acid base physiology response to ocean acidification of

RESEARCH ARTICLE

Acid–base physiology response to ocean acidificationof two ecologically and economically importantholothuroids from contrasting habitats, Holothuriascabra and Holothuria parva

Marie Collard & Igor Eeckhaut & Frank Dehairs & Philippe Dubois

Received: 3 April 2014 /Accepted: 24 June 2014 /Published online: 17 July 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Sea cucumbers are dominant invertebrates in sev-eral ecosystems such as coral reefs, seagrass meadows andmangroves. As bioturbators, they have an important ecologi-cal role in making available calcium carbonate and nutrientsto the rest of the community. However, due to their commer-cial value, they face overexploitation in the natural environ-ment. On top of that, occurring ocean acidification couldimpact these organisms, considered sensitive as echinodermsare osmoconformers, high-magnesium calcite producers andhave a low metabolism. As a first investigation of the impactof ocean acidification on sea cucumbers, we tested the impactof short-term (6 to 12 days) exposure to ocean acidification(seawater pH 7.7 and 7.4) on two sea cucumbers collected inSWMadagascar,Holothuria scabra, a high commercial valuespecies living in the seagrass meadows, and H. parva,inhabiting the mangroves. The former lives in a habitat withmoderate fluctuations of seawater chemistry (driven by day–night differences) while the second lives in a highly variableintertidal environment. In both species, pH of the coelomicfluid was significantly negatively affected by reduced seawater

pH, with a pronounced extracellular acidosis in individualsmaintained at pH 7.7 and 7.4. This acidosis was due to anincreased dissolved inorganic carbon content and pCO2 of thecoelomic fluid, indicating a limited diffusion of the CO2 to-wards the external medium. However, respiration and ammo-nium excretion rates were not affected. No evidence of accu-mulation of bicarbonate was observed to buffer the coelomicfluid pH. If this acidosis stays uncompensated for when facinglong-term exposure, other processes could be affected in bothspecies, eventually leading to impacts on their ecological role.

Keywords Sea cucumbers .Holothuria parva .Holothuriascabra . Ocean acidification . Acid–base regulation .

Echinoderms

Introduction

Sea cucumbers are major components of reefs, seagrassmeadows and mangrove ecosystems. They are responsiblefor a range of processes such as nutrient recycling (Hamelet al. 2001; Uthicke 2001a,b; Uthicke and Klumpp 1998), andcarbonate recycling with nearly 50% of the calcium carbonatedissolution occurring at nighttime on coral reefs being attrib-utable to sea cucumber digestive processes (Hammond 1981;Jansen and Ahrens 2004; Plotieau et al. 2013; Schneider et al.2011, 2013; Vaucher 2012). The recycling of nutrients canlead to increased community net and gross production byincreased availability of ammonium for primary producers(Uthicke and Klumpp 1998). In mangrove ecosystems, halfof the nutrient and carbon stocks are trapped in the sedimentsand made available to the trees by the bioturbation of benthic(Lovelock and Ellison 2007) organisms. Knowing that thegrowth of mangroves is nutrient-limited (Lovelock andEllison 2007), the supply brought by the bioturbation of sea

Responsible editor: Philippe Garrigues

Electronic supplementary material The online version of this article(doi:10.1007/s11356-014-3259-z) contains supplementary material,which is available to authorized users.

M. Collard : P. DuboisLaboratoire de Biologie Marine, Université Libre de Bruxelles, 50avenue F.D. Roosevelt, 1050 Brussels, Belgium

M. Collard (*) : F. DehairsLaboratory for Analytical, Environmental and Geo-Chemistry, EarthSystems Science research Group, Vrije Universiteit Brussel,Pleinlaan 2, 1050 Brussels, Belgiume-mail: [email protected]

I. EeckhautBiology ofMarine Organisms and Biomimetics, University ofMons,23 Place du Parc, 7000 Mons, Belgium

Environ Sci Pollut Res (2014) 21:13602–13614DOI 10.1007/s11356-014-3259-z

http://dx.doi.org/10.1007/s11356-014-3259-z

cucumbers present in those ecosystems is not negligible.These activities are not only beneficial to the ecosystemswhere the sea cucumbers live but also to those in close vicinity(for instance, seagrass beds near mangroves) as the quantity ofmaterial exchanged between mangroves and the other habitatsis at least half due to the biological activity (Lovelock andEllison 2007). Carbonate recycling occurs by dissolution ofcalcium carbonate in the gut of sea cucumbers. This process isdue to the respiratory carbon dioxide and secretion of diges-tive acids. Respiratory CO2 results in increased dissolvedinorganic carbon (DIC), which in turn leads to a decrease oftotal alkalinity (AT); these chemical changes togetherinduce a pH decrease of the gut content (Schneideret al. 2013). Subsequently, the dissolution of sedimentaryCaCO3contained in the gut increases AT and DIC, the formerpartially buffering the pH decrease (Schneider et al. 2011,2013). It was suggested that the added AT releasedduring defecation may impact chemistry of coral reefs withlong seawater residence time (Schneider et al. 2011, 2013).

Some of these sea cucumbers also have a high commer-cial value, sometimes reaching hundreds of dollars perkilogram of trepang (end-product of the processing of seacucumber tegument) (Conand 2008; Hamel et al. 2001;Purcell et al. 2013, 2014; Rasolofonirina et al. 2004).They represent a source of very high income for localfishermen in many countries, including Madagascar wherein 2001, 5–11 kg (wet weight) of sea cucumbers washarvested by fisherman and by day, with a total of 3,702fishermen estimated in 1996 (Anderson et al. 2011a,b;Eriksson et al. 2012; Rasolofonirina et al. 2004).Consequently, sea cucumbers are largely overfished andoverexploited in many countries of the world leading todeclines of populations of about 81 % of the commercialspecies (Conand 2008; Purcell et al. 2013, 2014). Thisoverexploitation may have major consequences on theecosystems as the role of these invertebrates is decreasedand the recycling of important nutrients and elements isreduced (Przeslawski et al. 2008). In recent years, maricul-ture of sea cucumbers was launched in different tropicalcountries (Eeckhaut et al. 2008; Eriksson et al. 2012;Purcell et al. 2013). In Madagascar, holothuriculture wasdeveloped in order to create sustainable harvesting andcommercialization of the sandfish H. scabra. The growthof this sea cucumber to the commercial size is done in seapens by local fishermen (Eeckhaut et al. 2008; Erikssonet al. 2012). They thus represent a financial income for thecompany but also for local fishermen and allow a reducedfishing pressure on the natural populations (Eeckhaut et al.2008; Eriksson et al . 2012; Purcell et al. 2013;Rasolofonirina et al. 2004).

In addition to this overexploitation, climate change isoccurring with an increased sea surface temperature anddecreased oceanic pH (Orr 2011). The dissolution of

atmospheric CO2 in seawater results in an increased con-centration of protons and bicarbonate ions and a de-creased concentration of carbonate ions. These phenome-na together are collectively known as ocean acidification(OA) (Orr 2011). It is believed that echinoderms, thegroup including sea cucumbers, will be highly affectedby OA and its consequences (i.e., hypercapnia and acido-sis) as they are osmoconformers, high-magnesium calcitecalcifiers, and have a low metabolism (Dupont et al. 2010;Melzner et al. 2009; Weber 1969). However, recent stud-ies have shown that adults of other echinoderms havephysiological abilities to withstand OA (Appelhans et al.2012; Collard et al. 2013a,b, 2014; Moulin et al. 2014;Stumpp et al. 2012). Whereas some sea urchins seem tobe able to tolerate OA by an increased buffer capacity ofthe coelomic fluid (CF; main internal fluid in echino-derms), which compensates for acidosis (Collard et al.2013a, 2014; Moulin et al. 2014; Stumpp et al. 2012),studied sea stars do not control acidosis of the CF butmaintain other physiological processes at the same levelas individuals maintained in control seawater (Appelhanset al. 2012; Collard et al. 2013b; McElroy et al. 2012;Nguyen and Byrne 2014; Schram et al. 2011).

In a previous study, we showed that the pH andbuffer capacity of the sea cucumber CF were similarto that found in sea stars (Collard et al. 2013a). If OAleads to hypercapnia and acidosis within the internalfluids of sea cucumbers, this could not only impactnatural populations and thus their role in the ecosystemfunctioning (recycling) but also their growth within thesea pens situated in the open sea and thus the commercialoutput of the mariculture of sea cucumbers, includingH. scabra in Madagascar. However, despite their high com-mercial value, no information is available on the potentialimpact of OA on sea cucumbers.

Therefore, in this study, we investigated the impact of OAon the acid–base physiology of two sea cucumbers, the highcommercial value species Holothuria scabra Jaeger, 1833,living in the seagrass meadows of Madagascar, and H. parvaKrauss, 1885, inhabiting the mangroves of SW Madagascar.The former lives in a habitat with moderate fluctuations ofseawater chemistry. It was reported that the metabolic activityof the seagrasses, in which H. scabra may be found, couldbuffer OA (Hendriks et al. 2014). However, no study hasinvestigated to what extent this influence occurred.Furthermore, the current decline of these meadows worldwidequestions further their future ability to counteract OA(Hendriks et al. 2014) and a recent study at a CO2 vent siteshowed that some seagrasses do not cope well with reducedseawater pH, but the response is highly species-specific(Apostolaki et al. 2014). The second species, H. parva, livesin a highly variable intertidal environment on the edge of amangrove. Parameters of the CF (pH, buffer capacity as AT,

Environ Sci Pollut Res (2014) 21:13602–13614 13603

DIC, isotopic signature of the DIC) were measured as well asthe metabolism of the individuals (oxygen uptake and ammo-nium excretion rates).

Materials and methods

Sampling sites and aquarium systems

Due to overfishing, H. scabra is now very rare on the reefs ofMadagascar. Consequently, juveniles (9±2 gww, n=54) wereacquired from the mariculture facilities of Indian OceanTrepang (Toliara, Madagascar) in May 2013. Conditions inthe outdoor ponds at collection time (daytime) were as fol-lows: temperature 27 °C, salinity 28 and pH (total scale) 8.2.Adults of the species H. parva (28±9 gww, n=36) werecollected 1 week later in the mangroves just behind the seacucumber farm in Belaza, Madagascar (20 km south ofToliara). The specimens were collected under rocks at lowtide when totally emerged. The conditions of the interstitialwater were as follows (daytime, low tide): temperature27.5 °C, salinity 20 and pH (total scale) 7.3; at the samemoment, conditions in the nearby open sea were as follows:temperature 27 °C, salinity 32 and pH (total scale) 8.0.Moreover, temperature and salinity values in the Tolaria Bayvary between 22 °C and 35.5 °C and 29 and 35 PSU, respec-tively, over the year (Delroisse et al. 2013; Lavitra et al. 2006;Rasolofonirina et al. 2005; Vaïtilingon et al. 2003, 2004).Also, day–night differences in temperature of about 1 °C weremeasured during April–May 2014 (G. Tsiresy, M. Schaltz andI. Eeckhaut, unpublished data).

Both species were brought back to the Institut Halieutiqueet des Sciences Marines of Toliara, Madagascar, and main-tained there. The system included 18 independent 25-l tanks,each provided with constantly aerated natural seawaterpumped from the bay of Tolaria (at least 25 % of the waterwas renewed daily) and 2–3 cm of sediments (as feeding takesplace in the first 2 cm of the sediments; Plotieau et al.2013)from the mariculture farm of Belaza. Temperature rangedfrom 24 °C to 25 °C and salinity was around 33.5 in all tanks(Table 1). The conditions used in the present experiment arefrequently encountered by both species (see above) and werechosen as a compromise between those two species. Theseconditions are close to the open sea conditions encountered byH. parva during high tide and by cultured juveniles H. scabra(the culture sea pens are located right outside the mangrovesand thus have conditions similar to that of open sea valuesmeasured forH. parva). The sea cucumbers were left 3–4 daysin those conditions before any decrease of pH. Furthermore,the behavior of juvenileH. scabrawas reported to not changeat temperatures ranging from 24 °C to 27 °C and salinitiesabove 25 (Mercier et al. 1999).

To assess water quality, the concentration in nitrates(NO3

−) and ammonium (NH4+) were measured in the different

tanks. Samples of 12 ml of seawater were taken, filteredthrough a 0.22-μm filter (Millipore) and frozen immediatelyat −20 °C until further analysis. The samples were analyzed atthe laboratory in Brussels, Belgium, by an automatic colori-metric method using a QuAAtro nutrient analyzer coupled to aXY-2 auto sampler (Seal Analytical, Mecquon, WI, USA).The calibration was done using solutions of known concen-trations of KNO3 ranging from 0 to 80.14 μmol l−1 for thenitrates and NH4Cl ranging from 0 to 149.18 μmol l−1 forammonium. Concentrations of nitrates ranged from 23.48 to52.93 μmol l−1 and those of ammonium ranged between 1.53and 3.43μmol l−1 throughout the experiment. The two specieswere maintained in independent triplicated tanks for eachtreatment with five specimens per tank for H. parva and eightper tank for H. scabra (the two species were maintainedseparately). Three times a day, pH, electromotive force (emf)and temperature were measured in all tanks with a MetrohmpH-meter (826 pH mobile, combined glass electrodeMetrohm 6.0228.010) calibrated with CertiPUR® buffer so-lutions pH 4.00 and 7.00 (Merck, Darmstadt, Germany).Salinity was also measured three times a day using a salinom-eter Multi 340i (WTW, Germany). All measurements wereconverted to total scale according to DelValls and Dickson(1998)’s method with TRIS/AMP buffers (kindly provided bythe Biogeochemistry and Earth System Modeling Laboratoryof the Université Libre de Bruxelles, Belgium).

According to the predictions for the global open ocean(Caldeira andWickett 2003, 2005), and as no local predictionsare available for Madagascar, the targeted pH values were 7.7and 7.4. As interstitial seawater pH decreases to 7.3 duringlow tide for H. parva, seagrass respiration can lead to de-creases in pH of up to 0.5 units at night (Invers et al. 1997;Marba et al. 2006; Hofmann et al. 2011; Saderne et al. 2013)and as both species are also exposed to open ocean conditions,these values are ecologically relevant for the two speciesinvestigated. These values were reached (gradual decrease of0.1 pH unit per day; total duration 5–6 days) and maintainedby bubbling CO2. The flow of CO2 was regulated by anautomatic computer-controlled system Aquastar (iksComputerSysteme GmbH, Karlsbad, Germany), which wascalibrated against the Metrohm measures three times a day.Effective pH values were significantly different in the threepH treatments for both species (pANOVA<10

−3, pTukey<10−3 in

all cases, species tested separately) and are reported in Table 1.Total exposure time was of 12 days for H. scabra and 6 daysfor H. parva (counting from the moment the conditions wereestablished). Short and different times of exposure were due totime limit constraints.

In order to determine AT, seawater samples (6 ml) werecollected every 6 days in all tanks, immediately filteredthrough 0.22-μm filters (Millipore) and poisoned with

13604 Environ Sci Pollut Res (2014) 21:13602–13614

Tab

le1

Water

andcarbonatesystem

parameters(m

ean±SD

)

Species

Nom

inalpH

Effectiv

epH

TS

A TDIC

pCO2

CO2

HCO3−

CO32−

ΩCa

ΩAr

Totalscale

°CPS

Ummol

kgSW−1

mmol

l−1

μatm

μmol

kgSW−1

Holothuriaparva

7.4

7.41

±0.08

24.7±0.6

33.4±0.1

2.88–2.80

2.84–2.79

2,435–3,865

69–83

2,690–2,644

82–65

2.0–1.6

1.3–1.0

7.4

7.44

±0.11

24.7±0.7

33.4±0.1

2.79–2.86

2.71–2.84

1,838–2,721

52–79

2,557–2,691

98–70

2.4–1.7

1.6–1.1

7.4

7.41

±0.06

24.6±0.8

33.4±0.1

2.88–2.90

2.86–2.83

2,883–2,008

82–58

2,712–2,679

70–95

1.7–2.3

1.1–1.5

7.7

7.68

±0.06

24.9±0.6

33.4±0.1

2.74–2.83

2.63–2.68

1,481–1,247

42–36

2,474–2,513

113–134

2.8–3.3

1.8–2.1

7.7

7.68

±0.11

24.7±0.8

33.4±0.1

2.79–2.86

2.74–2.71

2,138–1,195

61–35

2,592–2,529

86–141

2.1–3.4

1.4–2.3

7.7

7.66

±0.08

24.8±0.8

33.4±0.1

2.83–2.92

2.68–2.81

1,276–1,654

36–48

2,508–2,652

136–113

3.3–2.8

2.2 –1.8

8.0

7.88

±0.03

24.9±0.9

33.4±0.1

2.84–2.70

2.60–2.47

822–760

23–22

2,389–2,270

191–181

4.7–4.4

3.1–2.9

8.0

7.94

±0.06

24.8±0.8

33.4±0.1

2.84–2.84

2.55–2.58

629–719

18–21

2,303–2,357

230–204

5.6–5.0

3.7–3.3

8.0

7.93

±0.05

24.8±0.9

33.4±0.1

2.80–2.79

2.52–2.55

637–727

18–21

2,279–2,329

223–197

5.5–4.8

3.6–3.1

Holothuriascabra

7.4

7.34

±0.22

24.2±1.2

33.5±0.2

2.97

±0.03

2.99

±0.05

3,599±500

105±15

2,829±38

60±8

1.5±0.2

1.0±0.1

7.4

7.43

±0.06

24.1±1.3

33.5±0.2

2.88

±0.04

2.87

±0.04

2,727±147

80±3

2,718±35

71±4

1.7±0.1

1.1±0.1

7.4

7.33

±0.10

24.0±1.3

33.5±0.2

2.83

±0.09

2.84

±0.12

3,213±699

94±22

2,684±105

60±12

1.5±0.3

1.0±0.2

7.7

7.67

±0.06

24.3±1.2

33.5±0.2

2.88

±0.05

2.76

±0.05

1,500±57

43±1

2,597±46

120±1

2.9±0.0

1.9±0.0

7.7

7.59

±0.15

24.1±1.3

33.5±0.2

2.84

±0.06

2.73

±0.07

1,591±548

46±16

2,562±85

117±34

2.9±0.8

1.9±0.5

7.7

7.67

±0.08

24.0±1.4

33.6±0.3

2.77

±0.05

2.65

±0.05

1,399±112

41±2

2,495±45

118±5

2.9±0.1

1.9±0.1

8.0

7.92

±0.04

23.9±1.4

33.6±0.3

2.74

±0.04

2.49

±0.02

690±64

20±2

2,277±4

196±19

4.8±0.5

3.1±0.3

8.0

7.93

±0.04

24.0±1.5

33.6±0.3

2.74

±0.02

2.49

±0.01

676±45

20±1

2,268±16

198±15

4.8±0.4

3.2±0.3

8.0

7.94

±0.05

24.0±1.4

33.6±0.3

2.74

±0.12

2.48

±0.12

643±63

19±2

2,258±118

205±5

5.0±0.1

3.3±0.1

ForH

.scabraaquaria,n=39

forp

H,tem

peratureandsalin

ity(m

easuredthreetim

esadayfrom

D0toD12),3forallotherparam

eters(A

Tmeasuredon

days

0,6and12).For

H.parva

aquaria,n=21

forpH,

temperature

andsalin

ity(m

easuredthreetim

esadayfrom

D0to

D6),A

Tmeasuredon

days

0(firstvalue)

and6(secondvalue).P

aram

etersof

thecarbonatesystem

weremeasuredfrom

A TandpH

T

Ttemperature,S

salin

ity,A

Ttotalalkalinity,D

ICdissolvedinorganiccarbon,Ω

Casaturatio

nstateof

calcite,Ω

Arsaturatio

nstateof

aragonite


mercury chloride (HgCl2; 7 % w/w). These samples weremaintained at 4 °C in the dark until further analysis (analysisdone at the laboratory in Brussels, Belgium). On the samedays, 3 ml seawater were taken from each tank and transferredto gas-tight Exetainer tubes (Labco Limited, UK) and poi-soned with HgCl2 (7 % w/w) for further analysis of DIC andits isotopic signature (analysis done at the laboratory inBrussels, Belgium). These Exetainer glass tubes were main-tained at 4 °C and in the dark until further analysis.

Measurements in seawater

The DIC samples were prepared following the method ofGillikin et al. (2007) adapted as described by Collard et al.(2014). Briefly, a headspace of 0.75 ml helium was created inthe Exetainers using a gas-tight syringe and 50 μl of 99 %phosphoric acid were injected. The tubes were left on anagitating plate for at least 12 h before analysis. We used theprotocol combining the measurements of DIC content andcarbon isotopic ratio developed for sea urchin CF (Collardet al. 2014). Sample CO2was injected through a GC portmounted in front of the reduction column of an ElementalAnalyzer (Flash 1112 series EAThermo) coupled online via aConflo III to an isotope-ratio mass spectrometer (Delta V,Thermo) (Gillikin and Bouillon 2007). The DIC calibrationwas done using NaHCO3 (Sigma-Aldrich) solutions of knownconcentrations ranging from 0 to 11 mmol l−1, and plottingDIC concentration versus area of the total signal peak (‘areaall’ in mV) of CO2detected by the mass spectrometer. Error onthe measurements of standard certified material provided byAndrew G. Dickson’s Oceanic Carbon Dioxide QualityControl Laboratory was of 3.9±2.7 % (mean±SD, n=12).For the carbon isotopic ratio, the samples were calibratedagainst the standard NBS-19 (δ13C=+1.95‰) and data arereported as ‰VPDB using the conventional delta notation:

δ13C ¼ Rsample=Rstandard– 1� � � 1; 000;

where R=13C/12C.The AT of the seawater was determined by means of a

potentiometric titration method adapted to small volumes sothat the measurements were carried out in the same way asthose for the CF (see below) and could be compared (Collardet al. 2013a; Gran 1952). Briefly, a potentiometric titrationwas realized on a 0.5 ml sample by adding first 5 μl of 0.1 MHCl (Merck) with 0.7 M NaCl and then 1 μl at a time. Aftereach addition, the sample was agitated and pHmeasured usinga 3-mm-diameter glass microelectrode (reference 6.0224.100;Metrohm, Darmstadt, Germany). The AT-SW was then calcu-lated using Gran’s function (Gran 1952). The measures of ATwere corrected by using standard certified material provided

by Andrew G. Dickson’s Oceanic Carbon Dioxide QualityControl Laboratory.

Aragonite and calcite saturation state values (Ω) as well aspCO2 and the concentrations of the carbonate system compo-nents were calculated from AT, pH (total scale), salinity andtemperature data using the software CO2SYS (Pierrot et al.2006) with the dissociation constants for carbonate fromMehrbach et al. (1973) refitted by Dickson and Millero(1987), and for KSO4 from Dickson (1990) (Table 1).

Physiological parameters

For both species, pH (pHCF) and total alkalinity (AT-CF)were measured in the CF on two individuals per tank ondays 0 and 6, and also 12 for H. scabra. DIC of the CF(DICCF) was measured in H. parva only (due to limitedavailable volume of CF for H. scabra) for two individualsper tank on days 0 and 6.

pHCF and AT-CF were measured according to the methoddescribed by Collard et al. (2013a). Briefly, 1 ml was takenfrom the sea cucumber by cutting through the tegument on theventral side right behind the mouth and taking care to not haveany stomach content in the sample (as it contains carbonatesediments) and were transferred into an Eppendorf tube(Animals were sacrificed and never used twice). The pH wasimmediately measured using the microelectrode describedabove for seawater samples. As CF has a composition closeto that of seawater (Santos et al. 2012; Stickle and Diehl1987), pH was transformed to total scale as for seawatersamples (DelValls and Dickson 1998). Once the initial pHwas measured, 0.5 ml was transferred to a second Eppendorftube and AT-CF measured immediately. The method for AT-CF

measurement was the same as previously described for sea-water. For the DIC measurement, 4 ml of CF were extractedfrom a sea cucumber. The CF was transferred into twoEppendorf tubes filling them up completely. In order to re-move coelomocytes, the tubes were centrifuged at 2,000×gfor 5 min (Microcentrifuge Rotilabo, Carl Roth, GmbH+Co.,Karlsruhe, Germany) while keeping the original tank watertemperature. Thereafter, the CF was transferred into a 3-mlExetainer tube (Labco Limited) and HgCl2 was added (7 %w/w) to avoid any remaining biological activity. Tubes werethen sealed and kept at 4 °C until further analysis (same asdescribed above for seawater). The obtained values for AT-CFwere corrected by using standard certified material providedby Andrew G. Dickson’s Oceanic Carbon Dioxide QualityControl Laboratory. Aragonite and calcite saturation statevalues (Ω) as well as pCO2 and the concentrations of thecarbonate system components were calculated from DICCF

and/or AT-CF, pHCF (total scale), salinity and temperature datausing the software CO2SYS (Pierrot et al. 2006) as forseawater.


Using the measured values of DICCF for H. parva, weinvestigated the possibility of calculating DICCF from AT-CFand pHCF with CO2SYS as for seawater. As it was previouslyshown for other echinoderms (sea urchins) that this methodoverestimates DICCF because of buffering compounds otherthan the CO2–bicarbonate system (Collard et al. 2013a;Moulin et al. 2014), we compared the values obtained bymeasurement and calculations for the species in which wehad both (Table 2) before applying the calculation method tothe data of H. scabra. Measures for the treatment pH 7.7 onday 6 were left out of the analysis as there was an alkalinityanomaly in the measurements of the CF which induced un-derestimations of DICCF. We compared the means obtainedfor the different pH treatments and found no significantdifferences between measured values of DICCF and thosecalculated from AT-CF and pHCF (paired t-test, p=0.352).Therefore, DICCF was calculated from AT-CF and pHCF forH. scabra.

Metabolic parameters

Oxygen uptake and ammonium excretion rates measurementswere carried out on two individuals and the seawater of eachtank after 3 and 9 days of exposure for H. scabra and after3 days for H. parva. Firstly, the sea cucumbers (or seawater)were placed in sealed Plexiglas respirometry chambers (50 mlfor H. scabra and 800 ml for H. parva) filled with water fromthe experimental tank, equippedwith an optode oxygen sensor(PreSens, Regensburg, Germany) and covered with blackplastic. A magnetic stirrer, protected from the sea cucumber

by a mesh cage, was placed at the bottom of the chamber inorder to homogenize the oxygen concentration in the wholechamber. The oxygen saturation of the water was measuredevery 5 min for a total time of 1 h using a Fibox 3 PC-controlled fiber-optic oxygen meter (PreSens) and data wasacquired with Fibox 3 software v602 (PreSens) (calibrationand salinity corrections were done following manual instruc-tions). At the end of the measurements, the wet weight of thesea cucumber was measured with a Page Profi scale (Soehnle,Backnang, Germany; precision: ±1 g). The oxygen uptake ratewas calculated using the slope of the linear regression ofseawater oxygen content against time. This value was normal-ized by the seawater volume (based on the seawater densitycalculations from Millero and Huang 2009 and Millero andPoisson 1981) and the wet weight of the animal. The O2

measurements for the sea cucumbers were corrected by thatof the controls with only seawater from the same tank as thesea cucumber. The rate of oxygen uptake was expressed inμmol of O2 per hour and per gram of wet weight. Secondly, atthe beginning and the end of the oxygen uptake measurement,samples of 12 ml of seawater were taken for NH4

+ measure-ments, filtered through a 0.22-μm porosity filters (Millipore)and frozen immediately at −20 °C until further analysis. Thesamples were analyzed as described previously for seawater.The calibration was done using solutions of known concen-tration of NH4Cl ranging from 0 to 149.18 μmol l−1. Themeasurements for the sea cucumbers were corrected by that ofthe controls with only seawater from the same tank as the seacucumber. Ammonium excretion rate is expressed as μmol ofNH4

+ per hour and grams of wet weight.

Table 2 Carbonate system parameters (mean±SD) of the coelomic fluid of H. parva calculated from AT-CF/DICCF and pHCF using CO2SYS

Day Nominal pH pHCF T S DICCF AT-CF pCO2 CO2 HCO3− CO3

2− Ω Ca Ω Ar

Total scale °C PSU mmol l−1 mmol kgSW−1 μatm μmol/kg SW

Calculated from pHCF and DICCF

0 7.4 7.29±0.08 25.3±0.1 33.3±0.0 3.22±0.21 3.19±0.22 4,034±678 114±19 3,044±196 65±16 1.6±0.4 1.0±0.3

7.7 7.44±0.06 25.3±0.1 33.2±0.2 3.00±0.07 3.04±0.08 2,647±365 75±10 2,842±68 85±12 2.1±0.3 1.4±0.2

8.0 7.74±0.09 25.2±0.1 33.3±0.0 3.05±0.21 3.23±0.22 1,336±294 38±8 2,839±194 170±34 4.2±0.8 2.7±0.5

6 7.4 7.28±0.03 24.3±0.0 33.4±0.0 3.49±0.26 3.45±0.26 4,376±450 127±13 3,299±248 66±6 1.6±0.2 1.1±0.1

7.7 7.41±0.04 24.3±0.1 33.3±0.2 3.06±0.24 3.08±0.25 2,856±202 83±6 2,896±225 78±12 1.9±0.3 1.2±0.2

8.0 7.62±0.04 24.3±0.1 33.5±0.0 2.79±0.29 2.90±0.29 1,602±261 47±7 2,632±277 115±13 2.8±0.3 1.8±0.2

Calculated from pHCF and AT-CF

0 7.4 7.29±0.08 25.3±0.1 33.3±0.0 3.26±0.24 3.23±0.26 4,073±610 115±17 3,082±231 66±17 1.6±0.4 1.1±0.3

7.7 7.44±0.06 25.3±0.1 33.2±0.2 2.93±0.20 2.97±0.20 2,581±372 73±11 2,774±185 83±14 2.0±0.3 1.3±0.2

8.0 7.74±0.09 25.2±0.1 33.3±0.0 2.98±0.31 3.16±0.28 1,327±388 38±11 2,781±306 165±18 4.0±0.4 2.6±0.3

6 7.4 7.28±0.03 24.3±0.0 33.4±0.0 3.45±0.30 3.41±0.30 4,316±293 125±9 3,263±287 66±9 1.6±0.2 1.0±0.1

7.7 7.41±0.04 24.3±0.1 33.3±0.2 2.60±0.13 2.62±0.13 2,441±270 71±8 2,464±125 66±6 1.6±0.2 1.1±0.1

8.0 7.62±0.04 24.3±0.1 33.5±0.0 3.11±0.17 3.23±0.18 1,780±207 52±6 2,930±163 128±12 3.1±0.3 2.1±0.2

n=6 for all except on day 6 for pH 7.4 and 8.0 where n=4 and day 6 pH 7.7 where n=5

AT total alkalinity, DIC dissolved inorganic carbon content, Ω Ca saturation state of calcite, Ω Ar saturation state of aragonite


Statistical analysis

All ANOVAs models were built according to the recommen-dations of Doncaster and Davey (2007). Sample size variesthroughout the experiment (n=4, 5 or 6) and sampling due tosome CF samples being contaminated with carbonate sedi-ments from the gut or samples being lost during analysis. Thenumber of samples for each physiological or metabolic pa-rameter measured, species and day is reported in Table S1.pHCF, ΔAT (H. parva only) and DICCF were all analyzedusing cross-factored nested model III ANOVAs (tank, randomfactor, nested into seawater pH, fixed factor, and time, crossedfixed factor) followed by Tukey tests for multiple comparisons(using the appropriate mean square as error). This modelallows taking into consideration that the individuals sampledin the same tank are not true replicates (MS of the seawater pHeffect is divided by that of the tanks nested in seawater pH tocalculate the F-ratio instead of the model error; see Table S2).Due to missing data, ΔAT of H. scabra was analyzed usingmeans per aquarium in a two-way crossed factor ANOVA(pH, fixed factor, time, fixed factor) followed by Tukey testsfor multiple comparisons. δ13C was analyzed using a three-factor ANOVAmodel with repeated measures on cross factors(tank, random factor, nested into seawater pH, fixed factor,time, crossed fixed repeated factor, and sample — CF orseawater — crossed fixed repeated factor) followed byTukey tests for multiple comparisons. The measured andcalculated AT-CF and DICCF for H. parva were compared witha paired t-test. Respiration and ammonium excretion rateswere analyzed using cross-factored nested model IIIANOVAs (tank, random factor, nested into seawater pH, fixedfactor, and time, crossed fixed factor) followed by Tukey testsfor multiple comparisons for H. scabra and two-factor nestedmodel III ANOVAs (tank, random factor, nested into seawaterpH, fixed factor) followed by Tukey tests for multiple com-parisons for H. parva. All correlation analyses were carriedout using simple correlation of Spearman with associatedBonferroni probabilities. All tests were realized using thesoftware Systat 12 (Systat Software Inc., USA). All ANOVAtables are presented in Table S2.

Results

No mortality occurred during the experiments in both species.

Physiological parameters

H. parva showed a significantly reduced pHCF for both pHtreatments compared to the control but also a significantdifference between the two low pH treatments (Fig. 1a; pHpANOVA=0.001, pTukey≤0.043). This significant effect wasfurther reinforced by a significant correlation between

pHCF and pHSW (r=0.899, p<10−3). pHCF was not signif-icantly affected by time for individuals maintained atpH 7.7 and 7.4 (pH×time pANOVA=0.056, pTukey≥0.986),but it did significantly decrease between 0 and 6 days ofexposure for control individuals (pTukey=0.034). Total al-kalinity of the CF (expressed as ΔAT=AT-CF−AT-SW)showed an anomaly in the measurements for individualsfrom the treatment pH 7.7 on day 6 with a very low ΔAT(Table 2). In order to avoid confounding effects, we inves-tigated the possibility of calculating AT-CF from measuredDICCF and pHCF (all data except pH 7.7, D6; Table 2). Wefound no significant differences between measured valuesof AT-CF and those calculated from DICCF and pHCF (pairedt-test, p=0.316). Therefore, we used the calculated AT-CFfor further analysis. There was no significant differencebetween individuals maintained at the different treatments(pH pANOVA=0.196; Fig. 1b) or according to time (timepANOVA=0.976); the interaction was also not significant(pH×time pANOVA=0.129). The correlation analysis be-tween ΔAT-CF and pHSW was not significant (r=−0.171,p=0.349). The DIC content (Fig. 1c) was significantlyhigher for individuals maintained at pH 7.4 compared tothe two other treatments (pH pANOVA=0.011, pTukey≤0.025) but no significant difference appeared in betweenthese (pTukey=0.389). Time had no significant effect (timepANOVA=0.515), as well as the interaction between pH andtime (pH×time pANOVA=0.111). This is further supportedby a significant correlation between DICCF and pHSW (r=−0.450, p=0.010). The relationship between DICCF andpHCF was also significant (r=−0.414, p=0.019). Finally,we compared the CF carbon isotopic signature (δ13CCF)and that of the SW in which the individuals were main-tained (δ13CSW) (Fig. 2). The δ13CCF was significantlydifferent from δ13CSW (sample pANOVA = 0.045).However, the slopes of the relationships between δ13CCF/

SW and pHSW were not different (homogeneity of slopes,p=0.924). There was no significant effect of pH, day, theinteraction between pH and day, and the interaction be-tween pH, day and sample (pANOVA=0.113, 0.085, 0.644and 0.619, respectively). Nonetheless, there was a signifi-cant correlation between δ13CCF and pHSW (r=0.551, p=0.001) and the correlation between δ13CCF and δ

13CSW wasalso significant (r=0.755, p<10−3).

H. scabra showed a significantly reduced pHCF for in-dividuals maintained at both low pH treatments comparedto control individuals (Fig. 1d; pH pANOVA=0.001, pTukey≤0.009) but these did not significantly differ from one an-other (pTukey=0.090). This significant effect was furtherreinforced by a significant correlation between pHCF andpHSW (r=0.857, p<10−3). pHCF was not significantly af-fected by time (time pANOVA=0.382). ΔAT-CF (Fig. 1e) wasnot affected by pHSW (pH pANOVA=0.134), but did differsignificantly according to time (time pANOVA=0.001) with


Fig. 1 Physiological parameters of the coelomic fluid according to time.a Coelomic fluid pH (mean±SD; total scale) of H. parva. b Deltaalkalinity (mean±SD;ΔAT; AT of the coelomic fluid−AT of the seawater)ofH. parva. cDissolved inorganic carbon (mean±SD; DIC) ofH. parva.dCoelomic fluid pH (mean±SD; total scale) ofH. scabra. eΔAT (mean±

SD) ofH. scabra. fDIC (mean±SD) ofH. scabra. D days of exposure toexperimental pH (after a gradual decrease). Treatment pH 8.0: solid line,round markers; treatment pH 7.7: dashed line, triangle markers; treat-ment pH 7.4: dashed and dotted line, square markers. The number ofsamples is reported in Table S1

Fig. 2 δ13C (mean±SD; ‰) ofDIC of the coelomic fluid ofH. parva and of thecorresponding tank seawateraccording to seawater pH (n=3for seawater; the number ofsamples for coelomic fluid isreported in Table S1)


days 0 and 6 being significantly different from day 12(pTukey≤ 0.014) but not different in between them(pTukey=0.287); the interaction between pH and time wasnot significant (pH×time pANOVA =0.480). The correlationanalysis betweenΔAT-CF and pHSW was not significant (r=−0.245, p=0.097). DICCF (Fig. 1f) was significantly higherin individuals maintained at pH 7.4 compared to thosefrom the two other treatments (pH pANOVA=0.001,pTukey≤0.003) but no significant difference appeared inbetween these (pTukey=0.060). This is supported by a sig-nificant correlation between DICCF and pHSW (r=0.525,p<10−3). The relationship between DICCF and pHCF wasalso significant (r=−0.848, p=0.001). Time had a signifi-cant impact on DIC content for this species with values onday 0 being significantly lower from those on day 12 (timepANOVA=0.031, pTukey=0.039), while day 6 is not signifi-cantly different from any of the two other days (pTukey≥0.073).

Metabolic parameters

In order to assess the impact of OA on the metabolism ofthe sea cucumbers, oxygen uptake and ammonium excre-tion rates were measured. For H. parva, oxygen uptakerate was not significantly affected by the different pHtreatments (Fig. 3a; pH pANOVA=0.929). Ammonium ex-cretion rate was also unaffected by pHSW (Fig. 3b; pHpANOVA=0.334).

H. scabra showed no significant effect of pHSW (pHpANOVA=0.326) or time (time pANOVA=0.967) on oxygenuptake rate (Fig. 3c). Ammonium excretion rate was alsounaffected by pHSW or time (Fig. 3d; pANOVA=0.237 and0.421, respectively).

Discussion

Reduced seawater pH (pHSW) resulted in a significantlylowered CF pH (pHCF) in both species, H. scabra andH. parva, from about 7.5–7.7 for control individuals to 7.2–7.3 for treatment individuals. In the case ofH. parva, pHCF ofindividuals maintained at pH 7.4 was significantly lower thanpHCF of individuals maintained at pH 7.7 but this did notoccur in the case ofH. scabra. This decreased extracellular pHcould have consequences on the general physiological andmetabolic processes occurring in the sea cucumber as it maylead to changes in intracellular pH regulation, and a decreasein the latter, as seen for other invertebrates (Langenbuch andPörtner 2002; Reipschläger and Pörtner 1996), but also as itcould influence enzymes and their activities (Clarke 1998).

This decreased pHCF was accompanied by an increasedDICCF and thus an increased CO2concentration (Tables 2 and3), thereby indicating a state of hypercapnia of the CF for bothspecies when pHSW is reduced. Since echinoderms mostlydepend on a favorable gradient from the internal fluid to thesurrounding seawater to eliminate excess CO2 (mostly of

Fig. 3 Metabolic parameters of the sea cucumbers according to pHtreatment and time. a Oxygen uptake rate (mean±SD) of H. parva. bAmmonium excretion rate (mean±SD) ofH. parva. cOxygen uptake rate

(mean±SD) of H. scabra. d Ammonium excretion rate (mean±SD) ofH. scabra. The number of samples is reported in Table S1


metabolic origin) (Seibel and Walsh 2003), a reduced pHSW,and thus an increased CO2 concentration in seawater, wouldreduce this gradient. This results in an accumulation of CO2

inside the CF, leading to hypercapnia and, due to the dissoci-ation of CO2, to acidosis of the fluid. This is also what wasshown previously for starfish (Appelhans et al. 2012; Collardet al. 2013b; Dupont and Thorndyke 2012). For the seacucumbers studied here, the increased DIC is accompaniedby an increased pCO2 of the CF. It is noteworthy that thisincreased pCO2 is not proportional to the increase of theseawater pCO2, leading to an increased ΔpCO2. In the faceof OA and hypercapnia, it is important for animals to maintainthe ΔpCO2 with the environment to ensure elimination ofmetabolic CO2. The increased ΔpCO2 observed for bothspecies, H. scabra (from 1,049±554 μatm for individualsmaintained at control conditions to 1,647±813 μatm for thosemaintained at pH 7.4) andH. parva (from 798±353 to 1,793±389 μatm), indicates that the outward flux of CO2 is notmaintained but slowed in the face of hypercapnic surroundingseawater. This most probably causes the observed acidosis ofthe CF of the sea cucumbers. The cause of this reducedoutward flux of CO2 at reduced pHSW, once the ΔpCO2 isreestablished, is not clear. Gas exchanges between SWand CFare principally occurring due to the pumping of seawater inthe respiratory trees which can extend to three quarters of thebody length (Cherbonnier 1988; Lawrence 1987). However,no change in oxygen uptake according to pHSWwas recorded,suggesting that gas exchange was not directly affected. pHSW

could also influence the behavior of the sea cucumbers.H. parva is an intertidal species which is totally emerged atlow tide and stays half burrowed in sand under rocks.When inthis state, the individuals are swollen with seawater (princi-pally accumulated in the respiratory trees). This results in zeropossibility of gas exchanges with the environment until thetide rises again. On the other hand, H. scabra lives in a much

more stable environment where they are submerged at alltimes. However, it was shown previously that individuals ofH. scabra stay burrowed when the water column above thesediment surface is ≤10 cm high (Mercier et al. 1999). So,both species respond to stress conditions by burrowing whichin turn reduces or suppresses gas exchanges. If low pHSW

induces the same behavior, this could explain the increasedΔpCO2. The oxygen uptake rate of both species did notchange with pHSW, indicating a tolerance of the metabolicactivity to short-term exposure. The ammonium excretion ratewas also unaffected by reduced pHSW. Nonetheless, it shouldbe taken into account that these measures were carried out onsea cucumbers removed from the sediment and placed in cleanseawater at the respective pHSW. This indicates that the po-tential oxygen uptake rate is not significantly affected bypHSW but this does not exclude the aforesaid behavioralchanges. The influence of pHSWon behavior of sea cucumbersdeserves further studies.

For H. parva, the carbon isotopic signals (δ13C) of the CFwere not significantly different from that of the SW they weremaintained in. So, although the CO2 gradient is still frominside to outside (as pCO2CF is higher than pCO2SW), thesignal of the CF reflects that of the environment. The contri-bution of DIC from the SW is thus not negligible. Seawaterenters the sea cucumber in two ways. First, it is ingestedtogether with the sediments into the gut, and Holtmann et al.(2013) hypothesized the existence of an electrogenic HCO3

−

transport from the gut lumen into the CF in a sea urchin.Secondly, seawater fills the respiratory trees. The O2 andCO2 exchanges occur through the membrane of terminalvesicles of the respiratory trees using a favorable gradient.The latter is sustained by constant renewal of the SW in therespiratory trees (Farmanfarmaian 1966). If the respiratorytree wall is permeable to HCO3

− and/or is provided, togetherwith the gut wall, with anion transporters allowing the entry of

Table 3 Carbonate system parameters (mean±SD; n is indicated in between parentheses) of the coelomic fluid of H. scabra calculated from AT-CF andpHCF using CO2SYS

Day Nominal pH pHCF T S DICCF AT-CF pCO2 CO2 HCO3− CO3

2− Ω Ca Ω Ar

Total scale °C PSU mmol l−1 mmol kgSW−1 μatm μmol/kg SW

0 7.4 7.21±0.06 22.7±0.3 33.6±0.0 2.86±0.19 2.79±0.17 4,226±719 128±21 2,686±174 43±3 1.0±0.1 0.7±0.1

7.7 7.39±0.08 23.3±0.1 33.8±0.2 2.63±0.17 2.64±0.17 2,626±596 78±18 2,493±161 62±11 1.5±0.3 1.0±0.2

8.0 7.55±0.03 21.7±0.1 33.9±0.0 2.32±0.21 2.38±0.20 1,534±226 48±7 2,197±193 76±5 1.8±0.1 1.2±0.1

6 7.4 7.20±0.07 25.2±0.1 33.2±0.2 3.24±0.32 3.17±0.32 4,948±849 141±24 3,051±304 52±10 1.3±0.3 0.8±0.2

7.7 7.32±0.04 25.3±0.0 33.3±0.0 2.63±0.15 2.62±0.14 3,080±473 87±13 2,490±139 56±3 1.4±0.1 0.9±0.1

8.0 7.62±0.18 25.0±0.2 33.3±0.0 2.56±0.30 2.66±0.28 1,640±900 47±26 2,399±291 111±40 2.7±1.0 1.8±0.6

12 7.4 7.26±0.03 24.3±0.1 33.3±0.2 3.36±0.51 3.31±0.48 4,441±937 129±27 3,172±473 60±5 1.5±0.1 1.0±0.1

7.7 7.38±0.07 24.4±0.1 33.4±0.0 3.12±0.35 3.13±0.37 3,163±413 92±12 2,957±329 75±20 1.8±0.5 1.2±0.3

8.0 7.56±0.04 24.3±0.1 33.5±0.0 2.94±0.28 3.02±0.30 1,920±204 56±6 2,773±264 107±18 2.6±0.4 1.7±0.3

n=6 for pH 7.4 day 6, pH 8.0 days 6 and 12; 5 for pH 7.4 day 12, pH 7.7 all days; 4 for pH 7.4 day 0, pH 8.0 day 0

AT total alkalinity, DIC dissolved inorganic carbon content, Ω Ca saturation state of calcite, Ω Ar saturation state of aragonite


DIC from the seawater to the CF, this would explain the δ13Cobserved which is that of the water and not an isotopicsignature reflecting respiratory CO2. This is further supportedby the significant correlation between pHSWand δ13CCF. Also,the correlation between δ13CSW and δ13CCF was significant.Indeed, the CO2bubbling method involves adding CO2 in theseawater from an industrial gas bottle. The gas from this bottlehas a more negative signature compared to that of naturalCO2. So at high pCO2, the carbon isotopic signature of SWis more negative, and this change in signal is proportional tothe quantity of CO2 added, leading to differences according topH (more CO2 has to be injected to have a lower pH).

The increased DICCF content could indicate an accumula-tion of bicarbonate ions form the outside environment tocompensate for the acidosis of the CF, as reported for seaurchins (Collard et al. 2014; Holtmann et al. 2013; Moulinet al. 2014; Stumpp et al. 2012). However, the measure of thebuffer capacity of the CF (using total alkalinity as a proxy) didnot reveal significant differences according to treatments.Even though there is an increase in AT-CF during the courseof the experiment for H. scabra, this increase is also noted forcontrol individuals and is thus not linked to a response todecreased pHSW (also confirmed by the not significant corre-lation in between pHSWandΔAT-CF). Therefore, accumulationof bicarbonate within the extracellular fluid in order to com-pensate for acidosis is probably not occurring in the twostudied species.

Both species showed remarkably similar responses to lowpH although juvenile H. scabra were here compared withadult H. parva. Therefore, the juveniles of H. scabra, usedto a little fluctuating environment, do not seem to be morevulnerable to OA than adult H. parva from a highly variableintertidal habitat. It was previously suggested that the envi-ronment in which echinoderms live could give an indicationon their possible tolerance to OA, with temperate intertidalspecies being better equipped to withstand those changes(Clark et al. 2009). At least for the two tropical speciesconsidered in the present study, this is not the case. Pendingstudies onmore species, our results suggest that the short-termphysiological responses of tropical Holothuria sp. could bequite similar across stages and species.

Conclusions

At short term (6 to 12 days) the two studied species of seacucumbers did not compensate the acidosis of their CF causedby seawater acidification. On the contrary, the ΔpCO2 (be-tween CF and SW) even increased. This response was verysimilar in both species despite their very contrasted naturalhabitats. The metabolic parameters, such as respiration rateand ammonium excretion rate, seemed not to be affected but abehavioral effect cannot be excluded, as well as an effect of

uncompensated extracellular pH on regulation of internal pHwhich, on the long term, could lead to physiological andmetabolic effects.

Acknowledgments M. Collard is holder of a Belgian FRIA grant. Ph.Dubois is a research director of the National Fund for Scientific Research(FRS-FNRS; Belgium). We thank the “Institut Halieutique et des Sci-ences Marines” of Toliara, Madagascar and the staff of the institution fortheir welcome and help, particularly, R. Rasoloforinina, G. Todinanaharyand G. Tsiresy. We thank the FRS-FNRS for the travel grant to Mada-gascar. We would also like to thank Professor L. Chou for providing theTRIS and AMP buffers, C. Massin for determining the holothurianHolothuria parva, M. Schaltz for field measurements and G. Seghers,G. Caulier and B. Danis for their help and support during the experiments.Finally, we thank D. Verstraeten and N. Brion for their help with theanalyses.

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