with Goodall et al. (1990), further studies should be also performed 496

in a open controlled natural environment (where acoustical field is not influenced by walls such as 497

in the small tanks) to increase the information about the effects of noise on the behavioural and 498

physiological response. 499

500

ACKNOWLEDGEMENTS 501 502 This work was supported by Grants to VM ex 60% from University of Palermo and by the project 503

BIOforIU - PONa3_00025 and RITMARE of IAMC-CNR UO Capo Granitola. 504

505

506

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Liberge, M., Barthélémy, R.M. (2007). Localization of metallothionein, heat shock protein 659

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Lorenzon, S., Pasqual, P., Ferrero, E. A. (2002). Different bacterial lipolysaccharides as 668

toxicants and stressors in the shrimp Palaemon elegans. Fish Shellfish Immun. 13, 27-45. 669

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McMahon, B. R., Stuart, S. A. (1999). Haemolymph gas exchange and ionic and acid-based 674

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hemato-immunological parameters in the shrimp Farfantepenaeus paulensis submitted to 691

environmental and physiological stress. Aquaculture 214, 19–33. 692

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on blood glucose regulation in two decapod species. Braz. J. Med. Biol. Res. 34, 75- 80. 702

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Biology of freshwater crayfish. Oxford: Blackwell Science. 236–257. 742

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comparison of b-1,3-glucan binding protein from the white shrimp Penaeus Iannamei. 751

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crab Cancer pagurus during emersion stress. J. Exp. Biol. 199, 1579–1585. 754

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Decapoda). Comp. Biochem. Physiol. A 114, 105-109. 760

761

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Legends 762

763

764

Fig. 1. Schematic representation of experimental tanks equipped with an underwater loudspeaker 765

and a video camera placed above the centre of the tank. In the control tank, the acoustic stimuli 766

were not emitted by the loudspeaker. 767

768

Fig. 2. Oscillogram (top), spectrogram (middle) and power spectrum (bottom) of the linear sweep 769

emitted 1 m from hydrophone. Oscillogram: pressure (Pascal) vs. time (second). Spectrogram: 770

frequency (kHz) vs. time (s). The intensity is reflected by the gray scale. dB re 1 µParms, 1024-771

sample FlatTop window. Power spectrum: dB re 1 µPa rms (time window 10 sec) vs. frequency (k 772

Hertz). 773

774

Fig. 3. Significant differences in the numbers of behavioural events were only observed between 775

control and test groups during the acoustic stimulus period. Number of fights (A); number of tail 776

flips (B); number of specimens in movement (C); number of encounters (D). Data are expressed 777

as the mean ± SD. Pre = the hour preceding the acoustic stimulus; During = during the acoustic 778

stimulus (30 min) given to the test groups (no acoustic stimulus was administered to the control 779

groups); Post = the period immediately following the stimulus (30 min). 780

781

Fig. 4. Circulating haemocytes from Procambarus clarkii. Light (a, c, e; May-Grϋnwald-Giemsa) 782

and scanning electron microscopy results (b, d, f). (a, b) Granulocytes; (c, d) semigranulocytes; (e, 783

f) hyaline cells. Bars: (a, b) 6 µm; (c, d) 5 µm; (e, f) 3 µm. 784

785

Fig. 5 Effect of the acoustic stimuli on expression levels of the protein Hsp70 in Procambarus 786

clarkii. A: representative western blot of Hsp70 levels in 3 specimens from each group (one for 787

each experimental trial). B: Integrated optical density histogram (IDV) of the Hsp70 protein 788

bands. The data represent the means ± SD (n=15 control and n=15 test specimens). 789

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Table 1. Total haemocyte count (THC) and differential haemocyte count (DHC) in the 1

haemolymph of the control and test groups. The data represent the means ± SD (n=15 control and 2

n=15 test specimens). Significant differences between the control and test groups (acoustic 3

stimulus) are shown (** P < 0.01; *** P <0.001). 4

5 6

7

8

Collection THC Granulocytes

(%)

Semigranulocytes

(%)

Hyaline

(%)

Control Group 4.7x106±0.4x105 57.5±2.2 22.5±3.3 20±2.4

Tests Group 2.2x106±0.5x105*** 30±1.4*** 11.3±0.3** 58±6.2***

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Table 2. Plasma glucose, osmolarity, total protein levels and haemagglutination titre in the 1

haemolymph of Procambarus clarkii exposed to an acoustic stimulus. The data represent the 2

means ± SD (n=15 control and n=15 test specimens). Significant differences between the control 3

and test groups (acoustic stimulus) are shown (** P< 0.01). 4

5

6

7

8

9

10

11

Collection Glucose

(mg/dl)

Osmolarity

(mOsm/kg)

Total protein

(µg/µl)

Haemagglutination titre

RRBC SRBC

Control Group 468±19 400±16 1.18±0.1 6.3±2.1 7.2±1.6

Tests Group 575±34** 395±19 0.95±0.2 3.1±1.0** 3.3±0.9**

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