Effects of mechanical disturbance and salinity stress on bioenergetics and burrowing behavior of

32 Pages·2017·1.44 MB·English

©2018.PublishedbyTheCompanyofBiologistsLtd|JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
RESEARCHARTICLE
Effects of mechanical disturbance and salinity stress on
bioenergetics and burrowing behavior of the soft-shell clam
Mya arenaria
FouziaHaider1,EugeneP.Sokolov2andInnaM.Sokolova1,*
ABSTRACT fluctuations in salinity, temperature, oxygen content, pH and
pollutants in the sediment and overlaying water. Due to their
Bioturbationofsedimentsbyburrowingorganismsplaysakeyrolein
sedentary lifestyle, bivalves have limited capacity to escape the
the functioning of coastal ecosystems. Burrowing is considered an
unfavorable conditions and rely on physiological mechanisms to
energeticallyexpensiveactivity,yettheenergycostsofburrowingand
surviveinthechangingenvironment (BergerandKharazova,1997;
thepotentialimpactsofmultiplestressors(suchassalinitystressand
Grieshaberetal.,1994;Sokolovaetal.,2015,2011).Physiological
waveaction)onbioenergeticsandburrowingperformanceofmarine
adjustmentstoenvironmentalstressareenergeticallycostlyandmay
bioturbators are not well understood. We investigated the effects
resultintrade-offsbetweenthemaintenanceandotherfitness-related
of mechanical disturbance and salinity stress on the burrowing
functions,suchasactivity,growthandreproduction(Sokolova,2013;
behavior, aerobic capacity and energy expense of digging in a
Sokolova et al., 2012). Understanding the impacts of multiple
common marine bioturbator, the soft-shell clam Mya arenaria from
stressors on the bioenergetics of marine bioturbators is therefore
the Baltic Sea (control salinity 15). Mya arenaria showed large
importantforpredictingthepotentialeffectsofenvironmentalshifts
individual variability in the burrowing efficiency, with an average of
∼7%ofthebodyenergyreservesusedperburial.Clamswithhigher on survival and performance of their populations and on the
ecologicalservicestheyprovideasecosystemengineers.
mitochondrial capacity and lower energy expenditure per burial
showed higher endurance. Acclimation for 3–4weeksto low (5) or Fluctuating salinity is an important stressor in shallow habitats,
fluctuating (5–15) salinity reduced the burrowing speed and the including estuaries, coastal lagoons and semi-enclosed seas
(McLuskyand Elliott,2004). Salinity regime can fluctuate due to
number of times the clams can rebury but did not affect the
theseasonalorshort-termchangesinprecipitation,freshwaterrun-
mitochondrial capacityof thewhole bodyor the gill. Acclimation to
off and evaporation. Furthermore, alteration of the hydrological
thefluctuatingsalinityshiftedthepredominantfueluseforburrowing
cycle due to the anthropogenic climate change results in the
fromproteinstolipids.Ourdataindicatethatthereducedburrowing
fresheningofthesurfaceseawatersandhigherfrequencyofextreme
performance of clams under the salinity stress is not due to the
precipitationevents(Duracketal.,2012).Insemi-enclosedbrackish
limitationsofenergyavailabilityoraerobiccapacitybutmustinvolve
seaswithextensivedrainageareas,suchastheBalticSea,thismay
other mechanisms (such as impaired muscle performance). The
resultinmajorchangesofthesalinityregimeduetotheincreased
reductionintheburrowingcapacityofclamsduetosalinitystressmay
run-off (Vuorinen et al., 2015). Fluctuating salinity can affect
have important implications for survival, activity and ecological
marinebivalvesbyshiftingtheosmoticbalanceoftheirtissuesand
functionsoftheclamsinshallowcoastalecosystems.
negatively impacting key cellular processes and the integrity of
KEYWORDS:Bioturbation,Energycosts,Hyposalinity,Fluctuating organelles and cell membranes (Berger and Kharazova, 1997;
salinity,Mitochondria,Aerobiccapacity,Energyreserves,Proteins, Prosser,1991).Ashort-termsurvivalofextremesalinitiesmaybe
Lipids,Glycogen achievedbybehavioralavoidancesuchasretractingdeeperintothe
sediment or closing the shell to prevent contact with the ambient
INTRODUCTION water;however,thisbehaviorlimitsfeedingandgasexchangeandis
Burrowing benthic organisms (bioturbators) serve as ecosystem notsustainableinthelong-term(Sokolovaetal.,2000a,b;Truchot,
y
engineers in soft-bottom marine ecosystems affecting 1993).Acclimationofmarinebivalvestochangingsalinityinvolves g
biogeochemistry of marine sediments, pelagic–benthic coupling regulation of the intracellular osmolarity to achieve balance with o
l
o
andsecondaryproduction(Meysmanetal.,2006).Marinebivalves, the external salinityand preventswelling or shrinkingof thecells i
B
suchasasoft-shellclamMyaarenaria,arekeybioturbatorsinsoft- (BergerandKharazova,1997;PierceandAmende,1981;Yancey,
l
bottomecosystemsaroundtheworld(Powersetal.,2006;Strasser, 2005). In osmoconformers such as mollusks, intracellular a
t
1998) as well as important sentinel organisms in marine osmolarity is mainly maintained by controlling the amino acid n
e
environmental monitoring (Borja et al., 2000; Lu, 2005). Benthic concentrations(Davenport,1985;HawkinsandHilbish,2009).As m
bivalves are commonly exposed to multiple stressors, including the ambient salinity increases, the intracellular pool of free amino i
r
acids(FAAs)increasesduetotheproteinbreakdown,andassalinity e
p
declines,theFAAsarecatabolizedorexcreted,therebymaintaining x
1DepartmentofMarineBiology,UniversityofRostock,18059Rostock,Germany. E
the intracellular fluid isosmotic to the environment (Berger and
2DepartmentofAppliedEcology,UniversityofRostock,18059Rostock,Germany. f
Kharazova, 1997; Pierce and Amende, 1981). The protein o
*Authorforcorrespondence([email protected]) breakdown and subsequent loss of FAAs may incur considerable al
energy costs to osmoconformers in fluctuating salinity n
I.M.S.,0000-0001-6681-6529 r
environments (Hawkins and Hilbish, 2009) potentially impairing u
o
Received19October2017;Accepted13December2017 otherenergydemandingfunctionssuchastheburrowingactivity. J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
submarinegroundwaterdischarge(Matthäus,1995).Myaarenaria
Listofabbreviations can tolerate salinities from 4.5 to 35 and temperatures from
AM assaymedia −2°C to 28°C (Strasser, 1998) and is commonly exposed to the
BSA bovineserumalbumin mechanical disturbance and dislodgement by the waves and tidal
CCCP carbonylcyanidem-chlorophenylhydrazine currents (Emerson and Grant, 1991; Redjah et al., 2010; St-Onge
COX cytochromecoxidase etal.,2007).Wehypothesizedthatthecombinedeffectsofsalinity
E energyexpendedforonediggingcycle
stress and repeated burrowing will cause bioenergetic stress in
E totalconsumedenergy(ETScapacity)
c M. arenaria, and that elevated energy costs caused by the
EGTA ethylenebis(oxyethylenenitrilo)tetraaceticacid
E totalenergyreserve(combustionenergyequivalent physiological adjustments to low or fluctuating salinity will
s
ofproteins,lipidsandcarbohydrates) impairburrowingactivityoftheclams.Totestthishypothesis,we
ETS electrontransportsystem acclimated clams to different salinity regimes [normal salinity
FAAs freeaminoacids (NS) 15 low salinity (LS) 5 and fluctuating salinity (FS) regime
FS fluctuatingsalinity(5–15)
involving daily salinity cycles between 15 and 5. Clams were
Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid
subjecttomechanicaldisturbancebyexposingthemonthesurface,
HM homogenizationmedia
INT 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl and their ability to rebury and the average burial time were
tetrazoliumchloride determined.Wealsoassessedtheeffectsofsalinity,disturbanceand
LEAK protonleak their combination on tissue energy reserves (proteins, lipids and
LS lowsalinity(5) carbohydrates) and aerobic capacity of the clams. Our study
N numberoftimesclamscoulddiguntilexhaustion
ex provides insights into the physiological mechanisms and energy
NS normalsalinity(15)
costs of the combined effects of salinity stress and mechanical
OXPHOS oxidativephosphorylation
disturbanceonburrowingofM.arenariaandhasimplicationsfor
PMSF phenylmethylsulfonylfluoride
RCR respiratorycontrolratio understanding the potential effects of these stressors on the
SUIT substrate–uncoupler–inhibitortitration performance of a keystone marine bioturbator in shallow coastal
TMPD N,N,N′,N′-tetramethyl-p-phenylenediamine habitats.
ToD timeofdisturbance(numberofdiggingbouts)
MATERIALSANDMETHODS
Animalcollectionandmaintenance
Soft-shellclams(Myaarenaria,Linnaeus1758,10–70mmshell
Shallowcoastalhabitatsexperiencehighsedimentdynamicsdue length) were collected from Schnatermann, a shallow water area
totheexportfromtheland,stormsandtheactionofwavesandtides near Rostock, Germany, and transported in seawater to the
(Carniello et al., 2014). Sediment reworking can expose UniversityofRostock.Theclamswereplacedinawashedsandy
bioturbators, resulting in mortality due to predation or washing sediment in recirculating aquarium systems at 15±0.5°C and
off up the shore, or bury them under additional sediment layers salinity15±1for2weeks.Thiswasclosetothetypicalsalinityat
therebypreventingfeedingandgasexchange(EmersonandGrant, thestudysite(12atthetimeofcollection,10–20throughoutthe
1991;Emersonetal.,1990;Powilleitetal.,2009;Probert,1984).To year). A randomly selected group of clams was gradually
survive these changes, bioturbators need to actively burrow to acclimated to LS (5) by reducing salinity at the rate of 1 day–1
achieve the depth that allows them access to water and food yet until the target salinity of 5 was reached. Clams were then
provides protection against predators and/or abiotic extremes acclimatedfor3–4weeksatoneofthefollowingsalinityregimes:
(Powilleit et al., 2009; Zwarts and Wanink, 1989). Burrowing is (1) constant NS 15; (2) constant LS 5; and (3) FS with the daily
consideredanenergeticallyexpensivemodeoflocomotion(Brown, salinitychangesbetween5and15.IntheFStreatment,thesquare
1979; Dorgan et al., 2011; Willmer et al., 2000). The direct salinity wave was applied as follows: 23h at salinity 15, 1h at
estimates of the energy expense of burrowing are rare for marine salinity10,23hatsalinity5,and1hatsalinity10,repeatedfor3–
organisms but the available data indicate high energy costs of 4weeks.ClamswerefedadlibitumwithDTsPremiumBlendLive
burrowing (Ansell and Trueman, 1973; Dorgan et al., 2011; Phytoplankton (Coralsands, Wiesbaden, Germany) every other
Meysman et al., 2006; Trevor, 1978). Earlier studies showed that day(4mlper60–70gofclambiomass).
deviation of salinity from a species-specific optimum may impair
y
burrowingbehaviorofsediment-dwellingorganisms(Lardiesetal., Burialtime g
o
2001; Przeslawski et al., 2009). However, the physiological Toassesstheburrowingperformanceandexerciseenduranceofthe l
o
mechanismsunderlyingtheeffectsofsalinityontheburrowingof clams, M. arenaria were exposed to repeated mechanical i
B
marine bioturbators are not well understood, and the bioenergetic disturbance (every 45min) and their burial time (i.e. the time
l
a
consequences of the combined salinity stress and repeated required to escape from the sediment surface) and the number of
t
mechanical disturbance (such as may occur due to the wave times the clams could rebury before becoming exhausted were n
e
actionorsedimentreworking)arenotknown,limitingourabilityto determined. The behavioral trials were conducted at 15°C and m
make predictions about the impacts of these stressors on survival salinity 15 (for clams acclimated at the NS and FS) or salinity 5 ri
e
andecologicalfunctionsofbioturbators. (for clams acclimated at LS). Clams (shell length 26.9±0.6mm,
p
Theaimofourstudywastodeterminethecombined effects of N=67) were placed in shallow tanks filled with thewashed sandy x
E
salinitystressandmechanicaldisturbanceonthebioenergeticsand sediment and allowed to bury for 45min. The behavior was
f
burrowing behavior of the soft-shell clam M. arenaria. Mya recordedevery30s,andtheburialtimewasdeterminedasthetime o
arenaria is abundant in the shallow waters of the southwestern thatelapsedfromtheclam’sdilationofthefoot(indicatingthestart al
n
BalticSeawhereitfrequentlyexperiencessalinityfluctuationsdue of digging) to the time the clam disappeared under the sediment
r
totheexchangeofwaterbetweentheNorthandtheBalticSeaand surface(Fig.1A).After45mintheclamsthatdugintothesediment u
o
due to the freshwater inputs from precipitation, land run-off and weregentlypulledup,exposedandlefttoburyforanother45min. J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
A KCl, 100mmoll−1 NaCl, 30mmoll−1 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (Hepes, pH 7.5) and 8mmoll−1
ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Gill
mitochondriafromclamsacclimatedtotheLSregimewereisolated
using low-osmolarity HM containing 50mmoll−1 sucrose,
100mmoll−1 KCl, 50mmoll−1 NaCl, 30mmoll−1 Hepes (pH
7.5) and 8mmoll−1 EGTA. All HM were supplemented with
protease inhibitors [1mmoll−1 phenylmethylsulfonyl fluoride
(PMSF)and2μg ml−1aprotinin].Adjustmentoftheosmolarityof
the HM to the acclimation osmolarity was needed to isolate high-
quality mitochondria as assessed by the respiratory control ratio
12:22 h 13:09 h
(RCR)(Table1)andmitochondrialintegritytestusingcytochrome
c addition. Gill tissues were homogenized in ice-cold HM using
200 B
severalpassesofaPotter–Elvenhjemhomogenizerat200rpm.The
homogenatewascentrifugedat4°Cand2000gfor4mintoremove
n)150 R2=0.693 celldebris,andthesupernatantwascentrifugedat4°Cand8500gfor
mi 8min to obtain a mitochondrial pellet. The mitochondria were
e ( washed once in the corresponding HM, collected by brief
m100
centrifugation (8500g for 5min) and resuspended in ice-cold
al ti storagemediacontaining140mmoll−1taurine,130mmoll−1KCl,
Buri 50 10mmoll−1 NaCl, 30mmoll−1 Hepes, 10mmoll−1 KH2PO4,
10mmoll−1 glucose, 1mmoll−1 MgCl and 1% of fatty acid
2
free bovine serum albumin (BSA). Mitochondrial functions were
0 assessedusinghigh-resolutionrespirometryinoneofthetwoassay
10 20 30 40 50 60 media(AM):(1)high-osmolarityAM(450mOsm,correspondingto
Size (mm) salinity15):140mmoll−1sucrose,130mmoll−1KCl,10mmoll−1
Fig.1.TheeffectsofclamsizeonthediggingactivityofM.arenaria NaCl, 30mmoll−1 Hepes, 10mmoll−1 KH2PO4, 10mmoll−1
measuredasthetimeneededtoburyafterthedisturbance. glucose, 1mmoll−1 MgCl2, pH 7.2; and (2) low-osmolarity AM
(A)Representativestillframesofthevideorecordingshowingclamsatthe (150mOsm, corresponding to salinity 5): 60mmoll−1 KCl,
beginningoftheburrowingcycle(i.e.immediatelyafterthedisturbance)and 30mmoll−1 Hepes, 10mmoll−1 KH PO , 10mmoll−1 glucose,
2 4
after45min.Clamsthatcompletelyreburiedafter45minaremarkedbytheoval 1mmoll−1MgCl ,pH7.2.
shapesintheleftstillframe.(B)Size-dependentactivityassessedatthenormal 2
Oxygen consumption of isolated mitochondria was measured
salinity(NS)(15)intheclamsacclimatedtotheNSregime.Thelinerepresentsa
using Oxygraph-2k high resolution respirometer (Oroboros,
quadraticregression;thegoodness-of-fitisshowninthegraph.
Innsbruck, Austria) at 15°C. Two-point calibration for 0% and
100% air saturation was conducted. The following substrate–
This cycle was repeated 4–5 times (exercised group) or until the uncoupler–inhibitortitration(SUIT)protocolwasused:5mmoll−1
clams were unable to bury within the 45min interval (exhausted pyruvatewith2mmoll−1malatetostimulateComplexI(NADH-
group). Notably,once theclams wereunder thesediment surface, linked)respiration;10mmoll−1succinatetostimulatetheelectron
theirbehaviorcannotbeobserved.However,thedepthfromwhich flow through Complex II; 2.5mmoll−1 ADP to achieve state 3
the clams were retrieved after the 45min burial cycle (∼3–7cm) (ADP-stimulated) respiration; 2.5µmol l−1 oligomycin to inhibit
indicates that they continued digging once in the sediment. The mitochondrial F ,F -ATPase and achieve state 4 respiration
0 1
preferred burial depth of the clams of the studied size range
(10–14cm)(Emersonetal.,1990;ZwartsandWanink,1989)has
Table1.Mitochondrialrespiratorystatesandderivedindicesusedto
notbeenachievedduringthe45minexperimentaldiggingintervals.
assessthemitochondrialfunctioninMyaarenaria
Controlclamsweremaintainedunderthesamesalinityregimesbut
withoutdisturbance.Aftertheexposures,thecontrol,exercisedand Respiratorystate
y
exhaustedclamsweredissected,shockfrozenintheliquidnitrogen orindex Reflectiveof: Determinedas: g
andstoredat−80°C. OXPHOS OXPHOScapacity Respirationinthepresenceof lo
o
ADP
i
LEAK Protonleak Respirationinthepresenceof B
Mitochondrialassays
oligomycin al
Mitochondria were isolated from the gills of M. arenaria using
ETS ETScapacity Respirationaftertheadditionof t
differentialcentrifugation(Ivaninaetal.,2016;Sokolova,2004).The n
anuncoupler(CCCP) e
gill is the main respiratory organ of M. arenaria; therefore, the COX Capacityofthe Respirationratesafteradditions m
energeticperformanceofthegillsisimportantfortheoverallaerobic cytochromec ofantimycinA,TMPDand ri
performanceofaburrowingclam.ClamsacclimatedtotheFSregime oxidase ascorbate e
p
were collected during the salinity 15 phase of the FS cycle, RCR Respiratorycontrol =OXPHOS/PL x
ratio E
and clams acclimated to the NS or LS regimes were collected at
COX/OXPHOS ReserveCOX =COX/OXPHOS f
their respective acclimation salinities. Briefly, gills from two o
capacity
clams (0.6–0.9g) were pooled and homogenized in 5ml of ETS/OXPHOS ReserveETS =ETS/OXPHOS al
n
mitochondrial homogenization media (HM). Gill mitochondria of capacity r
clamsacclimatedtotheNSandFSregimeswereisolatedusinghigh- u
OXPHOS,oxidativephosphorylation;ETS,electrontransportsystem;COX, o
osmolarity HM containing 100mmoll−1 sucrose, 200mmoll−1 cytochromecoxidase;TMPD,N,N,N′,N′-tetramethyl-p-phenylenediamine. J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
indicative of the proton leak; 7.5mmoll−1 carbonyl cyanide For determination of the carbohydrate and protein contents,
m-chlorophenylhydrazone(CCCP)touncouplethemitochondrial ∼50mgoftissuepowderwasmixedwithultrapurewatercontaining
electron transport system (ETS); 1µmol l−1 rotenone to inhibit 0.1%Triton(1:10tissuemasstovolume).Cellswerelysedbythree
electron flux through Complex I; 2.5µmol l−1 antimycin A to rapidfreeze–thawcyclesof5minat−80°C,followedby5minina
inhibitelectronfluxthroughComplexIII;0.5mmoll−1N,N,N′,N′- 37°C water bath and centrifuged at 3000g for 3min at room
tetramethyl-p-phenylenediamine (TMPD) and 2mmoll−1 temperature.Thesupernatantwasusedforcarbohydrateandprotein
ascorbate to stimulate activity of Complex IV (cytochrome c measurements. Carbohydrate concentrations were measured using
oxidase, or COX); and 20mmoll−1 KCN to inhibit COX. phenol–sulfuric acid method (Masuko et al., 2005). Samples or
Mitochondrial respiratory states and control indices (Table 1) glucosestandards(200µl)weremixedwith600µlofconcentrated
werecalculatedasdescribedelsewhere(Estabrook,1967;Gnaiger, H SO , immediately followed by the addition of 120µl of 5%
2 4
2012).Allmitochondrialassayswerecompletedwithin60minafter phenol. Samples were incubated for 5min at 90°C, cooled and
the isolation. Protein concentrations were measured in absorbance measured at 492nm to calculate the carbohydrate
mitochondrial suspensions using a Bio-Rad Bradford protein content (in glucose equivalents). Protein concentrations were
assay (Bio-Rad, Hercules, CA, USA), and corrected for the BSA measured at 595nm using Bio-Rad Bradford Protein Assay Kit,
contentoftheAM.Mitochondrialrespirationrateswereexpressed accordingtothemanufacturer’sinstructions(Bio-Rad)withBSAas
asµmolO min−1g−1protein. a standard. The standard curves for lipids, carbohydrates and
2
proteinsshowedgoodlinearity(R2=0.99).Totalenergyreservewas
Determinationofenergyreserves calculated by transforming the measured protein, lipid and
EnergyreservesandETScapacitywereassessedinthefootandin carbohydrate into energy equivalent using their respective energy
therestofthebodyofM.arenaria.Thefootmassrepresents<1%of of combustion: 24kJg−1 for proteins, 39.5kJg−1 for lipids and
thetotalbodymassoftheclam(theaveragewetmassesofthefoot 17.5kJg−1forcarbohydrates(Gnaiger,1983).
and the body were 10.0±0.8mg and 1.13±0.09g, respectively,
N=44–69),andthereforethefootremovalhasnegligibleeffectonthe Whole-bodyETScapacity
estimateofthetotalenergyreserveofthebody.Lipid,carbohydrate ETS activity was measured using 2-(4-iodophenyl)-3-(4-
and protein concentrations were measured using colorimetric nitrophenyl)-5-phenyl tetrazolium chloride (INT) tetrazolium
methods. Lipid content was measured using chloroform–methanol reduction assay as described elsewhere (De Coen and Janssen,
method (Van Handel, 1985). Approximately 50mg of tissue 1997; Owens and King, 1975). Whole bodyof clams was ground
powder (ground under the liquid nitrogen) was added to 3ml of under the liquid nitrogen and homogenized with ETS-B solution
chloroform:methanol mixture (1:1, v:v) and incubated for 5min [153µmol l−1 MgSO , 1.5mgml−1 polyvinylpyrrolidone and
4
with periodic vigorous mixing. The mixture was centrifuged at 0.2% (v:v) Triton X-100 in 0.1moll−1 Tris-HCl, pH 8.5] at 1:50
3000gfor4minatroomtemperature.Two-hundredandfiftyµlof (w:v)ratio.Thehomogenatewascentrifugedat3000gfor10minat
samplesorstandards(soybeanoilinacetone)weretransferredto 4°C,andthesupernatantwasusedtoassesstheETSactivityasthe
culturetubesandheatedto100°Cfor∼10–12minuntilthesolvent rate of reduction of INT–tetrazolium to formazan at room
wasevaporated.One-hundredµlof98%sulfuricacid(H SO )was temperature (22.5°C). Briefly, 28.5µl of tissue homogenate was
2 4
added to thedrysample, mixed and heated at100°C for 10min. added to 85.5µl of the buffer [0.13moll−1 Tris-HCl and 0.3%
The sample was cooled down to room temperature, mixed with (w/v) Triton X-100; pH 8.5] and 29µl of NAD(P)H solution
2.4mlofvanillinreagent(600mgvanillinin100mlofhotwater (1.7mmoll−1NADHand250µmoll−1NADPH).Fifty-sevenµlof
and400mlof85%phosphoricacid)andincubatedfor5min.The 8mmoll−1INT–tetrazoliumwasaddedtostartthereactionandthe
absorbance was measured at 490nm using a SpectraMax M2 absorbancewasmeasuredat490nmfor15minat2minintervals.
microplate reader (Molecular Devices GmbH, Biberach-an-der- To account for potential non-mitochondrial reduction of
Riß,Germany). INT–tetrazolium, blanks wererunforeach sample bysubstituting
Table2.ANOVA:effectoftheexercisegroup,salinityregimeandtheirinteractiononburialtimeandbioenergeticsonMyaarenaria
Parameters Group Salinity Group×salinity Shellsize ToD
Burialtime F1,37=2.31 F2,37=6.41 F2,37=0.34 F1,37=10.09 n.d. y
P=0.137 P=0.004 P=0.715 P=0.003 g
o
Proteincontent F2,P47<=01.30.0813 F2,P47==04..05165 F4,P47==04..05043 F1,P47==01..22675 F1,P47==02..13289 ol
i
Lipidcontent F =11.44 F =10.40 F =1.10 F =3.24 F =0.44 B
2,45 2,45 4,45 1,45 1,45
P<0.001 P<0.001 P=0.369 P=0.079 P=0.509 al
Carbohydratecontent F =2.39 F =1.91 F =1.36 F =0.53 F =1.25 t
2,50 2,50 4,50 1,50 1,50 n
P=0.102 P=0.158 P=0.261 P=0.470 P=0.268 e
Totalbodyenergyreserves F =17.64 F =2.72 F =0.80 F =4.04 F =3.66 m
2,48 2,48 4,48 1,48 1,48
P<0.001 P=0.076 P=0.534 P=0.050 P=0.062 ri
ETScapacity F =2.79 F =12.61 F =5.06 F =3.50 F =2.72 e
2,47 2,47 4,47 1,47 1,47 p
P=0.072 P<0.001 P=0.002 P=0.068 P=0.106 x
E/E F =3.51 F =4.33 F =1.94 F =0.57 F =1.35 E
s c 2,46 2,46 4,46 1,46 1,46
P=0.038 P=0.019 P=0.120 P=0.454 P=0.251 f
o
Groups:control,exercisedandexhausted.Salinityregimes:normal,lowandfluctuatingsalinity.Sizeoftheclamshellandthenumberoftimestheclamswere l
a
disturbed(ToD)wereusedascovariates.Proteins,lipidsandcarbohydratescontent,aswellthetotalenergyreservesandelectrontransportsystem(ETS) n
capacityweredeterminedinthewholebodyoftheclamsafterthefoot(<1%ofthebodymass)wasremoved.F-ratios(withdegreeoffreedomandtheerrorshown r
u
asasubscript)andP-valuesfortheeffectsaregiven.Significanteffects(P<0.05)arehighlightedinbold.n.d.,notdetermined.Es/Ec,ratioofenergyreservesto o
thepotentialenergyexpenditure. J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
2µl of 1mmoll−1 rotenone (an inhibitor of the mitochondrial Table3.ANOVA:effectofsalinityregime,assayosmolarityandtheir
ComplexI)and27µmoll−1of5moll−1KCN(aCOXinhibitor)for interactiononrespirationofthegillmitochondriaofMyaarenaria
theNAD(P)Hsolution.ETSactivitywascalculatedbysubtracting
Assay Salinity×assay
the blank slopes from the reaction slopes for each sample. Parameters Salinity osmolarity osmolarity
Specific ETS activity of the tissues (µmol O min−1 mg–1 wet
2 RCR F =4.02 F =18.82 F =2.92
tissue mass) was calculated based on the extinction coefficient of 2,P26=0.030 1,P26<0.001 2,P26=0.072
formazan of 15.9lmol–1 cm−1 and corrected for the light path OXPHOS F =0.68 F =1.82 F =0.51
2,26 1,26 2,26
(0.729cm). The potential energy expenditure (E) was calculated P=0.517 P=0.190 P=0.606
c
based on the ETS flux assuming the stoichiometric equivalent of LEAK F2,26=0.06 F1,26=0.70 F2,26=0.19
1μmolformazanto0.5μmolO andusingoxyenthalpicequivalents P=0.940 P=0.410 P=0.829
2 COX F =1.47 F =2.41 F =0.35
for combustion of an average lipid, glycogen and protein mixture 2,26 1,26 2,26
P=0.248 P=0.132 P=0.709
(484Jmmol−1O )(Gnaiger,1983;Verslyckeetal.,2004).
2 ETS F =0.40 F =2.66 F =0.66
2,26 1,26 2,26
P=0.673 P=0.115 P=0.523
Statistics COX/ F =4.16 F =1.40 F =1.52
2,26 1,26 2,26
Effects of salinity regime and exercise on bioenergetics traits of OXPHOS P=0.027 P=0.247 P=0.238
M.arenariaweretestedusingANCOVAwithsalinityregime(NS, ETS/ F2,30=2.86 F1,30=5.18 F2,30=1.07
OXPHOS P=0.073 P=0.030 P=0.355
LSandFS)andexercisegroups(control,exercisedandexhausted)
asfixedfactorsandsizeoftheclamsandtheamountoftimesithas F-ratios(withdegreeoffreedomandtheerrorshownasasubscript)andP-
reburied prior to sampling as covariates (Table 2). Effects of valuesfortheeffectsaregiven.Significanteffects(P<0.05)arehighlightedin
bold.RCR,respiratorycontrolratio;OXPHOS,oxidativephosphorylation;
acclimation salinity and assay osmolarity on the mitochondrial
LEAK,protonleak;COX,cytochromecoxidase;ETS,fluxofelectrontransport
functionswastestedusinggeneralizedlinearmodelANOVAwith
system.
salinityregime(NS,FSandLS)andassaybufferosmolarity(150
and 450mOsm) as fixed variables (Table 3). The assumptions of
normaldistributionandhomogeneityofvariancesweretestedforall salinityregimegroup,dividedbythenumberofthediggingbouts.
dependent variables using Shapiro–Wilk and Levine tests, Becausetheamountofenergyreservescouldnotbemeasurednon-
respectively. Statistical outliers (0–2 per group) were detected invasively,averageenergycontentofthereserves(calculatedfrom
using stem-and-leaf plot and removed from subsequent analysis. the body content of proteins, lipids and carbohydrates) of the
Followingtheoutliers’removal,alldatawerenormallydistributed undisturbed clams from the same salinity regime group was used
andthevarianceswerehomogeneous(P>0.05).Themeansforthe asaproxyforthestartingamountofenergyreservesavailabletothe
burial time, tissue levels of energy reserves and ETS activity are clam.Cook’sdistancetestwasused todetectpotentialoutliers;two
correctedtotheaveragecommonsizeofclams(26–27mm)andthe significantoutliersweredetectedandremovedfromtheNS-acclimated
averagenumberoftimestheclamsweredisturbed(n=4)basedon group. Autocorrelation of the data was tested using Durbin–Watson
theANCOVAanalyses. test; no autocorrelation was detected. Because the effect of the
To determine the effects of the salinity regime, energy costs of clam size (L) on the energy cost of digging (E) was not significant
diggingandclamsizeonthenumberoftimestheclamcanrebury (P=0.866), it was excluded from the final analysis. Due to the
duringtherepeateddisturbance,amultipleregressionanalysiswas significant effects of the salinity regime on the numberof timesthe
used.Intheinitialmodel,clamsize(shelllength,L),salinityregime clamswereabletorebury,linearregressionsdescribingtherelationship
(S, encoded 1, 2 and 3 for NS, LS and FS, respectively) and the betweentheenergycostsofdiggingandthenumberoftimestheclams
amount of energy expended during a single dig (E) were used as can rebury were constructed separately for each salinity group, and
explanatory variables. The energy cost of digging (E) was testedforsignificantdifferencesintheinterceptsandslopes.
calculated as a difference between the amount of energy reserves StatisticalanalyseswereconductedusingIBM®SPSS®Statistics
remainingintheclam’sbodyafterrepeateddiggingandtheaverage v.22.0.0.0(IBMCorp.,Armonk,NY,USA)andGraphPadPrismv.
amountofenergyreservesintheundisturbedclamsfromthesame 7.02(GraphPadSoftwareInc.,LaJolla,CA,USA)software.
A B
40 Exercised * 12 y
Exhausted b g
es 10 a o
Burial time (min) 233505 a a b b b o. of digging cycl 468 b a,b mentalBiol
N
i
r
e
20 2
p
Normal Low Fluctuating Normal Low Fluctuating x
E
Salinity regime
f
o
Fig.2.EffectoftheacclimationsalinityonthediggingperformanceofM.arenaria.(A)Themeanburialtimesofclamsacclimatedatdifferentsalinities. l
a
(B)Theaveragenumberoftimestheclamswereabletoreburybeforeexhaustion.Themeanswerecorrectedtotheaverageclamsizeacrossallgroups n
(26mmand24mminAandB,respectively).Differentlettersindicatethevaluesthataresignificantlydifferentamongdifferentsalinityregimeswithinthe r
u
sameactivitygroup(exercisingorexhausted)(P<0.05);ifthecolumnssharealetter,therespectivevaluesarenotsignificantlydifferent(P>0.05).Asterisks o
indicatethevaluesthataresignificantlydifferentbetweentheexercisingandexhaustedgroupsatthesamesalinityregime(P<0.05).N=6–8. J
5
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
150 mOsm 450 mOsm Fig.3.Effectofhigh(450mOsm)
andlow(150mOsm)osmolarityon
A B
–1 –1ming) 1250 *a a a a a –1 –1ng) 180 a a a a a a tgdphhiilfeolfsesmrpoeihftnooctrclysahlamaoltinisnodniatrici(eaOcsllX.ifmu(PAnaH)ctOeOtidSoxi)nadtcsaattihipnveaetchitey,
µmol O2 10 a mol O mi2 46 (rc(eBEys))topapciprrhaoprttoaoomrrneyenlectcoarnekotsxr(eoLidrlEvarAeastKeeio)l(eCr(caROttrCeoX,Rn)()Cta,rac)(Dtnivs)iptyo,rt
HOS ( 5 µAK ( 2 saypsptaermen(tErTeSse)rcvaepcayctiotycharnodm(eF)c
XP LE oxidase(COX)capacity.Different
O 0 0 lettersindicatethevaluesthatare
Normal Low Fluctuating Normal Low Fluctuating significantlydifferentamongclams
acclimatedatthedifferentsalinity
regimes(P<0.05).Asterisksindicate
5 C *a 40 D tdhifefevraelnutebsetthwaeteanremsitigonchifiocnadnrtilay
1) measuredalhighandlowosmolarity
4 *a b a 1– g 30 a withinthesamesalinityacclimation
b –min *a a a group(P<0.05).N=4–7.
R 2 a
C3 O 20 a
R a mol
µ
2 X ( 10
O
C
1 0
Normal Low Fluctuating Normal Low Fluctuating
E F
1.5 * 1.6
a a
a a
1.4 b
S o
PHO1.3 a a b S rati1.4 a a a
X T
S/O1.2 X/E b
T O1.2
E C
1.1
1.0 1.0
Normal Low Fluctuating Normal Low Fluctuating
Salinity regime
RESULTS theirexhaustion(Fig.2B).IntheconstantLS,clamscouldrebury4–
Timetobury 5timeswhereasintheNStheycanreburyupto9–10timesbefore
y
The average time required to bury after the disturbance differed exhaustion.Asimilar(albeitstatisticallynon-significant)trendfor g
o
betweentheclamsofdifferentsizes(R2=0.693,P>0.05).Generally, lowerendurance(i.e.fewernumberoftimesthatclamscanrebury) l
o
largerclamswereslower,withclams>40mmshelllengthrequiring wasfoundintheclamsacclimatedtotheFScomparedwiththeNS i
B
over1–2htobury(Fig.1B).Therelationshipbetweentheclamsize clams(Fig.2B).
l
a
andthetimerequiredtoburywasnon-linear,andthespeedofburial
t
was similar for clams of ∼15–40mm shell length (Fig. 1B). Mitochondrialfunctionsinthegill n
e
Therefore,onlyclamsinthesizerangeof∼20–40mmwereusedin Acclimationtodifferentsalinityregimesaffectedtheabilityofthe m
thebehavioralandbioenergeticstudies. gillmitochondriaoftheclamstoperform atdifferentosmolarities ri
ClamsacclimatedattheLS(5)orFS(5–15)tookalongertimeto (Fig. 3). In clams acclimated to NS (15), the oxidation e
p
burythantheclamsacclimatedattheNS(15)(Fig.2A).Therewas phosphorylation (OXPHOS) capacity and COX activity of gill x
E
nosignificantdifference in theburial time betweentheexercising mitochondria were suppressed when measured in the low-
f
andexhaustedgroupsattheNSandLS.AttheFS,theaverageburial osmolarity media (150mOsm) compared with the high- o
time was significantly longer in the clams that were allowed to osmolarity media (450mOsm) (Fig. 3A,D). Proton leak rates al
n
reburyuntilexhaustioncomparedwiththeexercisedgroupcollected werenotaffectedbytheassayosmolarityingillmitochondriaofNS
r
after3–4cyclesofdigging(Fig.2A).Acclimationtotheconstant clams (Fig.3B).Asaresult, theRCRofNSclams’mitochondria u
o
LS(5)alsohadasignificanteffectonthenumberofreburialsbefore was lower at 150mOsm, indicating reduced mitochondrial J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
Control Exhausted
A B C
–1Protein (mg g) 1234500000 a a b a a,b a –1Lipids (mg g) 1234500000 a a a a µ–1ohydrates (mole g) 246000 a a b a,b a,b b
n.d. b
0 0 Car 0
Normal Low Fluctuating Normal Low Fluctuating Normal Low Fluctuating
Salinity regime
Fig.4.EffectsofthesalinityregimeanddiggingonenergyreservesinthefootofM.arenaria.(A)Proteins,(B)lipidsand(C)carbohydratesinthefoot
ofM.arenariacalculatedpergramofwetmass.Themeanswerecorrectedfortheaverageclamsize(26–27mm)foralltraits.Differentlettersindicatethe
valuesthataresignificantlydifferentamongthesalinityregimeswithinthesameactivitygroup(controlorexhausted);ifthecolumnssharealetter,therespective
valuesarenotsignificantlydifferent(P>0.05).Asterisksindicatethevaluesthataresignificantlydifferentfromthecontrolgroupatthesamesalinityregime
(P<0.05).Dataforthelipidcontentintheclamsacclimatedtothelowsalinityaremissingduetothesampleloss(n.d.).N=6–8.
efficiencycomparedwith450mOsm(Fig.3C).Incontrast,inclams theCOX/ETSratiolowerinthemitochondriaofclamsacclimatedat
acclimatedtoLSorFS,gillmitochondriaperformedequallywellat LS and measured at 150mOsm, reflecting the relatively high ETS
150 and 450mOsm (Fig. 3). When compared at 450mOsm, key fluxinthisgroupcomparedwiththeircounterpartsfromtheNSand
mitochondrial functions including the OXPHOS capacity, proton FSregimes.
leak,COXactivityandRCRwerenotaffectedbythesalinityregime
atwhichtheclamswereacclimated(Fig.3).At150mOsm,thegill Energyreserves
mitochondriaofLSandFSclamsgenerallyoutperformedthoseof Theenergycontentpergramtissuewassimilarinthefootmuscle
NSclams(Fig.3). andtherestoftheclambody(2.54±0.13kJg−1,N=26versus2.00±
TheapparentreservecapacityofETS(theratiooftheETScapacity 0.07kJg−1,N=18,respectively).Inthefoot,∼35–40%ofthetotal
to the OXPHOS rates) was relatively small (20–38%) in clam gill energy was contained in the proteins, ∼50–60% contained in the
mitochondria(Fig.3E),indicatingthatETSactivityisagoodestimate lipids and 4–5% in carbohydrates. In the rest of the body, the
oftheATPsynthesiscapacityinthisspecies.Similarly,theapparent contributions of proteins, lipids and carbohydrates to the total
reserveofthemitochondrialCOXcapacity(theratioofCOXtoETS energy reserves were ∼60%, 30–35% and 6–8%, respectively.
flux)was16–42%(Fig.3F).TheETS/OXPHOSratiowashigherand Acclimation to the constant LS (5) led to a slight but significant
Control Exercised Exhausted Fig.5.Effectsofthesalinityregime
andrepeateddiggingonthetotal
3.0 A B bodyenergyreservesofM.
1) arenaria.(A)Totalenergyreserves,
–g 60 a (B)proteins,(C)lipidsand(D)
gy reserves (kJ 122...505 a *a *a a *a *a a *a *b –1Protein (mg g) 40 a *a *a *a *b b a b cmttfchhoaaaeerlrcsbaaausollvvl.haeetTrytrraeaahdidtggersaeep.mteeDctiesrmliafagofmeenrfasrsMesmowni.zfteoaedlrfereie(swt2tntceue7aorrtrsmbribraaeimonncdd)ctyeieacdan(td4feo)r
er thevaluesthataresignificantly
n
E differentamongsalinityregimes y
1.0 20 withinthesameactivitygroup g
Normal Low Fluctuating Normal Low Fluctuating o
(control,exercisingorexhausted);if l
o
thecolumnssharealetter,the
i
respectivevaluesarenotsignificantly B
30 C 80 D different(P>0.05).Asterisksindicate al
b –1g) thevaluesthataresignificantly nt
25 e differentfromthecontrolgroupatthe e
–1Lipids (mg g) 1250 a *a a a *a a *b *a µhydrates (mol 4600 a a,b *a b *a *a a,b b a sNa=m4–e8s.alinityregime(P<0.05). fExperim
10 o o
b
5 Car 20 nal
Normal Low Fluctuating Normal Low Fluctuating r
u
o
Salinity regime J
7
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
Control Exercise Exhausted
A B
0.8 40
–1g) *b a a
–1 0.6 a
mol O min2 0.4 a a a a a a a b E/E, dayssc 2300 a* a* *a *a *a *b
µ
S ( 0.2
T
E
0 10
Normal Low Fluctuating Normal Low Fluctuating
Salinity regime
Fig.6.Effectsofthesalinityregimeandrepeateddiggingonthewhole-bodyaerobiccapacityofM.arenaria.(A)Thewhole-bodyaerobiccapacitywas
assessedastheelectrontransportsystem(ETS)activityand(B)theratioofavailableenergy(energystored,E)toconsumedenergy(ETScapacity,E)corrected
s c
toacommonclamsize(27mm).Differentlettersindicatethevaluesthataresignificantlydifferentamongsalinityregimeswithinthesameactivitygroup(control,
exercisingorexhausted);ifthecolumnssharealetter,therespectivevaluesarenotsignificantlydifferent(P>0.05).Asterisksindicatethevaluesthatare
significantlydifferentfromthecontrolgroupatthesamesalinityregime(P<0.05).N=5–8.
increaseinthecontentoftheproteinsandcarbohydratesinthefoot repeateddiggingintheexercisingclamsatallthreestudiedsalinity
oftheclams,whereasacclimationtotheFShadnoeffectonthese regimes, and in the exhausted clams in the FS (Fig. 5C).
traits (Fig. 4). Lipid content in the foot was not affected by Carbohydrate reserves were ∼20–25% higher in the clams
acclimation to the FS (no data are available for the LS due to the acclimated to the constant LS compared with their counterparts
sampleloss)(Fig.4).Duetothesmallsizeofthefoot,theenergy fromtheNSorFSregimes(Fig.5D).Carbohydratereserveswere
contentofthefootwas<0.2%ofthetotalbodyenergyreserves.No significantlydecreasedafter theexhaustingexerciseintheclams
depletionofenergyreserveswasfoundinthefootofdiggingclams attheNSandconstantlyLSbutnotinthoseacclimatedtotheFS
(datanotshown). (Fig.5D).Overall,theclamsacclimatedtotheNSorconstantlyLS
Repeateddiggingresultedinasignificantdeclineoftheenergy covered ∼68–69% of the energy requirements for digging from
reservesofthetotalbodyoftheclamsregardlessoftheacclimation the protein reserves, ∼26–27% from the lipids and ∼3–5%
salinity(Fig.5A).Notably,clamsacclimatedtodifferentsalinity from the carbohydrate reserves. In contrast, clams acclimated to
regimes depended on different types of energy reserves to fuel the FS covered ∼27% of the energy cost of digging from the
digging activity. The body protein reserves were significantly protein reserves, ∼71% from the lipids and ∼2% from the
lower(by∼20–30%)intheundisturbedclamsacclimatedtotheFS carbohydrates.
regimecomparedwiththeircounterpartsacclimatedtotheNSor
constantly LS (Fig. 5B). During repeated digging, the body AerobicETScapacity
contentoftheproteinsdeclinedintheclamsacclimatedtotheNS Acclimation salinity has no significant effect on the total ETS
orconstantlyLSbutnotinthoseacclimatedtotheFS(Fig.5B).In activityinclamtissues(Fig.6A).ClamsacclimatedtoFSshowed
contrast, the lipid reserves were higher (by ∼30–50%) in the anincreaseinthetissueETScapacityfollowingexercise,whichwas
undisturbedclamsacclimatedtotheFScomparedwiththosefrom especiallypronouncedintheexhaustedgroup(Fig.6A).Repeated
theNSandLSregimes(Fig.5C).Thelipidcontentdecreasedafter digginghadnoeffectonthetotalbodyETScapacityintheclams
15 A 1.0 B
NS
o. of digs completeduntil exhausted150 LFSS µ–1 –1S (mol O ming)2 0000....2468 entalBiology
N T m
E
0 0 ri
0 100 200 300 0 200 400 600 e
p
Energy cost (J g–1 per dig) x
E
Fig.7.Therelationshipbetweentheelectrontransportsystem(ETS)capacity,buryingperformanceandtheenergycostofburyinginM.arenaria. of
(A)Relationshipbetweenthenumberoftimesaclamwasabletoreburypriortoexhaustionandtheamountofenergyspentforonedig.NS,normalsalinity; l
a
LS,lowsalinity;FS,fluctuatingsalinity.Theslopesofthelinesfromclamsacclimatedatthedifferentsalinitiesarenotsignificantlydifferent(P>0.05).The n
interceptsoftheregressionlinefortheNSgroupissignificantlydifferentfromthosefortheLSandFSgroups(P<0.05).N=5–6.(B)Relationshipbetweenthe r
u
whole-organismETScapacityandenergyexpenditureperdigforexercisingandexhaustedclams.Theslopesandinterceptsoftheregressionlinesforthe
o
clamsacclimatedatdifferentsalinitieswerenotsignificantlydifferent(P>0.05),andthereforeasingleregressionwasconstructedforalldata.N=42. J
8
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
acclimatedtoNSorLSregimes.Theratioofenergyreservestothe size), in agreement with the earlier findings (Breum, 1970). Once
potentialenergyexpenditure(E/E )wasnotaffectedbythesalinity thediggingcommenced,itproceededrapidly,andtheclamswere
s c
regime but declined after the repeated exercise in NS, LS and FS fullyburiedwithin20–32min,dependingontheclamsize.Indirect
clams(Fig.6B). estimatesofenergyexpenseofdigging(basedonthedepletionof
energyreserves)showthatonediggingcycleofM.arenaria(with
Energycostofrepeateddigging an average duration of 25–35min) requires 0.109±0.02Jmg−1
The number of times aclam could repeatedly dig until exhausted bodymasscorrespondingto6×10−5Jmg−1s−1.Thesevaluesare
wasnegativelycorrelatedwiththeenergycostofreburial(expressed comparable to those reported for other burrowing bivalves
inJg−1wetmassburial–1)(Fig.7A).Dependingontheacclimation (5×10−4 J s−1 and 3.6×10−4 J s−1 for Donax denticulatus and
salinity, 69–77% of the variation in the number of reburials until Donax incarnatus, respectively) (Ansell and Trueman, 1973) and
exhaustionwasexplainedbythevariationintheenergyexpenditure higher than in gastropods (1.48×10−8 J mg−1 s−1 for Bullia
per dig. The multiple regression analysis showed that both the digitalis) (Brown, 1979) and polychaetes (4.8×10−10 and
salinity regime (S) and the amount of energy expended during a 3.3×10−9 J mg−1 s−1 for Cirriformia moorei digging in gelatin
singledig(E)significantlyaffectedthenumberoftimestheclams andsediment,104.3×10−7,3.1×10−7and2.8×10−7Jmg−1s−1for
could dig until exhausted (N ): N =11.22–1.21×S–0.02×E Nephtys cirrosa, Nereis diversicolor and Arenicola marina,
ex ex
(adjusted R2=0.739; P<0.01 for the constant, S and E). The respectively, digging in sediment) (Dorgan et al., 2011; Trevor,
relationship between the energy cost of an individual dig and the 1978). The daily energy requirement for burial of a surf
numberoftimestheclamcouldreburywasindependentoftheclam D. incarnatus (which repeatedly buries to maintain its vertical
size in the studied size range (P>0.05 for the effect of size in the position in the sand and to move along the shore during the tidal
multiple regression analysis). The regressions of the number of cycle)wasestimatedat>30%oftheroutinemetabolism(Anselland
timestheclamscouldreburyontheenergyexpenditureperburial Trueman,1973).Therespectiveestimateofthedailyenergybudget
forclamsacclimatedtothedifferentsalinityregimeshadthesame for digging is not available for M. arenaria; however, our data
slopes (P>0.05) but different intercepts (P<0.05) (Fig. 7A). This indicatethatevenasingleburialcycleincursaconsiderableenergy
indicates that at the same energy expenditure per dig, clams cost,usinguponaverage7%ofthetotalbodyenergyreservesof
acclimatedatLSandFScouldreburyfewertimesbeforebecoming M.arenaria.Interestingly,thebioenergeticestimatesofburrowing
exhaustedthantheirNScounterparts. terrestrial species (such as the rodents) also indicate high energy
Theenergyexpenditureperdigwasnegativelycorrelatedwiththe costs of the burrowing activity despite the differences in the
ETS capacity of the clams, so that the clams that have low ETS environmentalconditionsaswellastheburrowingphysiologyand
capacityspentrelativelymoreenergyperdig(Fig.7B).Theregression biomechanics compared with aquatic bioturbators (Lovegrove,
was significant (P<0.01) but the variation in the ETS capacity 1989; Vleck, 1979). Thus, the oxygen consumption rates of
explainedonly∼20%ofthevariationintheenergycostsofdigging. burrowing rodents Thomomys bottae, Cryptomys damarensis and
Heterocephalus glaber are 4–5 times higher during digging
DISCUSSION compared with the routine metabolism (Lovegrove, 1989; Vleck,
Acclimation salinity, body size and bioenergetics efficiency of 1979), indicatinghighenergycostsofburrowing.Takentogether,
digging behaviorare the important determinants of the burrowing these data support the notion that digging is an energetically
capacity of a common marine bioturbator M. arenaria. The expensiveactivityinaquaticaswellasterrestrialbioturbators.
burrowing performance of clams was negatively affected by LS Highenergycostsofburrowingimplythattheabilitytorebury
andFS(suchascommonlyoccursinestuariesandshallowcoastal after repeated disturbance may become limited when the energy
habitats)andpositivelycorrelatedwiththeindividualbioenergetic reservesareexhaustedbelowacertainthresholdlevel.Ourdatain
efficiency of digging. Regardless of the salinity regime, smaller M.arenariasupportthishypothesisshowingthatwhenthebody
M.arenariawerecapableofburrowingfasterafterthedisturbance energyreservesdroppedby20–27%,clamswerenolongerableto
comparedwiththeirlargercounterparts.Thesefindingsagreewith rebury.Notably,individualvariabilityintheenergeticefficiency
the results of earlier studies showing that smallerclams dig faster of digging was associated with the ability to sustain repeated
(Emersonetal.,1990;ChecaandCadée,1997)andmorefrequently digging activity in M. arenaria, so that the clamsthat spent less
(St-Onge et al., 2007) than the larger ones. This may reflect energyperunitbodymassinanindividualdiggingboutwereable
biomechanical constraints of digging due to the relatively smaller todigmoretimesbeforebecomingexhausted.Thisindicatesthat
y
foot size in the large clams and/or the possible bioenergetics the more energy-efficient diggers may have greater chances of g
o
constraints due to the greater resistance of the sediments for survivalinshallowhabitatsexposedtowindandwaveaction,and l
o
propelling a larger body (Checa and Cadée, 1997; Dorgan, 2015; supports the notion that sustained digging activity may be i
B
St-Ongeetal.,2007).Itisworthnoting,however,thatlargeclams energetically limited. Depletion of the whole-body energy
l
a
are likely to be less exposed to the disturbance due to the wave reserves during digging shows that repeated burrowing activity
t
actionbecauseoftheirgreaterburialdepthsothattheyonlybecome has global bioenergetic consequences for M. arenaria and may n
e
exposedduringthestrongeststorms(Redjahetal.,2010;Zaklanand result in trade-offs with other energy-demanding fitness-related m
Ydenberg, 1997; Zwarts and Wanink, 1989). Therefore, frequent functionssuchasgrowth,reproduction,immunedefenseorbasal ri
e
disturbanceisthemostrelevantstressorforthesmallerclamssuch maintenance.
p
asthose used in the present study that typically inhabit the upper Sustained digging of marine bioturbators including such clams x
E
10–15cm of the sediment (Emerson and Grant, 1991; Emerson requireshighaerobiccapacitytosupporttheelevatedATPdemand
f
etal.,1990;Redjahetal.,2010;ZwartsandWanink,1989). (Morley et al., 2007), and stressors that diminish the organism’s o
aerobicscope(suchasextremetemperaturesordecreasingoxygen al
EnergycostsofM.arenariadigging availability)maynegativelyimpacttheburrowingcapacityofclams rn
In all studied salinity regimes, M. arenaria started digging after (as shown in an Antarctic clam Laternula elliptica) (Peck et al., u
o
some delay after exposure (∼7–90min, depending on the clam 2007). Our study shows that the whole-body aerobic capacity J
9
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb172643.doi:10.1242/jeb.172643
(measured as ETS activity) is maintained at high levels (or even mitochondria different from those in the gills and other tissues;
increasesasobservedintheFSclamsexerciseduntilexhaustion)in however, this explanation appears unlikely because the
M. arenaria subjected to repeated exercise. Although the ETS mitochondrial responses to osmolarity change are similar in
capacitywasmeasuredinthewholebodyratherthantheexercising different tissues of marine bivalves (Ballantyne and Moon, 1985;
muscle,highaerobiccapacitymightassistintheglobalmobilization Ballantyne and Moyes, 1987a,b,c; Ballantyne and Storey, 1983,
of the energy reserves to support repeated digging. Notably, high 1984). Another possibilityisthelossofthecontractileproteinsin
ETS capacity was associated with higher energy efficiency (i.e. M. arenaria in response to salinity stress. This mechanism may
lowerenergyexpenditureperunitmass)ofdigginginM.arenaria partially explain the suppression of the burrowing activity in M.
(Fig. 6B). The mechanisms underlying this correlation are not arenariafromtheFSbutnotfromtheLSregime.FSisknownto
known;however,ourdataindicatethatbothhighcapacityofaerobic resultintheproteinbreakdowninmarineosmoconformerssuchas
metabolismandenergeticefficiencyofexercisemaybeimportant marinemollusks(BergerandKharazova,1997;Hilbishetal.,1982;
factorsforM.arenariasurvivalinwind-disturbedshallowhabitats. Pierce and Amende, 1981). During the salinity upshift, cellular
proteins are broken down to increase the cytosol osmolarity by
EffectsofsalinityregimeonbioenergeticsofM.arenaria accumulation of the FAAs and thereby prevent cell shrinkage.
burrowing Whensalinitydecreases,theFAAsaremetabolizedand/orexcreted
Acclimation atLS (5) or FS (5–15) increased the average amount torapidlylowerintracellularosmolarity.Therepeatedcyclesofthe
of time needed by the clams to rebury after a disturbance by up- and down-shifts of salinity thus result in the net protein loss,
7–15%.Theclamswerealso rapidlyexhausted(after5–6digging whichconstitutesasignificantfitnesscostofisosmoticcellvolume
cycles) in LS and FS regimes compared with their counterparts regulationinmarineosmoconformers(HawkinsandHilbish,2009;
maintainedatNSwhobecameexhaustedafter∼9diggingcycles. Hilbishetal.,1982).Constantsalinity(evenifitissuboptimal)does
This indicatesthatsalinitystressimpairstheburrowingspeedand nothavethesame effects ontheproteinbreakdown,andoncethe
staminaofM.arenaria.Similarly,hypoosmoticstress(salinity17) osmotic equilibrium with the ambient water is re-established,
increased the burial time of a brittle star, Ophiophragmus protein breakdown rates return to the steady state (Hawkins and
filograneus compared with NS (21) (Turnerand Meyer, 1980). A Hilbish,2009).Inagreementwiththismechanism,thetissueprotein
decreaseinsalinityfrom35to22suppressedtheburrowingactivity content of M. arenaria clams acclimated in the FS regime was
of a polychaete Capitella sp. (measured as the sediment area significantlylowerthanintheclamsacclimatedtotheconstantNS
covered by burrows), albeit this effect was partially reversed at orLS.
salinity16(Przeslawskietal.,2009).LSalsonegativelyimpacted A decrease in the amount of the protein reserves in the body
the burial depth of tropical infaunal bivalves Venus antiqua and caused by the acclimation to FS regime in M. arenaria was
Tagelusdombeiileadingtoshallowerburrowinginwinterwhenthe compensated by an increase in the lipid content so that the total
salinity was low (24) compared with the summer when the amount of the energetic reserves of the body did not change.
prevailingsalinitywas32.5(Lardiesetal.,2001).Takentogether, Whereasthemechanismsresponsiblefortheproteinbreakdownto
these data indicate that hyposalinity stress may negatively affect maintain intracellular osmolarity in marine bivalves are well
burrowingactivityofmarinebioturbators,decreasingthebiological understood (Deaton et al., 1984; Hawkins and Hilbish, 2009),
reworkingofthesedimentandincreasingtherisksofpredationand salinityeffectsonthelipidmetabolismarelessknown.TheEastern
displacementbywaves. oysters Crassostreavirginica acclimated at a reduced salinity had
ThenegativeimpactsoftheLSandFSonburrowingactivityof higher levels of phospholipids than their counterparts acclimated
M.arenariacannotbeexplainedbytheenergydeficitinducedby undertheNSregime(GlémetandBallantyne,1995).However,in
salinitystress.Thetotalamountofenergyreservesinclams’bodies marinefish(suchasAtlanticsalmonandturbot)tissuelipidcontent
wasnotaffectedbytheacclimationsalinity,andtherelativeamount increasedwithincreasingsalinity(Tocheretal.,1994),whereasina
ofenergy(20–27%)spentbeforetheclamsbecameexhaustedand euryhaline guppy Poecilia reticulatatransition from freshwater to
losttheabilitytoreburywassimilarinallgroupsregardlessofthe seawaterledtoadecreaseofthetotalbodylipidcontenteventhough
salinity regime. Furthermore, the relationship between the energy thelipidcontentincreasedlocallyinosmoregulatoryorgans(gills,
efficiencyofdiggingandthenumberoftimestheclamscanrebury digestive tract and kidney) (Daikoku et al., 1982). Although
beforeexhaustionwassimilarintheclamsacclimatedtotheNS,LS the underlying molecular mechanisms remain unknown, lipid
andFS,despitethegenerallylowernumberoftimestheLSandFS accumulationinM.arenariaacclimatedtotheFShasimplications
y
clamscouldrebury.Thisindicatesthattheburrowingperformance forthebioenergeticsofdigging.Whiletheenergycostofdiggingof g
o
of the LS and FS clams is impaired despite the similar energy clamsacclimatedtotheconstantsalinity5or15waspredominantly l
o
demandofburrowingatthedifferentsalinitiesandsuggestsadirect supported by the breakdown of proteins, digging activity of the i
B
negativeeffectoftheLSandFSofthecontractileapparatusofthe clamsacclimatedtotheFSwasmostlyfueledbycatabolismofthe
l
clams’ muscles. The mechanisms of such salinity-induced lipids. Contribution of carbohydrate breakdown to covering the a
t
impairment of the muscle properties are presently unknown. It is energydemandofdiggingwasminimalinallexperimentalgroups n
e
unlikelythatthisimpairmentinvolvesalossoftheaerobiccapacity ofM.arenariairrespectiveoftheacclimationsalinity.Thiswasan m
ofM.arenariatissues.Thewhole-bodyETScapacitywassimilar unexpected finding because in other animals (including birds, ri
e
amongallsalinityacclimationgroupsofM.arenaria,andalthough mammals,fishaswellastheactivemolluskssuchasscallopsthat
p
acutehypoosmoticstresssuppressedCOXactivityandreducedthe can swim to escape predators), vigorous aerobic exercise is x
E
coupling efficiency in gill mitochondria of M. arenaria, these predominantly fueled by carbohydrates such as glycogen (Sahlin,
f
effects were reversed and the normal mitochondrial function 2009; Spriet, 2014). However, the fuel preference for aerobic o
restoredaftertheprolongedacclimationintheLSorFSregimes. exercise depends on the metabolic environment and the relative al
n
Itisworthnotingthatthemitochondrialcapacitywasmeasuredin levelsofdifferenttypesofsubstratesintheworkingmuscle(Spriet,
r
thegill, which isthe main organ foroxygen uptake.Itis possible 2014), so that higher dependence on the lipid catabolism in the u
o
thatLSandFShavetissue-specificnegativeeffectsonthemuscle digging FS clams may reflect higher availability of this fuel type J
10