Comparative Biochemistry and Physiology, Part C 152 (2010) 433–443

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Comparative Biochemistry and Physiology, Part C

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Genotoxic, physiological and immunological effects caused by temperature increase,air exposure or food deprivation in freshwater crayfish Astacus leptodactylus

Olga Malev a, Maja Šrut b, Ivana Maguire b, Anamaria Štambuk b, Enrico A. Ferrero c,Simonetta Lorenzon d, Göran I.V. Klobučar b,⁎a School of Environmental Sciences, University of Nova Gorica, Vipavska 13, PO Box 301, Rožna Dolina, 5000 Nova Gorica, Sloveniab Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatiac Department of Life Science, University of Trieste, via Giorgieri 7, I-34127 Trieste, Italyd Department of Biological Oceanography, National Institute of Oceanography and Applied Geophysics, via A. Piccard 54, I-34151 S. Croce, Trieste, Italy

⁎ Corresponding author. Tel.: +385 1 4877700; fax: +E-mail address: gklobuca@zg.biol.pmf.hr (G.I.V. Klob

1532-0456/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.cbpc.2010.07.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 January 2010Received in revised form 15 June 2010Accepted 14 July 2010Available online 24 July 2010

Keywords:Astacus leptodactylusComet assayHaemolymphMicronucleus testStress

The aim of this research was to investigate influence of different environmental stressors, such astemperature increase, air exposure and food deprivation on DNA integrity of a bioindicator species,freshwater crayfish Astacus leptodactylus. DNA damage was measured in crayfish haemocytes using Cometassay and micronucleus test. Crayfish haemolymph was subsequentially sampled during their 7 days ofexposure to increased temperatures (25 and 30 °C) and during 24 h of air exposure. Both groups were alsomonitored through the following 7 days of recovery period. Food deprived crayfish were monitored over aperiod of 2 weeks. Alterations of measured physiological and immunological haemolymph parameters (THC,lactate, glucose and protein concentration) indicated stress response in exposed crayfish. However, only thestress induced by increased temperature significantly increased DNA damage in freshwater crayfish whilefood deprivation or air exposure did not cause a significant genotoxic effect.

385 1 4826260.učar).

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1. Introduction

The action of different environmental stressors, such as temper-ature change, air exposure, or food shortagemust be consideredwhenevaluating animals' response to pollution, as they can either adverselyaffect the physiological status and survival of the bioindicator species,or enhance their susceptibility to environmental pollutants (Morrisand Oliver, 1999). Genotoxicity continuously gains its importance inpollution biomonitoring programs considering the possible conse-quences it can have on organisms and populations inhabitingcontaminated environments (Belfiore and Anderson, 2001; Jha,2004). It is therefore important to resolve the magnitude of the effectthat environmental stressors can have on measurements of genotoxi-city biomarkers, such as Comet assay and micronucleus test, in orderto be aware of their possible contribution to the effects induced bychemical pollutants (Buschini et al., 2003).

Decapod crustaceans are particularly suitable for pollutionbiomonitoring programs as they are widely distributed and relativelyeasy to collect, they are mostly solitary bottom dwellers, which keepmuch of their bodies in contact with surrounding objects and tend toaccumulate pollutants in their tissues (Khan et al., 1995; Schildermanet al., 1999). Freshwater crayfish have often been used as model

organisms in ecotoxicological research (Benli et al., 2007; Wiggintonand Birge, 2007), however, there are lack of data on their use asbioindicator species. For this research the narrow-clawed crayfishAstacus leptodactylus has been chosen as a good representative of thefreshwater crayfish fauna. This species is mostly distributed in largerrivers of Eurasia and has not been considered threatened orendangered (IUCN, 2009).

It is believed that animals are stressed when a factor, stressor,causes their internal physiology to deviate from normal. Stressinduced by changes in environmental parameters: air exposure, fooddeprivation etc. requires homeostatic regulation that brings beha-vioural and physiological alterations in aquatic animals (Lorenzon,2005; Ridgway et al., 2006). Heat shock is known to influence DNAintegrity of fish Carassius auratus (Anitha et al., 2000), musselDreissena polymorpha (Buschini et al., 2003), and mice (Asanami andShimono, 1997). It has also been observed that prolonged exposure tohypoxic conditions can induce increased levels of DNA strand breaks inhumans (Møller et al., 2001; Lundby et al., 2003), while fooddeprivation caused significant increase in DNA damage in blood cellsof rats (de Souza Rocha et al., 2008) and in various internal organs ofmice (Higashimoto et al., 2009). Furthermore, there are exampleswhere these abiotic factors have increased genotoxicity of investigatedchemicals (Buschini et al., 2003).

Stress responses in crustaceans have been evaluated subjectively(behaviour, vigour, simple postural test) or expressed quantitativelyby measuring changes in physiological variables such as oxygen

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uptake, haemolymph characteristics and composition (glucose,lactate, total protein), hormones, and ions (Paterson and Spanoghe,1997; Lorenzon et al., 2007, 2008). In particular, glucose concentra-tion in crustaceans' haemolymph rises in response to a numberof stresses such as handling (Bergmann et al., 2001), emersion (Morrisand Oliver, 1999; Speed et al., 2001), disease and pollutants (Lorenzonet al., 1997, 2000). Lactate can accumulate in the haemolymphduring emersion and hypoxia (Durand et al., 2000; Lorenzon et al.,2008). Other hemato-immunological parameters involved inthe stress response following several environmental stressors arechanges in the total haemocyte count (THC), and total proteinconcentration (Jussila et al., 1999; Lorenzon et al., 1999; Perazzoloet al., 2002).

The Comet assay is a technique that has become one of the majortools for genotoxicity monitoring of the aquatic environments,specifically in invertebrates (Jha, 2004). It is a simple, sensitive andrapid technique for detection of DNA damage (single–double-strandbreaks, alkali labile sites or DNA–DNA and DNA–protein cross links) inindividual cells (Fairbairn et al., 1995), and therefore can be veryuseful in studies of genetic toxicology, especially eco-genotoxicology(Cotelle and Férard, 1999). The micronucleus test has been success-fully used for genotoxicity testing of environmental pollutants anddifferent chemicals since the late 1980s (Müller and Streffer, 1994;Evans, 1997). It detects both structural and numerical chromosomeaberrations. Micronuclei (MN) are small intracytoplasmic masses ofchromatin resulting from either chromosomal breakage during celldivision or chromosomes that are lagging in anaphase (Kirsch-Volderset al., 2000).

The main objective of this work was to evaluate the influence ofdifferent abiotic and biotic factors: increased temperature, airexposure and food deprivation on the DNA integrity of freshwatercrayfish A. leptodactylus by the means of Comet assay and micronu-cleus test. Furthermore, a battery of physiological and immunologicalparameters (haemolymph lactate, glucose, proteins and THC) wasused to evaluate the stress response of A. leptodactylus.

2. Materials and methods

2.1. Crayfish capture and maintenance

Specimens of narrow-clawed crayfish A. leptodactylus used in theexperiments were trapped with LiNi traps, specific cylindrical trapswith a tapering funnel at both ends (Maguire, 2002). The animalswere collected in the Mrežnica River (non polluted site), near thetown of Karlovac (45°29″N, 15°33″E) in the central Croatia betweenMarch and June 2007 and in March 2010. After capture, crayfish weretransported to the laboratory of the Department of Zoology,University of Zagreb where they were kept in 60 L flow-throughglass aquaria receiving filtered and thoroughly aerated water (19±1 °C), with a natural photoperiod condition.

Broken clay pots were placed as shelters at the bottom of theaquaria to reduce possible stress by mimicking the natural environ-ment. Crayfish were acclimatized in the aquaria for at least 2 weeksbefore the experiment and fed ad libitum every second day using foodtablets for bottom-feeding invertebrates (Hagen Nutrafin). Fifty onehard-shelled and apparently healthy crayfish both females and males(36.6±13.2 g live mass, 10.5±1.4 cm total body length) wereselected for the experiments.

2.2. Experimental design

Tests with four different conditions were conducted on groups of7–8 crayfish for each exposure. Control individuals (7-9 crayfish),were maintained in water (19±1 °C) in the glass aquaria throughoutthe experiment and sampled in the same intervals as the exposed

ones. All experimental groups except the food-deprived group werefed ad libitum every second day using food tablets.

2.2.1. Water temperature increasePrior to the exposure, the crayfish were acclimatized to experi-

mental conditions by increasing water temperature progressively(approximately 2 °C per day) in the aquaria to 25±1 °C and 30±1 °C.Water temperatures in the experimental tanks were maintained atconstant values using electronic thermostats. The crayfish were keptfor 7 days at the two temperatures and for the following 7 days theywere kept at temperature of 19±1 °C (recovery period) in 60 Laquaria. In every experiment, one third of the water (of appropriatetemperature) was renewed every 48 h. The specimens of each groupwere sampled at days 1, 3, 5 and 7 during the exposure period (25 °Cand 30 °C) and at days 1, 3 and 7 during the recovery period (19 °C).There was an additional sampling at the beginning of the experimentfor physiological and haemato-immunological parameters.

2.2.2. Air exposureCrayfish were taken from the aquaria, transferred into 2 polystyrene

boxes (4 crayfish per box) and exposed to atmospheric air for a period of24 h. When placed in air, the gills collapse and the crayfish suffercontinuously from systemic hypoxia and hypercapnia (Jackson et al.,2001; McMahon, 2001). Each box was lined with filter-paper soaked inwater to ensure themaintenance of relative humidity and kept at roomtemperature (19 °C). Animals were monitored by haemolymph bleed-ing at the beginning of the air exposure (0 h), at 2 h, 6 h, and 24 h afterthe emersion, and 1, 3, 5, and 7 days after re-immersion in the aquariawith the water at a temperature of 19 °C (recovery period). Only forphysiological and haemato-immunological parameters was there anadditional sampling at time 0 h.

2.2.3. Food deprivationAfter acclimatization, 8 crayfish were starved for 14 days. Animals

were monitored, by haemolymph bleeding at 1, 3, 5, 7 and 14 days offood deprivation. Also in this case, only for physiological andhaemato-immunological parameters was there an additional sam-pling at time 0 h.

2.3. Haemolymph sampling

Before the experiments, animals were marked for individualrecognition to allow sequential sampling of the haemolymph duringthe experimental period. Animals were blotted dry and haemolymphwas withdrawn from the pericardial sinus with a sterile 1 mL syringefitted with a 25G needle. Animals were bled ~600 μL haemolympheach time. The first 500 μL was distributed in different fractions fordetermination of haemolymph parameters: for Comet assay 100 μL ofhaemolymph with 50 μL anticoagulant solution (0.49 M NaCl, 30 mMtrisodium citrate, 10 mM EDTA; pH 6) (Lucić, 2004), 100 μL formicronucleus test, 300 μL haemolymph was centrifuged for 1 min at10,300×g and the plasma fraction was quickly frozen at −20 °C andstored until required for measurements of physiological parameters.Additional 100 μL of haemolymph was withdrawn for determinationof total haemocyte count (THC) with a sterile 1 mL syringe fitted witha 25G needle and filled with 900 μL of 1% formalin.

2.4. Determination of haemolymph parameters

2.4.1. Physiological parametersGlucose content was quantified with a reflectometer, One Touch®

II Meter (Lifescan, Milpitas, CA, USA) and commercial kit test strips(precision strip ±3% coefficient of variation in the tested range).

Lactate concentration of the haemolymph was obtained usingCDR-Lact photometer (CDR-Mediared, Florence, Italy) with reagentsin prefilled cuvettes.

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Total protein concentration was determined using Screen point(Hospitex Diagnostics srl. Florence Italy) amultichannel (420–590 nm),photometric reading system based on the Lowry method, clinicalchemistry analyser with reagents in prefilled cuvettes. Standardsolution calibration for each parameter was used between the samples.

2.4.2. Immunological parametersFor total haemocyte count (THC) after 24 h haemocyte fixation in

1% formalin solution, samples were analyzed using manual countingand the number of haemocytes per milliliters was quantified using aBürker haemocytometer taking into account the dilution of thehaemolymph during bleeding (Jussila et al., 1997; Giulianini et al.,2007; Lorenzon et al., 2007). Dark field and phase contrast observa-tions of the haemocytes were carried out under a phase contrast ZeissStandard RA Light Microscope.

2.4.3. The Comet assayThe Comet assaywas conducted as described by Pavlica et al. (2001)

with slight modifications. Aliquots of 100 μL haemolymph mixed withanticoagulant solution (2:1) and 0.8% agarose (lowmelting point, LMP)were placed on a normal 1% agarose precoated microscope slides. Acoverslip was added and agarose was allowed to solidify for 2.5 min inthe freezer. After removing the coverslip, the third layer of 0.5% LMPagarose was added and left to solidify for 3 min in the freezer. The cellswere lysed in freshly made lysing solution (2.5 M NaCl, 100 mM EDTA,10 mM Tris–HCl, 10% DMSO, 1% Triton X-100, pH 10) for 1 h at 4 °C.After rinsing with distilled water, the slides were placed on thehorizontal gel box, covered with cold alkaline buffer (0.3 M NaOH,1 mMEDTA, pHN13) and left for 15 min. The electrophoresiswas run inthe samebuffer at 35 V (1.16 V/cm) and 300 mA for 15 min at 4 °C. Afterelectrophoresis the slides were neutralised in the cold neutralisationbuffer (0.4 M TrisHCl, pH 7.5), 2×5 min, fixed in methanol:acetic acid(3:1) for 15 min and stored in the dark at room temperature. Prior toexamination the slideswere stainedwith10 μgmL−1 ethidiumbromideand examined using Opton Axioplan epifluorescence microscope fittedwith filter set 09 (excitation 520 nm, emission 610 nm). For each slide(per animal) at least 50 randomly chosen cells were analyzed. Acomputerized image analysis system, Komet 5 (Kinetic Imaging Ltd.)was employed to measure Comet parameters (the extent of DNAmigration expressed as percentage of DNA in tail; % tDNA). In order toreduce the influence of cytotoxicity on Comet values nuclei withextremely large tails (% tDNAN50) have been excluded from theanalysis. Such cells probably represent dead or dying cells, so themeasurement of increase in DNA migration in their absence is moreimportant for the evaluation of genotoxicity than an increase thatdepends on them (Hartmann and Speit, 1997; Kammann et al., 2000).Haemocyte viability, evaluated by the Trypan-blue exclusion test, wasconsistently above 95% in all crayfish used in Comet assay.

2.4.4. The micronucleus testSmears were prepared from 100 μL of neat haemolymph and left to

dry. Slides were stained with bisbenzimide 33258 (Hoechst) at finalconcentration 1 μg mL−1 for 5 min, and then rinsed out with redistilledwater and mounted in glycerol-McIlvaine buffer (1:1), pH 7. The slideswerekept in thedark at4 °Cprior toexaminingunder the epifluorescentmicroscope Olympus BX51 at 1000× magnification. On each slide, 800cells (per animal) were counted. Only intact cells with distinct nuclearand cellular membranes were scored. MN were identified according tothe following criteria: spheric cytoplasmatic inclusions with a sharpcontour, diametric smaller than one third of the nucleus, colour andtexture resembling the nucleus, and no contact with the nucleus(Majone et al., 1987; Kirsch-Volders et al., 2000).

2.4.5. StatisticsAll statistical analyses were performed using Statistica 7 StatSoft

for Windows package. Data are given as arithmetic means±standard

errors (SE). Statistics for Comet assay and micronucleus data wereperformed by Mann-Whitney U test and two levels were consideredsignificant: P≤0.05 (*) and P≤0.01 (**). Descriptive statisticalanalysis was used to characterize haemolymph parameters. Student'st test was used to test the null hypotheses that all treatment meanswere equal. A probability value in the statistical tests between thecontrol and experimental values (mean±SE) of less than 0.05 wasconsidered significant. Time scale of graphs is not proportional for abetter visual inspection of data and the size of the illustration. Theresults of our research have shown that there was no significantdifference (P≤0.05) between measured values considering male andfemale crayfish.

3. Results

3.1. Water temperature increase

3.1.1. Physiological parametersIn animals exposed at 25 °C, a significant increment in glucose

concentration compared to the control groupwasmeasured after 3 daysof exposure (P=0.0052). Glucose concentration returned to controlvalues from the seventh day of exposure onward (Fig. 1A). In the groupkept at 30 °C, the highest value (13.3±2.05 mg dL−1=0.74 mM) wasrecorded after 3 days of exposure (P=0.0064). The glycaemia remainedsignificantly (P≤0.05) above the resting value until the seventh day. Atthe end of the recovery period the glucose levels returned to controlvalues (6.7±0.89 mg dL−1 at 25 °C and 4.8±0.49 mg dL−1 at 30 °C).

Crayfish exposed at 25 °C exhibited the highest level of lactate inhaemolymph (9.61±3.05 mg dL−1=1.07 mM) after 24 h of expo-sure (Fig. 1B). Higher values (P≤0.05) were also maintained duringthe subsequent 2 days of exposure, followed by a gradual decrease toresting values of lactate concentration. At the end of the experimentaltrial lactate concentration was set on a value slightly higher (4.6±2.5 mg dL−1) than the ones at 0 h of 3.4±1.01 mg dL−1 (P=0.48). Inthe group of individuals exposed at 30 °C (3.6±0.93 mg dL−1 at 0 h),the maximum increment of lactate was obtained on the third day ofexposure (5.1±1.50 mg dL−1; P≤0.05), but this value was lower(PN0.05) than those of the animals in the group at 25 °C. After 3 daysof exposure a continuous decrease in the mean values of lactate isevident, with the lowest value of 2.16±0.54 mg dL−1 at the end of theexperimental trial (Fig. 1B).

The alterations in the total protein concentration in A. leptodactylusduring the exposure to higherwater temperatureswere similar to thosefor glycaemia and lactate measurements (Fig. 1C.). Crayfish exposed to25 °C showed a significant increment in total protein concentration thefirst, third and seventh day of exposure (P≤0.05) in comparison to thecontrol values, reaching the highest value of 4.21±0.17 g dL−1 on thethird day (P=0.0003). During the 1 week recovery period total proteinconcentrations returned to the control values. Crayfish exposed to 30 °Cshowed a gradual and significant (P≤0.05) increment in total proteinconcentration during the 7 days of exposure. The mean protein valuesmeasured in group exposed at 25 °C are mostly higher than the onesmeasured in the group exposed at 30 °C, but there is no statisticallysignificant difference between the two groups (PN0.05).

3.1.2. Immunological parametersCrayfish exposed to a water temperature of 25 °C showed an

increment of the THC values during the first 5 days (Fig. 1D). Thehighest and significant peak (P≤0.05) was present after 5 days ofexposure (7.50×105±1.30×105 cell mL−1), after which the THCvalues decreased and reached control values at the end of recoveryperiod. THC values in the crayfish exposed at 30 °C showed anincrease that was higher and appeared earlier than at 25 °C. Thehighest THC (1.08×106±3.24×105 cell mL−1) was observed alreadyafter 3 days of exposure.

Fig. 1. Time course of A) glucose, B) lactate, C) total protein, and D) THC values in the haemolymph of A. leptodactylus exposed for 7 days at 25±1 °C and 30±1 °Cwater temperaturefollowed by a 7 days of recovery period at 19±1 °C. Values are expressed as means±SE.

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3.1.3. DNA integrityNo significant increase in the % tDNA (P≤0.05) was observed after

3 days of exposure at 25 °C water temperature when compared to thecontrol value measured the same day. However, the mean % tDNA inhaemocytes of control crayfish on the third daywas the lowest controlvalue measured in the experiment and the recorded DNA damage inthe crayfish exposed at 25 °C for 3 days is within the range of values

observed in the control individuals during the whole 2 week period(Fig. 2A). For a better visualization of a baseline level of DNA damagein crayfish haemocytes a mean cumulative control of all the controlindividuals (4.03 % tDNA) is also given in Figs. 2A, 4A, and 6A.Unambiguously significant increase of DNA damage was observedafter 7 days (P≤0.05) of exposure to 25 °C and on the third day ofrecovery period at 19 °C after which the values of % tDNA significantly

Fig. 2. DNA damage in the haemocytes of A. leptodactylus exposed for 7 days to water temperatures of 25±1 °C and 30±1 °C and through the 7 days of recovery at 19±1 °Cmeasured by A) Comet assay and B) frequency of micronucleated haemocytes. *P≤0.05, **P≤0.01 denotes statistical difference from the respective daily control value for Cometassay.

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declined below the control values. Increased water temperature of30 °C induced strong and significant increase in % tDNA after 3 days ofexposure (P≤0.01). Another increase in the DNA damage wasmeasured following 24 h of recovery after which the levels returnedto the control values within the end of the recovery period (Fig. 2A).

Moderate, less than two-fold and statistically not significantinduction in frequency of micronucleated haemocytes was observed inthe crayfish after one (1.61±0.45‰) and 7 days (1.5±0.46‰) ofexposure to the temperature of 30 °C (Fig. 2B). Exposure of crayfish to25 °C yieldedhigherMN frequency only after 7 days of exposure (2.08±0.69‰)whichwas also not statistically significant. Control crayfish had amean frequency of 0.89±0.23‰ of micronucleated haemocytes. Asthere was no statistically significant induction of MN up to 7 days ofexposure to higher temperatures, we did not analyse their frequency inthe rest of the haemolymph samples.

3.2. Air exposure

3.2.1. Physiological parametersThe analysis of data concerning the level of glucose in crayfish

haemolymph shows an increase in the glycaemia during the airexposure of 24 h and a return to reference values during the re-immersion period (in water at 19 °C) for 7 days (Fig. 3A). EmergedA. leptodactylus showed a significant increase in haemolymph glucoseto 33.88±2.82 mg dL−1 reached after 2 h of air exposure (P≤0.05)(Fig. 3A). Glycaemia remained significantly (P≤0.05) above theresting values until 24 h after re-immersion in water. The glucoseconcentration in the haemolymph returned to the control values after3 days of recovery in water.

Air exposure resulted in a progressive increase in the meanconcentration of lactate compared to both initial value (0 h, P≤0.05),and respective control (P≤0.05) (Fig. 3B). Crayfish exposed toair reached the highest peak of lactate concentration (98.93±14.34 mg dL−1; P=0.0001), corresponding to lower pH, after 24 h ofexposure. Lactate concentration decreased significantly on the first

day of re-immersion, and reached the control values within 7 days ofrecovery.

The total protein concentration in the air exposed A. leptodactylusis also altered as in other physiological parameters (Fig. 3C). Asignificant increase in the total protein values during the 24 h of aerialexposure (P≤0.05) was observed and values remained significantlyhigher than the control during the 24 h after re-immersion (5.05±0.29 g dL−1; P=0.0001). The protein concentration decreased fromthe third day of re-immersion, but remained significantly higher thanthe physiological values (P≤0.05) throughout the recovery period.

3.2.2. Immunological parametersIn the air exposed crayfish THC increased rapidly after 2 h to

9.45×105±2.45×105 cell mL−1 and peaked after 6 h to 1.35×106±3.02×105 cell mL−1 (P≤0.05). THC values decreased to the controllevels during the re-immersion period (Fig. 3D).

3.2.3. DNA integrityMeasured values of % tDNA from haemocytes of air exposed

crayfish did not show significant increase in the DNA damage.However, a modest increase in % tDNA was observed after 6 h afterwhich the values gradually decreased. On the first and third day afterre-immersion the % tDNAwas lower than in control crayfish (Fig. 4A).Micronuclei inductionwas observed in A. leptodactylus after 24 h of airexposure but it was not statistically significant compared to controlcrayfish. The frequency of MN after 24 h air exposure was 1.61±0.23‰ (Fig. 4B).

3.3. Food deprivation

3.3.1. Physiological parametersIn the control group of crayfish, during 2 weeks, a slight non-

significant variation of glycaemia is present, probably related to themanipulation of crayfish during the haemolymph sampling (Fig. 5A).

Fig. 3. Time course of A) glucose, B) lactate, C) total protein, and D) THC values in the haemolymph of A. leptodactylus exposed to air for 24 h followed by the 7 days of recovery periodin water. Values are expressed as means±SE.

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In the food deprived crayfish the glucose levels are in the lower rangeof the values obtained from the control.

In the starved animals, during the first 5 days, lactate concentrationwas similar to those measured in the control animals and notsignificantly different from values observed at 0 h (Fig. 5B). Only twovalues were significantly different from those in the control group: atday 1 of starvation (4.18±0.65 mg dL−1; P≤0.05) and at day 7 with a

peak of 45.15±16.23 mg dL−1 (P≤0.05). The distribution of measuredlactate values during the 2 week starvation period presents an overallstable time course, except for a peak on the seventh daywhich is a resultof unexplained high concentrations of lactate in haemolymph of twoindividuals (61.7 and 79.7 mg/dL). The rest of the exposed animals onthe same day had lactate concentrations in the range of control valuesobserved during the whole period of the experiment.

Fig. 4. DNA damage in the haemocytes of A. leptodactylus during the 24 h air exposure and 3 days of recovery period in water measured by A) Comet assay and B) frequency ofmicronucleated haemocytes. * P≤0.05 denotes statistical difference from the respective daily control value for Comet assay.

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In the starved animals the total protein content in the haemolymphduring the whole period of the experiment was significantly higher(P≤0.05) than that of the animals in the control group (Fig. 5C).However, the values recorded at the fifth day (2.83±0.3 g dL−1) weresignificantly lower (P≤0.05) than the initial value (0 h). After 2 weeksof food deprivation the protein concentration was 3.48±0.27 g dL−1

and still significantly higher (P≤0.05) than the control value at the sametime.

3.3.2. Immunological parametersThe starved crayfish group exhibited an initial but not significant

increase in the number of circulating haemocytes on day 1 and 3 afterthe beginning of food deprivation. After that, from the fifth day, THCstarted to decrease reaching a minimum of 2.25×105±4.38×104 cellmL−1, significantly lower than the initial value and control (Pb0.05),at the end of the experimental period (Fig. 5D).

3.3.3. DNA integrityTwo weeks of complete food deprivation did not cause any

significant increase in DNA damage in haemocytes of A. leptodactylusmeasured by the Comet assay although a significant decrease wasobserved on the first day (Fig. 6A). Increase in MN frequency wasobserved in A. leptodactylus during the 2 weeks of complete fooddeprivation, but did not reach statistical significance (Fig. 6B).

4. Discussion

The use of biomarkers is an important approach in the biomoni-toring of the aquatic environments to evaluate the relationshipbetween exposure to environmental pollutants and increased long-term effects on individuals and populations. Apart from chemicalpollution, other stressors such as temperature variations or variationsin oxygen concentration can also adversely affect the physiologicalstatus and survival of bioindicator species (e.g. crustaceans) (Morris

and Oliver, 1999). Such stresses have been associated with weakeningof crustaceans, failure of all or part of the integrated homeostaticresponses leading to increase of physiological disturbance and death.In this work we assessed the genotoxicity of stress caused by differentabiotic and biotic factors such as temperature increase, air exposureand food deprivation on freshwater crayfish A. leptodactylus in orderto determine their significance in genotoxicity biomonitoring ofpolluted environments.

During the water temperature increase and the air exposure,measured physiological and immunological parameters demonstrat-ed significant alterations indicating ongoing stress in exposedcrayfish. High inter-individual variability of these parameters duringthe recovery period after exposure of crayfish to the differentstressors was also observed and is probably related to individualcapacity of recovery. Glucose concentration in the haemolymph ofA. leptodactylus increased during exposure to higher water tempera-tures (25 °C and 30 °C) and a similar response has also been observedin other crustaceans (Durand et al., 2000; Lorenzon et al., 2007).Results of haemolymph glucose in Maia squinado acclimated todifferent temperatures indicated that the concentration of glucose inhaemolymph is reduced at low temperatures, while short periods ofacclimation to high temperatures resulted in a higher haemolymphglucose level (Durand et al., 2000). Variation of glucose content wasalso observed in crustaceans after exposure to several pollutants(Machele et al., 1989; Fingerman et al., 1998) and to atmospheric air(Santos and Colares, 1986). The glycaemic values reported an evidentand significant increase also during aerial exposure of A. leptodactylus.Emerged crayfish showed a significant increase in haemolymphglucose already after 2 h of air exposure, and glycaemia remainedsignificantly above the resting values until 24 h after re-immersion inwater. The increment of glucose concentration is probably related tothe mobilization of energy stores during stressful conditions of loweroxygen availability, as a source of fuel for anaerobic metabolismresulting in the production of lactate (Ridgway et al., 2006).

Fig. 5. Time course of A) glucose, B) lactate, C) total protein, and D) THC values in the haemolymph of A. leptodactylus starved for 14 days. Values are expressed as means±SE.

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Lactate is the major end-product of anaerobic metabolism incrustaceans and its increase is indicative of anaerobic metabolism(Jackson et al., 2001; Ridgway et al., 2006). In this work the lactateconcentration in the haemolymph of A. leptodactylus is accordingly,most effectively influenced by the air exposure caused hypoxia, but tosome extent also by the temperature increase confirming the resultsreported for other crustacean species (Durand et al., 2000; Speedet al., 2001; Lorenzon et al., 2007).

In all three stressful conditions (temperature increase, airexposure, food deprivation) used in this research, the alteration ofthe haemolymph protein content was also present. The level ofproteins is an expression of crayfish adaptive characteristics and of

strategies for acclimatization and can provide valuable information toidentify their condition (Ozbay and Riley, 2002; Buckup et al., 2008).

The total haemocyte count (THC) reported an increase of the meanvalues during exposure to the high temperatures and air exposurewhile 2 weeks of fooddeprivation caused its decrease. The alterations ofthese physiological and immunological parameters during theexposure to increased water temperature and air are indication ofcrayfish actual stress condition and were accompanied by a decrease ofDNA integrity.

Exposure of crayfish to the higher temperature (30 °C) inducedsignificant increase in DNA damage, measured by the Comet assay, onthe third day of the experiment.We are of the opinion that a statistical

Fig. 6. DNA damage in the haemocytes of A. leptodactylus during the 14 days of food deprivation measured by A) Comet assay and B) frequency of micronucleated haemocytes.**P≤0.01 denotes statistical difference from the respective daily control value for Comet assay.

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significance of % tDNA increment measured on the third day ofexposure to 25 °C is a result of a very low DNA damage measured inthe control individuals on the same day and that the observed value of4.48% tDNA falls within normal variations of a baseline DNA damagein haemocytes of control crayfish (mean cumulative control=4.03±0.07 (SE) % tDNA). Therefore it could be assumed that the first strongand unambiguously significant increase in DNA damage due to theexposure to 25 °C was observed on the seventh day. In both appliedtemperatures these increases were followed by a decrease and,interestingly, a second increase in the % tDNA measured on the first(30 °C) and the third (25 °C) day of the recovery period. The secondincrease of the DNA damage could be caused by changes in the activityof the DNA repair process. Similar increase of the DNA damage duringthe recovery period was observed after 24 h exposure of freshwaterbivalve Unio pictorum to ethyl methanesulfonate (EMS) whereauthors also speculated that this was a result of DNA repair processes(Štambuk et al., 2008). Thermal stress has already been associatedwith genotoxic effects in aquatic organisms such as freshwater fish(C. auratus) and mussel (D. polymorpha) measured also by theComet assay (Anitha et al., 2000; Buschini et al., 2003). Further-more, it has been established that oxidative stress is an importantcomponent of the stress response during exposure of organisms toincreased temperatures (Heise et al., 2006; Lesser, 2006) probablycausing the DNA damage.

MN induction, although not statistically significant, was alsoobserved in crayfish exposed to the higher temperatures. Somestudies have reported thatmussel tolerance to biocides is regulated byseasonal changes (Van Benschoten et al., 1995) and by temperature(Buschini et al., 2003) because temperature-dependent metabolicrates canmodulate exposure to toxicants (Kilgour and Baker, 1994). Itis also known that baseline frequency of MN depends on a variety ofbiotic and abiotic factors and that in poikilothermic organisms, such ascrayfish, the metabolic rate (mitotic activity) is largely dependent onthe environmental temperature (Buschini et al., 2003; Rybakovaset al., 2009). Exposure of cells or organisms to thermal stress can

result in nucleus integrity alterations and adverse effects on thestructure and function of the centrosomes (Brown et al., 1996). Anithaand coworkers (2000) observed statistically significant increase in thefrequency of micronucleated erythrocytes of the goldfish C. auratus24 h after the heat shock. However, twomarine bivalves (Brachidontespurpuratus and Semimytilus algosus) exposed to mild thermal stress(23 °C) did not exhibit increases in the frequency of MN or nuclearabnormalities (Sotil et al., 2008).

Measured values of % tDNA from haemocytes of air exposedcrayfish indicated a moderate but statistically not significant increasein the DNA damage after 6 h of air exposure. Afterwards themeasuredpercentages of tail DNA decreased which was probably the result ofthe induction of adaptive mechanisms activity, including DNA repair.Interestingly, all measured physiological and immunological para-meters also exhibited their highest values after 6 h of air exposureindicating the highest level of stress. Hypoxic conditions (caused alsoby air exposure of crayfish) are apparently associated with theincreased production of reactive oxygen species in mitochondria andoxidative stress (Li and Jackson, 2002; de Almeida et al., 2007). DNAdamage following short (24 h) and prolonged (up to 8 weeks)hypoxic conditions was observed in humans (Møller et al., 2001;Lundby et al., 2003). Oxidative stress following exposure to hypoxiahas been confirmed through increased levels of lipid peroxidation(Nakanishi et al., 1995). However, the reason for the lack ofsignificantly higher DNA damage in haemocytes of crayfish exposedto hypoxic conditions could be due to their routinely encounters withwaters of reduced oxygen tension, resulting in a broad array ofbehavioural and physiological responses that allow crayfish to dealwith the stress (Reiber, 1995) and prevent higher DNA damage. Slightincrease in MN induction was also observed in haemocytes ofA. leptodactylus after 24 h of air exposure. As mice living underhypoxic conditions exhibited a statistically significant elevation inMNfrequency (Snyder and Diehl, 2005), this lack of sensitivity forA. leptodactylus, observed also in thermal stress, could be attributed toits large number of small chromosomes (368) (Silver and Cukerzis,

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1964), which is considered a disadvantage for the MN test. Increasingthe number of cells scored for micronucleus test would probablyincrease statistical significance of the data but due to the largeamounts of haemolymph needed for all the parameters investigatedand being aware of possible adverse effects of increased bleeding only100 μL was assigned for micronucleus test. On the other hand, thereare several studies using freshwater and marine invertebrates thatused even lower number of cells for micronucleus test and gavestatistically significant changes (Pavlica et al., 2000; Klobučar et al.,2003, 2008; Štambuk et al., 2008, 2009).

The values of glucose level in the starved group are mostly lowerthan the ones in the control group, showing a possible depletion ofsugars during starvation, used as a primary energy reserve for dealingwith the lack of food. A continuous increase in the concentration ofproteins in the haemolymph of starved crayfish, during the whole2 week period, in comparison to control individuals could suggest itsinvolvement as a reserve that provides energy in case of prolongedfood scarcity intervals (Sánchez-Paz et al., 2007). Starvation is alsoknown to affect the THC. It has been shown that food deprivationcauses a decline in total haemocyte counts in Homarus americanus(Stewart et al., 1967) and in the crab Carcinus maenas (Johnson,1980). In our study, the results of THC in the food deprived group ofcrayfish showed a decrease after the fifth day of starvation. However,2 weeks of food deprivation did not cause any significant genotoxiceffects while only a modest increase in the MN frequency wasobserved and it was approximately the same as those measured as aconsequence of the thermal stress and air exposure. Contrary to ourresults, partial or complete food deprivation in rats caused aprogressive increase of DNA damage measured by the Comet assay(de Souza Rocha et al., 2008). In their experiment partial (25%)chronic food deprivation caused an increase in the Comet score in thefirst 5 days, and a decrease thereafter reaching a basal level after50 days. The authors suggested that the increased DNA damage inorganisms exposed to acute partial or total food deprivation and up tothe fifth day of partial chronic food deprivation has been caused byan increase of production of free radicals and a reduction of thedefence mechanism system (de Souza Rocha et al., 2008). Apparently,freshwater crayfish are much better adapted to fasting thanmammals.

All the physiological and immunological haemolymph parametersused in this research confirmed their applicability to assess animalphysiological and metabolic status, sensitivity, and susceptibility todifferent stressors and can integrate the genotoxicity assessment ofenvironmental pollution. Our results indicated that the increasedtemperature may significantly increase DNA damage in freshwatercrayfish while food deprivation and air exposure under studiedconditions could be considered as a non-genotoxic stress. Bearing inmind the growing awareness that (eco)toxicology is merely a branch ofthe wider field of stress biology (Van Straalen, 2003), it is important toconsider, in every process of pollution biomonitoring, the possibility ofadditional genotoxic effects caused by abiotic factors other thanchemical pollution. Our results indicate that it is possible to use crayfishhaemocytes for genotoxicity assessment of freshwater environmentsand also underline the need for comprehensive research on the possibleinfluence of diverse biotic and abiotic factors on the DNA integrity inbiomonitor species.

Acknowledgements

This work was supported by the Republic of Croatia ScientificResearch Council within the framework of Project No. 119-0982934-3110. The authors would like to thank the personnel of theDepartment of Life Science (Zoology laboratory) at the University ofTrieste, Italy for analytical and technical assistance and also valuablediscussions.

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