Comparative Biochemistry and Physiology, Part A 162 (2012) 357–363

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Comparative Biochemistry and Physiology, Part A

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Sustained hydrostatic pressure tolerance of the shallow water shrimp Palaemonetesvarians at different temperatures: Insights into the colonisation of the deep sea

Delphine Cottin a,⁎, Alastair Brown a, Andrew Oliphant a, Nélia C. Mestre a, Juliette Ravaux b,Bruce Shillito b, Sven Thatje a

a University of Southampton, Ocean and Earth Science, National Oceanography Centre, Southampton, European Way, Southampton, SO14 3ZH, UKb UPMC Université Paris 6, CNRS UMR 7138, Systématique, Adaptation et Evolution, F-75005, Paris, France

⁎ Corresponding author at: Université Claude BernaLaboratoire d'Ecologie des Hydrosystèmes Naturels et Ataire de la Doua, 6 rue R. Dubois, 69622 Villeurbanne Ce

E-mail address: delphine.cottin@univ-lyon1.fr (D. Co

1095-6433/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.cbpa.2012.04.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 December 2011Received in revised form 7 April 2012Accepted 9 April 2012Available online 16 April 2012

Keywords:Deep seaEvolutionHSP70PressureShallow waterShrimpTemperature

We investigated the tolerance of adult specimens of the shallow-water shrimp Palaemonetes varians to sus-tained high hydrostatic pressure (10 MPa) across its thermal tolerance window (from 5 to 27 °C) usingboth behavioural (survival and activity) and molecular (hsp70 gene expression) approaches. To our knowl-edge, this paper reports the longest elevated hydrostatic pressure exposures ever performed on a shallow-water marine organism. Behavioural analysis showed a 100% survival rate of P. varians after 7 days at10 MPa and 5 or 10 °C, whilst cannibalism was observed at elevated temperature (27 °C), suggesting no im-pairment of specific dynamic action. A significant interaction of pressure and temperature was observed forboth behavioural and molecular responses. Elevated pressure was found to exacerbate the effect of temper-ature on the behaviour of the animals by reducing activity at low temperature and by increasing activity athigh temperature. In contrast, only high pressure combined with low temperature increased the expressionof hsp70 genes. We suggest that the impressive tolerance of P. varians to sustained elevated pressure may re-flect the physiological capability of an ancestral species to colonise the deep sea. Our results also support thehypothesis that deep-sea colonisation may have occurred during geological periods of time when the oceanicwater column was warm and vertically homogenous.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Today's deep-sea fauna are characterised by a high diversity ofspecies. Life at great depths requires adaptation to ecologically andphysiologically challenging conditions, such as high hydrostatic pres-sure, absence of sunlight, limited food availability, and generally lowbut stable temperatures (Somero, 1992a; Childress, 1995; Tyler,1995; Glover et al., 2010). Extant deep-sea fauna are thought tohave arisen through colonisation of the deep sea by shallow-waterspecies. There is general consensus that regional extinctions, drivenby climatic changes, and subsequent recolonisations of the deep seahave occurred numerous times over many geological ages (Jablonskiet al., 1983; Horne, 1999; Wilson, 1999). Consequently, extantdeep-sea fauna may consist of both ancient and relatively recentshallow-water lineages (Wilson, 1999; Raupach et al., 2009). This issupported by a growing number of molecular phylogenetic studies,which evidence that many shallow-water and deep-sea taxa demon-strate close relatedness (Distel et al., 2000; Jones et al., 2006; Hall andThatje, 2009; Raupach et al., 2009).

rd Lyon 1, UMR CNRS 5023,nthropisés, Domaine universi-dex, France.ttin).

rights reserved.

The cold temperatures prevailing in the deep sea are thought tolimit colonisation by shallow-water species, which are adapted tothe warmer conditions of the upper ocean (except at high latitudes).Colonisation of the deep ocean may therefore have occurred duringthe Mesozoic and early Cenozoic periods when the oceanic water col-umn was warm and isothermal (Hessler and Wilson, 1983; Young etal., 1997; Tyler and Young, 1998; Tyler and Dixon, 2000). It has alsobeen suggested that cold-adapted species at high latitudes mayhave colonised the deep sea through regions of deep-water formation(Tyler and Dixon, 2000; Thatje et al., 2005).

Recent studies have demonstrated impressive pressure and tem-perature tolerance in larvae and adults of some shallow-water spe-cies, supporting the potential for migration to greater depths(Young et al., 1995, 1997; Tyler and Dixon, 2000; Benitez-Villaloboset al., 2006; Aquino-Souza et al., 2008; Mestre et al., 2009; Oliphantet al., 2011). Both temperature and hydrostatic pressure are abioticfactors that strongly determine the distribution of marine species(Desbruyères et al., 1982; Hochachka and Somero, 1984; Somero,1992a, 1992b; Sarrazin et al., 1997; Lee, 2003; Tomanek andSandford, 2003; Brown and Thatje, 2011). These parameters affectthe development and survival of organisms and may therefore influ-ence the ability of a species to undergo range extensions in changingenvironments, and to colonise new habitats (Macdonald, 1997; Tylerand Young, 1998; Clarke, 2003; Brown and Thatje, 2011). Although

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studies have investigated the relevance of each of these factors inde-pendently, few experimental studies have focused on their combinedeffects, especially over longer periods of sustained hydrostatic pres-sure exposure (more than several hours) (e.g. Benitez-Villalobos etal., 2006; Aquino-Souza et al., 2008; Mestre et al., 2009; Oliphant etal., 2011).

In this paper, we investigated the combined effects of pressureand temperature over a sustained period (7 days) on adult Palaemo-netes varians (Leach, 1814) using both behavioural (survival and ac-tivity) and molecular (hsp70 gene expression) approaches. To ourknowledge, the experiments presented in this study are the longestsustained pressure exposures performed on a shallow-water speciesto date. P. varians is a coastal shrimp found in areas of salt-marsh inmany parts of Europe (Nugegoda and Rainbow, 1989; Nielsen andHagerman, 1998). Previous study has shown that this species is capa-ble of tolerating high hydrostatic pressures (critical pressure maxi-mum~15 MPa at 10 °C) and high thermal range (critical thermalmaximum~31 °C; critical thermal minimumb0 °C) at least underacute conditions (few minutes/hours) (Oliphant et al., 2011). In addi-tion, two HSP70 isoforms have already been characterised in this spe-cies (one inducible form and one constitutive form) (Cottin et al.,2010). These proteins are known to function as molecular chaper-ones, assisting in protein folding in response to a wide variety ofstressors (Sørensen et al., 2003; Mayer and Bukau, 2005). They areamong the most prominent proteins induced by exposure to elevatedtemperature, but several studies have also reported induction ofthese proteins following variation in hydrostatic pressure(Takahashi et al., 1997; Kaarniranta et al., 1998, 2000; Elo et al.,2003; Kaarniranta et al., 2003). The aim of this study was to testwhether P. varians, despite its shallow living, demonstrates a physio-logical tolerance to hydrostatic pressure that may reflect upon thephysiological capability of an ancestral species to colonise the deepsea.

2. Materials and methods

2.1. Specimen collection and acclimation

Adult specimens of P. varians (4 to 5 cm in total length) were col-lected from Lymington salt marshes (Hampshire, England. 50°45′N,1°32′W) using a hand-held net.

For short pressure exposures (6 h), shrimp were collected in June2010 and kept for 10 days at 10 °C in 10 L PVC-tanks in filtered sea-water (32 ppt salinity; 1 μm filtered) under continuous aeration.The photoperiod was 12 h:12 h light:dark and the animals were fedad libitum with fish flakes twice a week. Half of the water wasreplaced every 2–3 days with fresh 10 °C filtered seawater.

For sustained pressure/temperature exposures (7 days), animalswere collected in September 2010 and maintained in a running sea-water system in the aquarium of the National Oceanography Centre,Southampton (England). Prior to experimental treatments, animalswere kept in 10 L PVC-tanks in filtered seawater (32 ppt salinity;1 μm filtered) under continuous aeration and acclimated stepwise(at a rate of 2 °C per day) to the desired experimental temperatures(5, 10 or 27 °C) using temperature-controlled incubators with a pho-toperiod of 12 h:12 h light:dark. Animals were maintained at the ex-perimental temperature for a further three days prior toexperimentation; shrimp were not fed during this period.

2.2. Short pressure exposures (6 h)

Short pressure exposures were performed on a total of 80 P. var-ians specimens using the pressure vessel IPOCAMP in flow throughmode (20 L/h−1

flow rate) at four different absolute pressures:0.1 MPa (corresponding to atmospheric pressure; control specimens),5 MPa (corresponding to 500 m depth), 10 MPa (corresponding to

1000 m depth) and 15 MPa (corresponding to 1500 m depth). Thesepreliminary experiments were carried out (prior to sustained pres-sure exposures) to determine the survival and the heat-shock re-sponse of the animals after a short-term period (6 h) at elevatedhydrostatic pressure. For each experiment, 20 shrimp were placedin PVC cages (2 cages; 10 shrimp per cage) inside the pressure cham-ber at a constant seawater temperature of 10 °C (see Ravaux et al.,2003; Shillito et al., 2006 for schematic and description of IPOCAMP).The system was run at atmospheric pressure for 1 h prior to pressur-isation to allow acclimation and recovery from handling stress. Pres-sure was then increased by 1 MPa every 5 min until experimentalpressure was reached (after Oliphant et al., 2011). Animals weremaintained at experimental pressure for a total of 6 h. The tempera-ture of the flowing seawater (filtered at 1 μm) was measured con-stantly by temperature probes at the inlet and outlet of theIPOCAMP (±1 °C). More accurate measurements of both temperature(±0.1 °C) and pressure (±0.12 MPa) were obtained by placing anautonomous pressure and temperature data logger (SP2T4000, NKEinstrumentation) inside the pressure chamber in all treatments. Atthe end of each treatment the pressure vessel was depressurised(within a few seconds), shrimp were rapidly dissected and abdomenswere frozen in liquid nitrogen and stored at −80 °C for later molecu-lar analysis.

2.3. Sustained pressure/temperature exposures (7 days)

Sustained (7 days) pressure/temperature exposures of P. variansshrimps were carried out at a combination of temperatures (5 °C,10 °C, 27 °C) and absolute pressures (0.1 and 10 MPa) using the pres-sure vessel IPOCAMP, on a total of 180 individuals. These treatmentswere performed to evaluate the physiological tolerance of theshallow-water shrimp to high hydrostatic pressure over an extendedperiod and across its thermal tolerance window (after Oliphant et al.,2011). A total of 6 experimental treatments were carried out with 3replicates of each treatment. The high pressure condition (10 MPa)used for these sustained exposures was chosen according to previouswork (Oliphant et al., 2011) and according to the results of our pre-liminary short-term experiments which showed no mortality andno significant induction of stress proteins after a 6 h exposure at pres-sures≤10 MPa (see Results section, part 1.). The IPOCAMP was runfor at least 1 h prior to the start of each experiment to ensure thewhole system was maintained at the desired experimental tempera-ture. The water temperature was measured using a thermometerprior to experimentation. More accurate measurements of both tem-perature and pressure were obtained by placing a data logger insidethe chamber. All treatments were recorded for subsequent beha-vioural analysis. For each treatment, ten shrimp were placed in aPVC cage with an inclined lid, which was mounted on a tripod plat-form inside the pressure chamber. The platform elevated the cage,allowing a clear view via an endoscope inserted into a viewing portin the lid of the vessel (Shillito et al., 2006; Oliphant et al., 2011).The endoscope was connected to a video recorder and TV monitor.Following introduction of the animals to the pressure vessel, the sys-tem was run at atmospheric pressure for a further 1 h prior to pres-surisation to allow acclimation and recovery from handling stress.Pressure was then increased stepwise at the same rate as in theshort pressure exposure (i.e. 1 MPa every 5 min). Shrimp were keptinside the incubator for a period of 7 days (168 h) with a photoperiodof 12 h:12 h dark:light and without food supply. The video recorderwas set to record the first 15 min after the end of pressurisation,then 15 min at 10:00 h and 17:00 h every day. For the treatmentsperformed at atmospheric pressure, shrimp were placed in theIPOCAMP for 1 h (time for acclimation and recovery from handlingstress) and allowed a further 50 min (time of pressurisation for the10 MPa treatments) before recording began. At the end of each treat-ment, the pressure vessel was depressurised (within a few seconds)

Fig. 1. Expression levels of hsp70 form1 (inducible form; black columns) and hsp70form2 (constitutive isoform; grey columns) obtained, using real-time PCR, in adultPalaemonetes varians maintained for 6 h at different pressures (0.1, 5, 10, 15 MPa)and at a constant temperature of 10 °C. The expression level was normalised to the cor-responding RPL8 abundance. All amplifications were reproduced in triplicate andvalues correspond to the mean normalised expression (±s.e.m.) of at least 4 indepen-dent samples (n=4–5 individuals).

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and shrimp were rapidly dissected and abdomens were frozen in liq-uid nitrogen and stored at −80 °C for later molecular analysis.

2.4. Behavioural analysis

Behavioural analysis was conducted on P. varians maintained for7 days at a combination of temperatures (5 °C, 10 °C, 27 °C) and pres-sures (0.1 and 10 MPa) inside the IPOCAMP. Three replicates wereanalysed for each of the 6 pressure/temperature combinations. Beha-vioural responses of shrimp were determined for a period of 1 min,twice a day (10:00 am and 5:00 pm). These 1 min sequences wererandomly determined within the 15 min of recorded material. Indi-viduals were identified and their behaviour classified into two cate-gories, as follows:

‘Active movement’; when the shrimp walked or swam a distanceexceeding its own length in less than 30 s.‘Cannibalism’; when a shrimp was missing from the cage (infer-ring consumption by other shrimp).

‘Active movement’was previously found to be a useful indicator ofheat stress (Ravaux et al., 2003; Shillito et al., 2006) and has also re-cently been shown to be a good pressure-initiated stress indicator(Oliphant et al., 2011). In this study, it was therefore considered asa significant stress indicator. ‘Cannibalism’ represents the percentageof shrimp that have been eaten by conspecifics. Since cannibalism oc-curred during the experiments, ‘active movement’ was expressed asthe percentage of alive individuals actively moving during the 1-min analysis. To avoid any potential effect of a circadian rhythm of ac-tivity (Aguzzi et al., 2005; Jury et al., 2005) data from morning andevening analyses were pooled to obtain mean daily activity of the an-imals (Fig. 2). Other behaviours, such as ‘attacking’ (when one indi-vidual attacked another using its chelipeds), ‘jumping’ (shrimptypical, backwards-directed escape movement), ‘feeding’ (when anindividual fed on a dead body) or ‘moulting’ (when an individualshed its exoskeleton) were also observed. These behaviours were no-ticed but they were not included in our behavioural categories as theywere impossible to quantify.

2.5. Real-time PCR analysis of hsp70 genes

2.5.1. RNA extraction, DNAse treatment and reverse transcriptionTissues from shrimp abdomens, with their cuticle, were ground in

liquid nitrogen. The powder was homogenised in Trizol reagent (Invi-trogen) and total RNA was isolated according to the manufacturer'sinstructions. For each individual, 3 μg of total RNA was treated withDNase and reverse transcribed to DNA as previously described inCottin et al. (2010).

2.5.2. Real-time quantitative RT-PCRReal-time PCR was used to evaluate accurately the expression

levels of the hsp70 genes (hsp70 form1 and hsp70 form2), previouslyidentified by Cottin et al. (2010), in P. varians specimens exposed ei-ther to 6 h or 7 days at different pressure/temperature regimes. Allreal-time quantitative RT-PCR reactions were performed on the Light-Cycler® 480 Real-Time PCR Detection System (Roche, France) usingthe protocol described in Cottin et al. (2010). All primer pairs testedgenerated a single and discrete peak in the dissociation curve. A neg-ative control and a 5-fold dilution series protocol of pooled cDNAswere included in each run to construct a relative standard curve todetermine the PCR efficiencies and for further quantification analysis.All primer pairs gave amplification efficiencies of 90–100%. The ex-pression of hsp70 genes was normalised to geometric means of a ref-erence gene (RpL8), according to Cottin et al. (2010), and the meannormalised gene expression of each triplicate reaction was then cal-culated with the LightCycler® 480 software. Specific primers for

hsp70 form1, hsp70 form2 and RpL8 were described in Cottin et al.(2010).

2.6. Statistical analysis

Molecular data were analysed either by one-way ANOVA (forshort pressure exposures) using pressure as a fixed factor or bytwo-way ANOVA (for sustained pressure exposures) with pressureand temperature as fixed factors. Behavioural data, as proportionaldata, were arc-sine square root transformed and subjected to analysisby two-way repeated-measures ANOVA with pressure and tempera-ture as fixed factors and time as repeated measurements. These ana-lyses were followed by Tukey's HSD test for multiple comparisons. Allanalyses were performed using STATISTICA version 7.0 (Statsoft) andsignificance levels were pb0.05.

3. Results

3.1. Short pressure exposures (6 h)

3.1.1. Shrimp survivalNo mortality was observed for P. varians specimens maintained at

10 °C either during the acclimation period (10 days) in PVC-tanks orafter a 6 h-exposure at 0.1, 5, 10 or 15 MPa inside the IPOCAMP.

3.1.2. Expression analysis of hsp70 genesA significant effect of pressure was found on the expression of

hsp70 form1 (F3, 13=5.98; p=0.008; Fig. 1) whilst no significant ef-fect was observed on the expression of hsp70 form2 (F3, 14=0.1;p=0.95; Fig. 1). Generally, the expression of hsp70 form1 increasedwith increasing pressure from 1-fold at 5 MPa to 1.3-fold at 10 MPaand to 3.2-fold at 15 MPa compared to individuals maintained at at-mospheric pressure. However, multiple comparisons only revealed asignificant difference at 15 MPa (p=0.02).

3.2. Sustained pressure/temperature exposures (7 days)

3.2.1. Shrimp survival and cannibalismNo mortality was observed for P. varians specimens maintained at

5 and 10 °C at 0.1 or 10 MPa. However, cannibalism occurred duringthe experiments performed at 27 °C; at 0.1 MPa this began on day 5(7±4.2% of shrimp were missing) whereas at 10 MPa this began on

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day 2 (7±3.3% of shrimp were missing). At the end of the treatment(7 days), the percentage of shrimp that were cannibalised was 13±4.2% at 0.1 MPa and 37±4.8% at 10 MPa (Fig. 2).

3.2.2. Shrimp activity

3.2.2.1. Effect of temperature. The effect of temperature on ‘activemovement’ of P. varians was significant (ANOVA, F2, 12=57.5;pb0.001). Levels of ‘active movement’ for a single temperature, aver-aged across pressure and time, generally increased with increasingtemperature. The lowest mean level of ‘active movement’ occurredat 5 °C (2±3.4%); the highest occurred at 27 °C (85±6.7%; seeFig. 2). Mean levels of ‘active movement’ increased from 5 to 27 °C.The difference in mean level of active movement between tempera-tures was significant (pb0.05), except between 5 °C and 10 °C at0.1 MPa.

Moulting was observed at all temperatures. The animals then atethe cast exuvia. At 27 °C, shrimp were frequently observed attackingone another; at 5 °C and 10 °C this was less common.

3.2.2.2. Effect of pressure. The effect of pressure on ‘active movement’of P. varians was not significant (ANOVA, F1, 12=0.13; p=0.13).Levels of ‘active movement’ for a single pressure, averaged acrosstemperature and time, were not statistically different between 0.1and 10 MPa. Both the highest (85±6.7%) and lowest (2±3.4%) levelsof ‘active movement’ occurred at 10 MPa.

Moulting occurred at both 0.1 and 10 MPa; the animals then atethe cast exuvia. Shrimp were also observed attacking one another atboth pressures.

3.2.2.3. Combined effect of temperature and pressure. Combined, theeffect of the interaction between temperature and pressure on the beha-vioural category ‘active movement’ was significant (ANOVA, F2, 12=11.3; p=0.001). Generally, pressure exacerbated the effect of tem-perature on the activity of P. varians specimens. At 27 °C, the meanlevels of ‘active movement’, averaged across time, were higher at10 MPa (74±7.9%) than at 0.1 MPa (44±8.9%). At 5 °C, they werelower at 10 MPa (6±3.1%) than at 0.1 MPa (16±7.7%) (Fig. 2). Incontrast, at 10 °C, the mean levels of ‘active movement’ averagedacross time were very similar between 0.1 MPa (26±7.2%) and10 MPa (27±6.9%).

Fig. 2. Effect of temperature and pressure on the behavioural response (‘active movement’:Shrimp were maintained for 7 days at two different pressures (0.1 MPa: black markers; 10 Mof these 6 treatments was performed in triplicate. For each behavioural category, percentawere averaged for each day, data are thus presented as a mean of 6 replicates of n=10 spcage and was only indicated at 27 °C since this behaviour has not been observed at 5 and 1centage of alive individuals present inside the cage. As the first data point analysed was onlyavoid a possible stress effect due to pressurisation.

3.2.2.4. Effect of time. The effect of time on ‘active movement’ of P. var-ianswas significant (ANOVA, F5, 60=3.34; p=0.009). Levels of ’activemovement’ averaged across pressure and temperature, generally de-creased with time.

3.2.3. Expression analysis of hsp70 genes

3.2.3.1. Effect of temperature. A significant effect of temperature wasobserved on the expression of hsp70 form1 (F2, 18=12.57;p=0.0003; Fig. 3) and hsp70 form2 (F2, 21=9.73; p=0.001;Fig. 4). The expression level of hsp70 form1 was found to be signif-icantly higher at 5 °C (4.6-fold at 0.1 MPa and 7.7-fold at 10 MPa;p=0.002) and 27 °C (4.7-fold at 0.1 MPa and 5.5-fold at 10 MPa;p=0.02) compared to the level measured at 10 °C whereas no sig-nificant difference was observed between 5 °C and 27 °C (1-fold at0.1 MPa and 1.4-fold at 10 MPa; p=0.06). The expression level ofhsp70 form2 was found to be significantly higher at 5 °C comparedto the level measured at 10 °C (1.7-fold at 0.1 MPa and 1.8-fold at10 MPa; p=0.03) or at 27 °C (2-fold at 0.1 MPa and 3.4-fold at10 MPa; p=0.0009). No significant change was observed between10 °C and 27 °C (1.2-fold at 0.1 MPa and 1.7-fold at 10 MPa;p=0.26).

3.2.3.2. Effect of pressure. A significant pressure effect was observedon the expression of hsp70 form1 (F1, 18=24.67; pb0.0001;Fig. 3) whilst no significant effect was found on the expression ofhsp70 form2 (F1, 21=0.22; p=0.63; Fig. 4). The expression ofhsp70 form1 was generally higher in 10 MPa-exposed animals com-pared to individuals maintained at atmospheric pressure (0.1 MPa):4.6-fold at 5 °C; 2.8-fold at 10 °C and 3.2-fold at 27 °C. However,multiple comparison tests only revealed a significant difference at5 °C (p=0.002).

3.2.3.3. Combined effect of temperature and pressure. A significant in-teraction of temperature and pressure was observed on hsp70form1 expression level, indicating a different response to pressureaccording to temperature (F2, 18=5.52, p=0.01; Fig. 3). Indeed,the differential expression observed between 0.1 MPa and 10 MPaexposed-shrimp was only significant at 5 °C with a 4.6-fold induc-tion. In contrast, no significant interaction of temperature and pres-sure was observed on the expression of hsp70 form2 (F2, 21=0.56,p=0.06; Fig. 4).

circles; ‘cannibalism’: triangles) of adult Palaemonetes varians according to time (days).Pa: white markers) at each of three different temperatures (5 °C, 10 °C and 27 °C). Eachges (±s.e.m.) obtained during morning (10:00 am) and evening (17:00 pm) analysesecimens. ‘Cannibalism’ represents the percentage of shrimp that were missing in the0 °C. When cannibalism occurred, levels of ‘active movement’ were expressed as a per-5 min after the end of pressurisation, day 1 was excluded from the statistical analysis to

Fig. 3. Expression levels of hsp70 form 1 (inducible form) obtained by real-time PCR inadult Palaemonetes varians maintained for 7 days at different temperature (5, 10 or27 °C) and pressure (0.1 or 10 MPa) combinations. The expression level was normal-ised to the corresponding RPL8 abundance. All amplifications were reproduced in trip-licate and values correspond to the mean normalised expression (±s.e.m.) of at least 4independent samples (n=4–5 individuals).

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4. Discussion

4.1. Effect of sustained temperature/pressure regimes on the behaviouralresponse

The brackish-water shrimp, P. varians, is found in very shallowwater (0–10 m depth) where the seasonal fluctuations of environ-mental temperature can range from 0 °C to 33 °C (Lofts, 1956;Jefferies, 1964; Healy, 1997). This species has previously served as amodel for studying the effects of pressure and temperature onshallow-living fauna (Oliphant et al., 2011). To our knowledge, nopublished study has focused on the combined effect of pressure andtemperature on a shallow-water marine organism for the period oflength demonstrated here. We therefore report here a more ecologi-cally relevant study than previous works.

Our results showed that P. varians which is found in very shallowwater can tolerate sustained exposure to high pressure under lowtemperature conditions (from 5 to 10 °C), as demonstrated by the100% survival rate determined after 7 days of maintenance at a pres-sure of 10 MPa at temperatures of 5 and 10 °C. At higher temperature(27 °C), cannibalism was observed at both 0.1 and 10 MPa (seeFig. 2); however we were not able to determine whether shrimpwere killed by the other animals or were eaten once they were al-ready moribund or dead. Nevertheless, we observed shrimp attacking

Fig. 4. Expression levels of hsp70 form 2 (constitutive form) obtained by real-time PCRin adult Palaemonetes varians maintained for 7 days at different temperature (5, 10 or27 °C) and pressure (0.1 or 10 MPa) combinations. The expression level was normal-ised to the corresponding RPL8 abundance. All amplifications were reproduced in trip-licate and values correspond to the mean normalised expression (±s.e.m.) of at least 4independent samples (n=4–5 individuals).

each other with their chelipeds and feeding on each other, which in-dicates that specific dynamic action of feeding was not compromisedat 10 MPa (Thatje and Robinson, 2011). These behaviours were onlyobserved at 27 °C and may reflect increased rates of metabolism(and subsequently increased nutritional demands) at elevated tem-perature. IPOCAMP does not provide a mechanism to feed animalswhen the system is pressurised and therefore cannibalism representsthe only way for P. varians to meet elevated nutritional requirements.This is also supported by the significant increase in activity levels ob-served at 27 °C (Fig. 2). An increase in metabolic rate with increasingtemperature is a common response in ectotherms (for review seeClarke, 2003). Elevated oxygen consumption rates were also previ-ously found in P. varians shrimp exposed to elevated temperature(Oliphant et al., 2011).

The significant interaction between the effects of temperature andpressure, revealed by increased mean activity and cannibalism at ele-vated pressure and temperature (27 °C) and decreased mean activityat elevated pressure and low temperature (5 °C), suggest that highpressure exacerbates the effect of temperature on the behavioural re-sponse of P. varians. The application of pressure to shallow-water an-imals elicits hyperexcitability followed by convulsions or spasms bydirectly affecting the animal's neuromuscular system (Macdonaldand Gilchrist, 1978; Wilcock et al., 1978). Elevated swimming activityin response to high hydrostatic pressure has been observed previous-ly in adult P. varians (Oliphant et al., 2011) and in larvae or adults ofseveral other invertebrates under acute conditions (Hardy andBainbrige, 1951; Knight-Jones and Qasim, 1955; Wilcock et al.,1978; Gherardi, 1995). As suggested by Oliphant et al. (2011), suchbehaviour may indicate an escape response of P. varians to maintainits optimal bathymetric distribution. At low temperatures, thispressure-initiated stress response appears constrained. Similar com-bined effects of high pressure and low temperature have beenreported previously on embryonic development of other shallow-water invertebrates (Young et al., 1997; Benitez-Villalobos et al.,2006). The effect of pressure on the behavioural response of P. variansdecreases over time; the percentage of active animals observed at0.1 MPa and 10 MPa is very similar after 7 days at all temperatures(Fig. 2). This may suggest an acclimation of P. varians to pressureand temperature conditions. However, further studies showingchanges on membrane lipid composition or enzyme activities arenecessary to confirm this hypothesis.

4.2. Effect of sustained temperature/pressure regimes on the molecularresponse

Whichever the sustained pressure conditions applied to P. varians(0.1 or 10 MPa), a significant increase (5 to 8-fold) in hsp70 induciblegene expression was observed in both cold- (5 °C) and heat-exposedshrimp (27 °C) compared to animals maintained at reference temper-ature (10 °C) (Fig. 3). Although a significant difference was also ob-served at 5 °C in the expression level of hsp70 constitutive gene, thefold-inductions observed (b2-fold compared to reference animals)suggest a small up-regulation compared to the inducible form. An in-crease in hsp70 gene expression after a cold-shock exposure has beenpreviously reported in Drosophila species (Denlinger et al., 1991; Gotoand Kimura, 1998) and the beetle Leptinotarsa decemlineata (Yocum,2001). Indeed, many of the physiological effects of cold exposurethat are observed at a cellular level are similar to those seen inheat-stressed cells, including decelerated protein synthesis and cellcycle progression, reduced membrane permeability and changes incytoskeletal structure, as well as protein denaturation (Sonna et al.,2002; Airaksinen et al., 2003). This increase in hsp70 genes aftercold-exposure may indicate the occurrence of cellular damageunder sustained low temperature exposure in P. varians. An up-regulation of hsp70 form 1 has previously been found in P. variansafter a 1 h heat-shock at 28±2 °C and 0.1 MPa (Cottin et al., 2010).

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However, the expression level measured in 1 h heat-shocked shrimpwas three times higher (~15-fold) than the expression level observedin shrimp maintained for 7 days under similar temperature and pres-sure conditions. This difference in expression level may reflect an ac-climation to elevated temperature. Such an acclimation effect is alsosuggested by the significant decrease in active movement observedover time in P. varians specimens (Fig. 2).

The effect of sustained high hydrostatic pressure was found to sig-nificantly affect the expression levels of hsp70 inducible form. Never-theless, the level of hsp70 inducible gene obtained in individualsexposed to sustained high pressure at 10 °C (after 7 days: ~3-fold)appear in the same range as the level obtained in short-exposed ani-mals (after 6 h: ~1.5-fold). Generally, levels were higher in 10 MPa-exposed animals than in individuals maintained at atmospheric pres-sure. However, the differential expression between 0.1 MPa and10 MPa-exposed shrimp was only significant at low temperature(~5-fold induction at 5 °C), revealing a significant interaction be-tween pressure and temperature on the heat-shock response of P.varians. Exposure to elevated pressure (10–50 MPa) for a few hours(2 to 4 h) is known to induce protein dissociation and denaturationas well as disorganisation of cytoskeletons (Salmon and Ellis, 1975;Swezey and Somero, 1985; Balny et al., 1997; Takahashi et al., 1997;Bourns et al., 1998). Takahashi et al. (1997) indeed reported an induc-tion of HSP70 mRNA, which serves to reorganise cytoskeletons, after2 h of exposure at 5 and 10 MPa in human chondrocyte cells undercontrol temperature. Our results suggest that, under sustained highpressure conditions, cellular damages are more important at low tem-perature than at warmer temperature. Usually, the effects of temper-ature and pressure on biochemical systems are antagonistic. Anincrease in pressure has similar effects to a decrease in temperature,reducing kinetic energy and membrane fluidity (Behan et al., 1992;Balny et al., 1997; Pradillon and Gaill, 2007). Similarly, the effects ofincreases in pressure and temperature on proteins are antagonisticwithin ecologically relevant ranges; the denaturation temperatureof proteins increases with increasing pressure (Balny et al., 1997).Studies on bacteria have previously shown that pressure increase en-hances the thermostability of several proteins and enzymes (Moritaand Mathemeier, 1964; Suzuki and Tanigushi, 1972; Hei and Clark,1994; Summit et al., 1998). Our results may therefore indicate an ad-ditive effect of high pressure and low temperature on protein dena-turation whilst warmer temperatures tend to counteract the effectof high pressure on denaturation process. This present work showsthat molecular and behavioural studies are complementary, andthat the examination of multiple hierarchical levels of biological orga-nisation is essential to understand the mechanisms involved in pres-sure and temperature tolerance.

5. Conclusions

Here, we have demonstrated that the shallow-water shrimp, P.varians, can tolerate prolonged exposure to pressure found outsideits normal depth range. An attempt was also made to maintain 10specimens for a month at reference temperature (10 °C) and highpressure (10 MPa). Cannibalism was observed after 10 days (20% ofshrimp were missing) and 70% of animals remained alive inside theIPOCAMP after 28 days when this preliminary experiment wasended. This result confirmed that P. varians can survive prolonged ex-posure of up to 28 days at high pressure and 10 °C. Most shallow-water adult invertebrates studied to date exhibit pressure convul-sions or spasms at around 5–10 MPa depending on temperature andrate of compression, followed by a progressive immobilisation athigher pressure (Macdonald and Gilchrist, 1978; Wilcock et al.,1978; Sébert, 2002; Thatje and Robinson, 2011). Wilcock et al.(1978) demonstrated that compression to 10 MPa is lethal in theshrimp Crangon crangon if applied for 8 h. The impressive pressuretolerance of P. varians may therefore reflect the physiological

capability of an ancestral species to colonise the deep sea. Our exper-iments also demonstrate that pressure does not appear as a limitingfactor for the colonisation of deep-sea habitats, but that cold temper-atures (≤5 °C) combined with elevated pressure conditions may limitsuch invasion by reducing the activity of the animals and presumablyincreasing cellular damages. Our results therefore support the hy-pothesis that deep-sea colonisation may have occurred during pe-riods when the oceanic water column was warm and verticallyhomogenous (Hessler and Wilson, 1983; Young et al., 1997; Tylerand Young, 1998; Tyler and Dixon, 2000). It also suggests that the po-tential for deep-sea invasion is not limited to early life stages and thatcolonisation through adult form is also a possible scenario. Furtherstudies on the ability of P. varians to complete its life cycle underhigh-pressure conditions are instrumental to confirm this hypothesis.

Acknowledgements

This work was funded through a research grant (Abyss2100) fromthe Total Foundation to Sven Thatje. Alastair Brown and Andrew Oli-phant were supported through PhD studentships from the NaturalEnvironment Research Council and the University of Southampton,respectively. The qPCR analyses were performed at the real-timePCR platform (IFR 83 Biologie Integrative, Université Pierre et MarieCurie). We are grateful to Samuel Bornens for his help in qPCR ana-lyses and we also wish to thank Christophe Piscart for his help in sta-tistical analyses.

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