an interestingavenueforfuturephysiologicalresearch. encountered inanchialinehabitats.Thus,taxarepresent of potentiallycopingwiththefluctuatingsalinitiesthatare of geneexpressionandiontransportcapabilityinthegillsasameans possibly otheranchialinespecies)maintainshigh,constitutivelevels rubra *Author ([email protected]) for correspondence AL 36849,USA. for Environmental andClimate ChangeStudies, University, Auburn Auburn, Program, Auburn University,Program, Auburn AL36849,USA. Auburn, © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,2309-2320doi:10.1242/jeb.103051 Received 27January 2014;Accepted31March2014 1 maintain internalosmoticconcentrationsabovethoseoftheexternal Euryhaline crustaceanscanfunctionasstrongosmoregulators that H co-transporter, qPCR,Salt/iontransport,Transcriptome, V-type Euryhaline, Geneexpression,Gill,Na KEY WORDS:Argininekinase,Carbonicanhydrase,, maintaining amaximumosmoticgradientof~868 ~0–56‰, actingasbothahyper-andhypo-osmoregulator salinity range.Inthelaboratory, H.rubra survival andosmoregulatoryresponseswereexaminedoverawide near-continuous spatialandtemporalsalinitychanges.Giventhis, subsurface freshwaterandseawaterconnections)thatencounters ancestry fromtheislands’ anchialineecosystem(coastalpondswith atyid creeks onaseasonalbasis.Here,wedescribeosmoregulationinthe brachyurans withmarineancestriesinhabitingestuariesortidal waters. However, previousstudieshavemainlyexamineddecapod osmoregulatory adaptionsallowinghyper-osmoregulationindilute Studies ofeuryhalinecrustaceanshaveidentifiedconserved Justin C.Havird during salinitytransfers mitochondria-rich cellproliferationandhemolymphosmolality rubra Osmoregulation intheHawaiiananchialineshrimp RESEARCH ARTICLE Na upregulation duringsalinitytransferinosmoregulatorygills(i.e. transporters andsupportingenzymesthattypicallyundergo osmoconformer inseawater).Additionally, expressionofion functioning iniontransport(includingwhen osmoregulatory, withalargeproportionofeachindividualgill and vitalmitochondria-richcellstainingsuggestallgillsare during salinitytransfersrelativetoothercrustaceans.Silvernitrate freshwater. Furthermore,hemolymphosmolalitywasmorestable type H INTRODUCTION ABSTRACT Department of BiologicalSciencesandCellularMolecularBiosciences of Department + -ATPase + /K rubra + during similartransfers.TheseresultssuggestH.rubra + -ATPase, carbonicanhydrase,Na -ATPase andargininekinase)weregenerallyunalteredin (Crustacea: ):expressionofiontransporters, 1,2, *, ScottR.Santos , anendemicHawaiianshrimpoffreshwater + /K + -ATPase, Na + /K 2 + Molette BiologyLaboratory /2Cl tolerated salinitiesof 1,2 H. rubra

and RaymondP. Henry – mOsm cotransporter, V- + /K + kg acts asan /2Cl − 1 – H 2 (and O in H. cytoplasmic carbonicanhydrase(CA),whichproducesH electrochemical gradientforiontransportintothehemolymph, and 2012). Ofthese,NKA,whichestablishestherequired Charmantier etal.,2009;Henry2012;McNamaraandFaria, basolateral Na 1999), (3)inosmoregulatory versusrespiratory gills(Henry, 1984; (2) ingillsversusothertissues (e.g.Henry, 2001;Lucuand Flik, (Henry, 1984;HarrisandBayliss,1988)(butseePilleretal., 1995), higher activities:(1)ineuryhaline versusstenohalinecrustaceans Kays, 1986;Henry, 2001).Overall,theseenzymesgenerally have (e.g. Towle etal.,1976;HenryandCameron,1982a;Towle and been extensivelystudiedduring salinityacclimationincrustaceans basolateral Cl accomplished viaapicalco-transport,Cl context, Na supporting enzymes(Evansetal.,2005;Henry2012):in this the MRCsisaccomplishedviaasuiteofiontransporters and Ordiano etal.,2005;Huong2010).Activesaltabsorptionin Henry, 1987;Dicksonetal.,1991;McNamaraandLima,1997; individual gillsremainspecializedforrespiration(Wheatlyand MRCs andareinvolvedinosmoregulation,althoughsomeareasof al., 2008).However, allgillsincrayfishesandshrimpspossess (1–2 μm) respiratoryepithelium(Taylor andTaylor, 1992;Freireet contrast totheanteriorgills,whicharecharacterizedbyathin characterized byathick(10–20 mitochondria-rich cells(MRCs)oftheposteriorgills,whichare (35‰) tofreshwater(FW; near0‰). of traversingwidesalinityrangeslikethosespanningfromSW Hsueh etal.,1993).Thus,onlyosmotic/ionicregulatorsarecapable their lowerlimitistypicallyintherange16–18‰(Kinne,1971; marine osmoconformerscansurviveinsalinitiesaslow~10‰, 1972) withoutcompetitionfromstenohalinespecies.Althoughsome take advantageofthesehighlyproductiveenvironments(Gross, of environmentssuchasestuaries,allowingeuryhalinespeciesto physiological transitionenablessurvivalinthefluctuatingsalinity activated ineuryhalinecrustaceans(Henry, 2005).This to waterbelow~26‰,hyper-osmoregulatory mechanismsare medium (Henry, 2001;Henryetal.,2012).However, duringtransfer hemolymph osmolalitymirroringconcentrationsintheambient marine crustaceansactasosmoconformers,withinternal McNamara andFaria,2012).InSW, theoverwhelmingmajorityof by Péqueux,1995;Charmantieretal.,2009;Henry2012; Farmer, 1983)throughactivesalttransportacrossthegills(reviewed environment whenindiluteseawater(SW)(reviewedbyManteland exchange, Na HCO Osmoregulation inbrachyurancrabstakesplacethe 3 – 1 needed tosupportNa + absorption occursviaacombinationofapicalNa + – + /K /K channels (reviewedinFreireetal.,2008; + + /2Cl -ATPase (NKA), while Cl – co-transport, Na + /H μm) osmoregulatoryepithelium,in + and Cl Halocaridina – /HCO – /HCO + channels andthe 3 – 3 – – exchange, have exchange and absorption is + 2309 + and /H +

The Journal of Experimental Biology osmotic urine(Riegel, 1968;CameronandBatterton, 1978;Wheatly salinities whilemostmarineeuryhaline crustaceansproduceiso- produce hypo-osmoticurineto preventfurthersaltlossinlower marine counterparts(seeabove). Moreover, crayfishestendto clarkii euryhaline FW crayfishes NKA activityisalsouniformlydistributedacrossallgillsin the epithelia, whichhindersionloss(reviewedinHenryetal.,2012). low salinity, FW speciestypicallyhavelowconductance(‘tight’) conductance (‘leaky’),resultinginhighratesofdiffusive ionlossin example, whilemarinespeciestendtohavegillepitheliawith high marine andFW speciesalso exist withregardtoosmoregulation.For 2005). Additionalphysiologicalandbiochemicaldifferences between (HAT), inothermarine euryhalinecrustaceanspecies(Luquetetal., 2310 the Na have beenreportedforadditionalosmoregulatorygenes,including (Serrano etal.,2007;SerranoandHenry, 2008).Analogouspatterns salinity transfersin underwent a100-foldincreaseinexpressionduringhighto low Similarly, cytoplasmicCA (CAc;theosmoregulatoryisoformof CA) fold (Luquetetal.,2005)whentransferredfrom30‰to2‰. granulate) (Sakaietal.,2006)increasedNKA expression33-to55- Chasmagnathus granulatus(subsequentlyrenamed increases inexpression(Havirdetal.,2013).Forexample, higher tolowersalinitiesover1–3 osmoregulatory gillsfollowingsalinitytransfer, withtransfersfrom 2006). osmoregulatory gilllamellae(Neufeldetal.,1980;Lovett drives MRCproliferationandassociatedprocessesinthe During low-salinitytransfers,theneedforincreasediontransport Henry, 2005;Royetal.,2007;Torres etal.,2007;Lucu2008). (Henry andWatts, 2001;Henryetal.,2002;2003; high tolowsalinities(butconfinedtheosmoregulatorygills) Holliday, 1985;Böttcheretal.,1990),and(4)duringtransfersfrom RESEARCH ARTICLE The expressionofosmoregulatorygenesusuallyincreasesinthe A V-type H freshwater Eric’s Pond HAT FW 4-[4-(dimethylamino)styryl]- EP DASPMI K Na mitochondria-richcells Mike’s Pond NKA MRCs Makalawena3 KonaCoast KeawaikiBay MIKE KalaeloaUnit Joe’sMAKA3 Pond KONA IssacHale KIKI KBP CapeHanamanioa JP inductivelycoupledplasmaopticalemissionspectrometry IH ICP-OES HM HBlueHole carbonicanhydrase(corgisoform) BabyBear C arginine kinase CA(c/g) BH BB AK A Tap’s Pond seawater Skippy’s Pond TAP Restoration 1 SW PapaBear SKIP Pu’uhonuaoH RES1 Waianae BoatHarbor PUHO3A PB OWAI KCNa NKCC List of symbolsand abbreviations List of t + (Wheatly andHenry, 1987;Dicksonetal.,1991),unliketheir /K + /2Cl threshold cycle – + + co-transporter (NKCC)andtheV-type H /K /K + + /2Cl -ATPase + Callinectes sapidus -ATPase – cotransporter Pacifastacus leniusculus ōnaunau NationalHistoricalPark3A N-methylpyridinium iodide days inducingthegreatest and Carcinus maenas and Procambarus Neohelice + -ATPase Eriocheir sinensis mOsminspeciessuchas compared with~600 mOsmaboveambientmedium(Mantel andFarmer, 1983) ~370–450 osmoregulators inFW, maintainingahemolymphosmolalityat speciesalsotendtobeweakerhyper- and Henry, 1987).FW Hemolymph osmolalityof (supplementary materialMovie 1). the surfaceandbottominhabitats withverticalsalinitygradients observed movingfreelyinandoutofthislens,aswellbetween in somehabitatsfollowingrainfall(Fig. salinity waterextending2–3 thus, theaverageoftwowasused.Lastly, surfacelensesoflow were notidenticalforthesevenhabitatswherebothtaken; instrument-based andlaboratoryosmometersalinitymeasurements gradients, salinityincreasedmeasurablywithdepth(Fig. 1A).Forthe threehabitatswithvertical salinity acrossdepth;Fig. gradient fromthesurfacetobottom(i.e.theypossessed same habitats (e.g.spanning2.8–20.9‰),mostlackedadetectablevertical anchialine habitatsrevealedthatwhilevariationexistedbetween Measurements ofverticalsalinitygradientsfrom12Hawaiian SW (32‰;960±1.6 osmoconformer (i.e.958±30.6 Osmotic gradients duringsalinity transfer Osmotic gradients Haloclines inHawaiian anchialine habitats RESULTS of rubra detailed osmoregulatorystudieshaveyettobeperformedfor (Smith andWilliams, 1981;vonRintelenetal.,2012).However, adult atyidsoutsidethe‘anchialineclade’ areintolerantofSW water forlarvaldevelopment(Hunte,1979a;Hunte,1979b),most (Fryer, 1977),and(3)whilesomespeciesrequiresaltorbrackish Smith andWilliams, 1981);(2)noextantmarineatyidsareknown FW depositsofatyidsdatetotheCretaceous(Glaessner, 1969; Moreover, thefamilyAtyidaehasadecisivelyFW ancestryas:(1) from ~0–50‰inthelaboratory(Holthuis,1973;Maciolek,1983). 2–36‰ (Maciolek,1983)andcanbeacclimatedtosalinitiesranging (, Atyidae).Thisspeciesisfoundinsalinitiesrangingfrom (~10 organism oftheHawaiiananchialineecosystemissmall Brock etal.,1987).Themostcommonandabundantmacro- habitats foundthere(MaciolekandBrock,1974;1987; in theHawaiianIslands,with~600of~1000knownanchialine habitats haveaworldwidedistribution,theyaremostconcentrated tides (e.g.20‰over24hours;Maciolek,1986).Whileanchialine daily fluctuationsinsalinitycomparabletothoseestuariesdue 1996). Accordingly, organisms fromthesehabitatscanexperience and FW throughunderground connections(Holthuis,1973;Sket, connections totheopenocean,butwhichareinfluencedbybothSW ecosystem consistsofcoastalcavesandpondslackingsurface ecosystem representanotherinterestingopportunitytodoso,asthis 2005; Huongetal.,2010).Crustaceansfromtheanchialine in brachyurancrabs(McNamaraandLima,1997;Ordianoetal., Macrobrachium For example,studiesutilizingtheFW carideangenus further insightintotheevolutionofosmoregulatorymechanisms. studies toothertaxonomicorecologicalgroupscouldprovide their lifecycleindiluteSW orFW. Giventhis,extendingsuch euryhaline crustaceanswithmarineancestrythatonlyspendpartof The majorityofosmoregulatorystudieshavefocusedon H. rubra mm), endemicshrimp or anyotheranchialineorganism. Here,theosmoregulation The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 was examinedacrossmultipleorganizational levels. have revealedmechanismsthatcontrastwiththose (Cameron, 1978;Onken,1999). mOsm cm belowthesurfaceweremeasured H. rubra kg − 1

mOsm H 2 O) reflectedthatofan

chronically acclimatedto 1C). Notably, shrimpwere kg − 1 H 2 O). Therefore, C. sapidus Holthuis 1963

1B). Field and H.

The Journal of Experimental Biology 20‰; 927versus742 RESEARCH ARTICLE H et al.,2012).Aftertransferto15‰(450 salinity transferexperiments,asiscommonincrustaceans(Henry chronically SW-acclimated animalswereusedas‘controls’ for extreme case).(C)Profilesfromfivehabitatssurveyed~8 gradients, withsalinityincreasingdepth(from15‰to30‰inthemost depth. (B)Profilesfromthreehabitatsdepictingappreciableverticalsalinity anchialine habitats.(A)Manyhabitatsexhibitedlittlesalinityvariationwith Fig. hemolymph osmolalityremainedelevated(1000±10.8 at 5‰;727versus317 443 40–56‰), hemolymph osmolalityremained belowambient ambient medium.Fortransfers tohyper-saline conditions(i.e. versus ~30 osmolality remainedatlevelshigherthantheambientmedium (898 ~0–56‰. Aftertransferstolowersalinities(0–25‰),hemolymph quantify hemolymphosmolalityaftertransfersfrom32‰ to stabilized by2 4 2 osmolality was~868±60.3 hyper-osmoregulatory response(Fig. event, withevidenceforalensoffresherwaterextending2–3 surface. Forabbreviations,seeFig.

days post-transferandremainingatthatleveluntiltheendof the 2 a xeiet(i.2A).Becausehemolymphosmolality day experiment(Fig. O) 24

.Vertical salinitygradientprofilesidentifiedfromHawaiian 1. mOsm

h post-transferbeforedroppingto~700 Depth (cm) 200 160 120 300 250 200 150 100 80 40 90 80 70 60 50 40 30 20 10 50 mOsm kg 0 0 0 B C A − 1 01 025 20 15 10 5 0 01 20 15 10 5 0 01 02 035 30 25 20 15 10 5 0

days aftertransfer, thistimepointwas used to H PB rain rain MIKE kg 2 O at15‰;860versus585 − 1 mOsm H 2 O inFW; 825versus153 mOsm

mOsm kg BB rain BB TAP rain rain RES1

7. Salinity (‰) Salinity − 1 kg H kg KIKI HM MAKA3 2 − O at25‰),reflectingastrong − 1 1

H H 2B). InFW, hemolymph 2 2 O at10‰;703versus O higherthanthatofthe PUHO3A TAP RES1

mOsm mOsm mOsm

h afterarainfall mOsm PB BB MIKE

SKIP JP BH cm belowthe kg kg

kg mOsm − − − kg 1 1 1 H H − H 1 2 2 O by O at H kg 2 O), 2 O − 1 Silver nitrate(AgNO the highestexaminedsalinity(1428±9.5 (Fig. Fig. more anteriorgills(linearfixed-effects model, posterior gillstendedtohavea smaller fractionofareastainedthan treatments (linearfixed-effects model, the areafractionofeachgill stained betweenthe2‰and32‰ latter pointcomesfromnosignificantdifference beingdetectedin acclimated to2‰(Fig. between theanteriorversusposteriorgillsorshrimp among salinitiessuggestsallanimalswereintheintermoltstage. period aswelltherelativelyconstanthemolymphosmolalities osmolality, thelackofanyobservedmoltsduringexperimental stage wasnotmonitoredduringmeasurementsofhemolymph apparent visualdifferences inmorphologyorAgNO flattened, centralgillshaft(Freireetal.,2008).Therewere no plate- orleaf-likelamellaeextendingfrombothsidesof the gills arephyllobranchiate,whichistypicalofcarideanshrimps, with with 10–16pairsoflamellaepointinganteriorlyanddorsally. The 3A),each thoracomeres (i.e.thegills)asbeingheavilystained(Fig. acclimated to15‰identifiedthepleurobranchsof5th–8th hemolymph versus1692 salinity andacclimationtime. anterior versus posterior) respondedsimilarly to salinity. number effects ( Lastly, therewasnosignificantinteractionbetweensalinity andgill Ultrastructure, ion transport stainingandMRCs ingills iontransport Ultrastructure, rubra 4 are means±s.e.m.,N 56‰ foratleast2 acclimated to~0‰,5‰,10‰,15‰,20‰,25‰,32‰,40‰,45‰,50‰and

days (dashedlinerepresents15‰).(B)Hemolymphosmolalityfor –1

.OsmolalityofHalocaridinarubra 2. Hemolymph osmolality (mOsm kg H2O) 1000 1100 1000 1200 1400 chronically acclimatedto32‰(time=0 2B). Notably, hypo-osmoregulationwasstilldetectedateven 600 700 800 900 250 500 600 800 250 500 0 0 ~ ~ 0 0 0 2010 1800mOsmkg 1500 1200 900 600 300 0 02 04 060‰ 50 40 30 20 10 0 ~ ~ A The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 2h2 as4days 2days 24 h 12 h 6h 2h 0 h B

days (dashedlinerepresentstheiso-osmoticline).Values F =3–6 samplesofsixpooledanimalspersample. -drop test,P 3 Time since Time since transfer ) stainingofwholeshrimpchronically

3B) and32‰(Fig. mOsm Salinity (A) Hemolymphosmolality(solidline)for kg =0.24), suggestingallgills(i.e. − 1 hemolymph asafunctionof H

h) thentransferredto15‰for 2 P O medium).Althoughmolt 01;Fg 4A).However, =0.13; Fig. 3C). Supportforthis

mOsm P <0.01; Fig. kg 3 − staining 1 H. rubra –1 H 2311 4A). H 2 H. O 2 O

The Journal of Experimental Biology outer marginal canals(afterMcNamaraandLima,1997)(Fig. evenly throughouteachlamella,absentaroundtheirperimeters or within thelamellaereadilyvisible(Fig. gill lamellaeunderbothsalinities(Fig. 32‰ (Fig. and 32‰treatments( between theareafractionoflamellae thatfluorescedbetweenthe2‰ (Fig. (>80%) ofallgillsandlamellae fluorescedunderbothsalinities and werenotconfinedtoparticular gills.Instead,alarge fraction in thegillswhenshrimpwereacclimatedtoboth2‰(Fig. methylpyridinium iodide(DASPMI)revealedanabundanceofMRCs transport (Fig. stage orthatpreviousstagespossessgillsdonotundergo ion 2312 RESEARCH ARTICLE Zoea stages (Fig. low levelsofubiquitousstainingintheexoskeletonearlyzoeal Although there wasnosignificantdifference betweensalinity and heavystaininginZoea (Fig. undeveloped, withonlybudsofafewlamellaedetectedper gill Vital stainingusing 4-[4-(dimethylamino)styryl]- AgNO 4 4B,C). Therewerenostatistical differences ingills1and 2 3H). However, gillsweretheonlystructures withconsistent stage, suggestingeitherthatgillsdonotdevelopuntilthis 3 staining ofZoea 3J). Furthermore,populationsofMRCswereidentifiedin

3D–F). Clearlystainedgillswerenotobserveduntilthe 3G). Relativetoadultgills,Zoea t -test, 1 4 –Zoea . P >0.225 forallcomparisons;Fig. 4 larvae fromH.rubra

3K). MRCsweredistributed 3K,L), withvessels/lacunae 4 stage gillswere identified

3I) and

4B). 3L), N - (similar totheNKA of insertion neartheC-terminus,wereidentified.Thesmallerisoform KF650070). ForNKA,twoisoforms,differing bya27aminoacid untranslated regionsofthesegenes(GenBankKF650058- transcriptomic data,including,inmanycases,the5 arginine kinase(AK)were identifiedfromthe Homologs ofNKA,CAc,CA gisoform(CAg),NKCC,HAT and treatments intheposterior-most lamellaeexaminedofgill3( identified from theEastHawaiigenetic lineage. Full-length partial (228basepair, 22%oftotallength)CAgtranscriptwas were recoveredfromtheWindward Oahugeneticlineage,onlya amplify eitherisoform.While complete CAcandCAgtranscripts NKA quantitativereal-time(qRT)-PCR primersweredesignedto identified fromtheEastHawaii geneticlineage.Giventhis,the larger isoform(similartotheNKA of the EastHawaiiandWindward Oahugenetic lineages,whilethe Gene expression duringsalinitytransfer P area fractionthatfluorescedin2‰versus32‰forgill4( all comparisons;Fig. area fractionthatfluorescedin2‰versus32‰( P =0.097), theotherthreelamellaeofthisgillhadasignificantlylarger <0.049 forallcomparisons,Fig. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051

4C). Lastly, alllamellaehadasignificantlylarger Penaeus monodon larva treatedwithAgNO with AgNO chronically acclimatedto15‰andtreated in thegillsof staining ofmitochondria-richcells(MRCs) larva treatedwithAgNO Fig. orientation asinA. (i.e. az them intoasingle,three-dimensionalimage photos at2–12 J. ImagesI–L were createdbytakingmultiple of theimage.(L)Highmagnificationimage efferent vessel/lacunaeepithelianearthetop epithelia nearthebottomofimageand magnification. Noteafferent vessel/lacunae (K) Transverse section ofIunderhigh adult H.rubra cells aregreen).(J)SameasforI,butfrom staining ofMRCsusingDASPMI(positive acclimated to2‰andfollowing adult H.rubra (I) Confocallaserscanningmicrographof by redarrows).(H)Close-upofgillsfromG. lineage; Fig. lineage; Fig. lineage; Fig. lineage; Fig. with AgNO by redarrows).(B)Close-upofgillstreated as theprimarysiteofiontransport(indicated pleurobranchs ofthe5th–8ththoracomeres) larva treatedwithAgNO larva treatedwithAgNO compared withB.(D)Zoea in somecentrallamellaeoftheposteriorgill acclimation to32‰.Notethelackofstaining arrows). (C)SameasforB,butafterchronic (darkly stainedlamellaeindicatedbyred

.Silvernitrate(AgNO 3.

-stack). Alladultgillsareinthesame 4C). 3 3 , showinggills(i.e.the after chronicacclimationto2‰

7), withdevelopinggills(indicated 7). (G)Zoea 7). (F)Zoea 7). (E)Zoea acclimated to32‰. gill 4(i.e.mostposteriorgill) H. rubra.(A)Anadult μm incrementsandsplicing ) wasrecoveredfromboth C. sapidus)wasonly 3 3 3 3 3 2 4 (from theOWAI (from theOWAI (from theKBP (from theKBP t stage ofH.rubra stage ofH.rubra -test, stage ofH.rubra 1 3 stage ofH.rubra ) andvital in vivo P <0.046 for H. rubra ′ and3′ t t -test, -test,

The Journal of Experimental Biology of MRCs. lamellae ( 20 gillspersalinity).Therewasastatisticallysignificanteffect ofsalinityfor vital MRCsfrom P considered separately(indicatedbyanasterisk,Student’s unpaired acclimated to2‰or32‰(±s.e.m., RESEARCH ARTICLE expression inthe gilldidnotchangesignificantly duringtransfer and supportingenzymesinthe gills of the two,thusfacilitatingdesign ofprimersetsforqRT-PCR. sequences werenearly100%identical inthecodingregionbetween where transcriptswereobtained frombothgeneticlineages, Hawaii andWindward Oahugeneticlineages.Forthosegenes transcripts forbothHAT andAKwereobtainedfromboththeEast isoform) fromtheEastHawaiigeneticlineage.Finally, full-length identical inaminoacidsequenceacross98residuestothe other well asapartialtranscriptofsecondNKCCisoform (76% transcripts ofNKCCwereobtainedfrombothgeneticlineages as Fig. a statisticallysignificanthigherproportionofAgNO (linear fixed-effects model, effects model,P Letters indicatestatisticallysignificantgroupingsamonggills(linearfixed- =0.003). (B,C)PercentageofcentralfourlamellaestainedwithDASPMI for Salinity transfersdidnotalter the expressionofiontransporters

.Areafractionof 4. % Lamella fluorescent % Gill stained t 100 100 -test, P (A) PercentageofwholegillsstainedwithAgNO 100 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 0 0 0 <0.01). Althoughtherewasnotanoveralleffect ofsalinity H. rubra C <0.049 forallcomparisons)indicatedwithanasterisk. A B il1Gl il3Gill 4 Gill 3 Gill 2 Gill 1 a H. rubra * acclimated to2‰or32‰(±s.e.m., P Gill 1 il3Gill4 Gill 3 =0.13), gill4(i.e.themostposteriorgill)didhave * gills stainedbyAgNO a,b N =10 /20gillspersalinity). * H. rubra.Forexample,NKA * b,c 3 staining at2‰when * Gill 2 3 or vitalstaining * 3 from H.rubra N * =10 animals/ ,c 32‰ 2‰ * t -test, transfers (t Fig. ( either 24 among animalsacclimatedtothethreeexperimentalsalinitiesat expression didnotchangesignificantlyfromtheinitiallevelor with theinitiallevel24 (Fig. evident intheexpressionofNKCCandAKduringsalinitytransfers 45‰ or15‰(ANOVA, the gillsofanimalstransferredto2‰thanthose transfer. At24 45‰ (t decreasing ~30%7 tissue showedasinglesignificantchangerelativetotheinitiallevel, with theexceptionofa~2.5-foldincrease3 For NKCC,nosignificantchangesinexpressionwereobserved, P transfer, whenlevelsdecreasedinallthreesalinitytreatments( different fromtheinitiallevelinanytransferuntil7 expression ofNKA inthecontrol tissuewasnotsignificantly the shrimpbodyexcluding‘gillundercarriage’).Specifically, during salinitytransfersinthecontroltissue(i.e.remainderof significantly different betweensalinitytreatments(Fig. expression levelsreturnedtotheirinitiallevelandwerenot initial level48 levels decreasedto35%oftheinitialvalue24 levels werestatisticallysimilartoinitialat32‰whileNKA 7 post-transfer (remainingassuchuntiltheendofexperiment being statisticallyindistinguishablefrominitiallevelsby2 decreased to26%oftheinitialvalue( approaching 36‰,withhaloclines at~1 post-transfer foranyofthesixgenes(Fig. the controltissuebetweensalinitytreatmentsateither24 (Fig. the controltissuedidnotchangesignificantlywithanytreatment (ANOVA, of animalstransferredto2‰thanthose45‰or15‰ transfer, HAT levelsweresignificantlyhigher(~2.7-fold)inthegills than theinitiallevel( post-transfer to45‰,whereexpressionlevelswere50-foldlower in alltransfersbetween3and48 treatment (Fig. Overall, nochangesingillCAgexpressionweredetectedduringany from 32‰to15‰until24 were given),consistent withthesalinitygradients of15‰to30‰ always possessedverticalsalinity stratification(althoughnodata 1973) notedthatanchialinehabitats onMaui’s southerncoastnearly 1999; Iliffe, 2000;Pohlman,2011). Previously, Holthuis(Holthuis, habitats, withsurfacewaters at0‰andthose6 been recordedfromDalmatian, BahamianandAustraliananchialine documented. Forexample,strongverticalsalinitygradients have some anchialinehabitats,spatialchangesinsalinityhavealso been characteristic ofthisecosystem(Maciolek,1986;Sket,1996). In salinity duetotidalinfluencesareawell-knownanddefining natural environment.Foranchialinehabitats,temporalchanges in facilitated byknowingthesalinityregimestheyencounterin their Understanding theosmoregulatoryprocessesoforganisms canbe DISCUSSION t

00 o l;Fg 6A).A similar trendwasnotedforAK(Fig. <0.03 forall;Fig. -test, asps-rnfr i.5A).Duringthetransferto2‰,NKA days post-transfer;Fig. In mostcases,geneexpressionwasalsonotsignificantlyaltered 6D) whileCAgdecreased~50%by7 5B,C), withNKCCexpressionincreasing~2.6-foldcompared 6F). Overall,therewerenodifferences inexpressionlevels -test, P

=0.02; Fig. h or7 -test, The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 P P <0.01; Fig. =0.03) beforereturningtoinitiallevelsby7

h, NKA levelsweresignificantlyhigher(~2-fold)in 5E). Lastly, HAT expressiondecreasedsignificantly

days post-transfer, exceptfora76%reductionofthe h aftertransferto15‰( P <0.02 forall,Fig.

6B). Likewise,CAcexpressioninthecontrol days aftertransferto45‰( t -test,

h aftertransferto2‰.Inmostcases,CAc P 5F). By7 P <0.01). SimilartrendstoNKA were <0.01; Fig.

h aftertransfer, whenexpression

h, withthemostdrasticbeing24 6E). Finally t -test,

m (Sket,1996;Humphreys, days post-transfer, HAT 5F). Notably, at24

t

6). -test, days post-transferinall P

h aftertransferto15‰ =0.02) andreturnedto , HAT expressionin

P h aftertransferto =0.03; Fig. t -test,

5F).

h or7 days post-

days after

m depth P h post- =0.04, t 2313

-test,

days 5D). days 6C).

h

The Journal of Experimental Biology ~600 mOsmkg between theexternalmediumandtheirhemolymph of the bluecrab( ‘strong’ osmoregulatingcrustaceanswithamarineancestrysuchas 2010), includinganotheratyidspecies(Born,1968).However, 1980; HenryandWatts, 2001;ChungandLin,2006;Faleirosetal., previously studiedmarineeuryhalinecrustaceans(e.g.Zanders, (32‰), ittransitionstoosmoregulationatlowersalinities,similar to 2.8–30‰) theseshrimpnaturallyencounterinanchialinehabitats. rubra as welltemporally. Ofparticularinteresttothisstudyishow suggest the findingthat 2314 H no verticalsalinitystratification(Fig. rainfall event(Fig. of thehabitatsandsurfaceFW lenseswereapparentfollowinga SW) likelyoccurinthefissuresleadingtohypogealenvironment geographic region(Fig. reported herefordeeperhabitatsfromthesameislandand RESEARCH ARTICLE 370–450 similar FW ancestry, such ascrayfish,maintainagradientof 15‰ and0‰(Fig. H. rubra , hyper-osmoregulating capacityappearedtodecreasefor supporting thisconclusion(Havird etal.,2013).Alsoatypicalfor salinity transferforeightother euryhalinecrustaceanspecies documented, withameta-analysisofhemolymphosmolalityduring shrimp speciesmaybeamongthestrongestosmoregulators

2 Relative expression While O whentransferredtoFW (Fig. 0.2 0.4 0.6 0.8 1.0 1.2 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 2.5 0 0 0 copes withthewiderangeofenvironmentalsalinities(e.g. 01010200 150 100 50 0 01010200 150 100 50 0 01010200 150 100 50 0 E C A H. rubra mOsm H. rubra between 25‰and15‰before increasingagainbetween * * * a a , , , b b b * , b Callinectes sapidus −1 kg H. rubra H can encountervariablesalinityregimesspatially −

1C). Thus,whilemanyofthehabitatsexhibited acts asanosmoconformeratoceanicsalinities 1 2 O (Cameron,1978;Henry, 2001).Incontrast, H 32‰ (pre-transfer) 2B). Interestingly, othercrustaceanswitha 2 O andareconsidered ‘weak’ osmoregulators

1B). Furthermore,highsalinitywaters(i.e. maintains agradientof~868 Time after transfer (h) transfer after Time ) maintainanosmoticgradient 15‰ transfer

1A), theseenvironmentaldata 2B) suggeststhisanchialine 0.4 0.8 1.2 1.6 0.5 1.0 1.5 2.0 2.5 0 1 2 3 4 5 0 0 * 2‰ transfer 01010200 150 100 50 0 01010200 150 100 50 0 01010200 150 100 50 0 B F D * * * * , b , a , , * a b b , b

mOsm * * 45‰ transfer kg H. − 1 new salinity, only toswitchbacktheoriginal statewithinminutes prematurely ‘committing’ osmoregulatorypathways tocopewitha fluctuating salinities with thenewsalinityisadvantageous. Giventheconstantly chronic transfer, andactivatingosmoregulatorypathwaystocope Therefore, whentheyundergo asalinitychange,itislikely tobea spring migrationintotheestuary forbluecrabs(Warner, 1976)]. salinity aspartofannualmigrationsintheirnaturallifecycle [e.g. other crustaceanssuchasC.sapidus transfer (Fig. hemolymph osmolalitysimilartothatfoundinSW until2 by 12 hemolymph osmolalityby3 lower salinities.Forexample, hemolymph osmolalitytonewlevelsfollowingtransferfromSW to having anevolutionaryhistorytiedtoFW environments. pressure offluctuatingsalinitiesinanchialinehabitats,despite maintaining strongosmoticgradientsbecauseoftheselection potentially otheranchialineatyidshrimp,maybeuniquein by amemberofFW-adapted family. Thissuggests family, while likely representsasecondaryFW invasionbyamemberofmarine of 335–400 classified as‘weak’ osmoregulatorsandmaintainosmoticgradients (Mantel andFarmer, 1983).PreviouslystudiedFW atyidsarealso 550–700 in FW habitatsandmaintainsanosmoticgradientof mitten crab( 2004). Anexceptiontothis(besides Unlike h (HenryandCameron,1982b).Here,H.rubra mOsm H. rubra,mosteuryhalinecrustaceansrapidlydecrease The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 mOsm Eriocheir sinensis),whichspendsmostofitsadultlife 2A). Onepossibleexplanationforthisdifference isthat H. rubra kg V-type H associated isoformofcarbonicanhydrase(CAg)and(F) carbonic anhydrase(CAc),(E)themembrane- (C) argininekinase(AK),(D)thecytoplasmicisoformof salinity transfers. supporting enzymesinthegillsof Fig. 48 ATPase (NKA),(B)Na at 24 respectively) werealsoexamined24 32‰ to2‰and45‰(filledtrianglessquares, (open andfilledcircles,respectively).Transfers from othoc ANOVA withTukey post significant groupingsamongsalinitytreatments(two-way measurements (Student’s indicate asignificantdifference fromthepre-transfer treatment (seeMaterialsandmethods).Asterisks N single samplefromthe2‰,24 transfer. Symbolsrepresentmeanvalues(relativetoa =5–6 samplesof3–6pooledgillundercarriagesper − kg 1 h, and7days(168

.Relativeexpressionofiontransportersand 5. − H H. rubra h and7 1 represents aninvasionofaeuryhalinehabitat 2 H O (Onken,1999).However, thisspecies + -ATPase (HAT) wasquantified0,3,8,24and 2 O (Born,1968;Dhaouadi-Hassenetal.,

h aftertransferandreachesstablelevels

day timepointsrepresentstatistically encounters inanchialinehabitats, mRNA expressionof(A)Na C. sapidus

h) aftertransferfrom32‰to15‰ H. rubra)maybetheChinese + /K might ‘commit’ toaspecific + t /2Cl -test, P tests, P –

h treatment)±s.e.m., co-transporter (NKCC), <0.05 forall).Letters noticeably lowers <0.05 forall).

h and7 H. rubra H. rubra maintained

days after days post- following + /K + - , and

The Journal of Experimental Biology et al.,1989),andforstrongosmoregulators, AgNO surface areaforthemoderateosmoregulator and increased4-fold duringlowsalinitytransfer for transfer fromSW to12‰for from 35%ofthelamellaein osmoregulatinggillsto52%during RESEARCH ARTICLE maintaining thehighosmoticgradientseenin 2012), whichwouldreducediffusive ionlossandcontributeto typically having‘tight’ gillepithelia(reviewedinHenryetal., regularly encountered.ThisissupportedbyFW crustaceans salinities evenwhenathighasinnatureloware constantly activeosmoregulatorymechanismsforcopingwithlow has beenselectedagainst.Rather, thisspeciesappearstomaintain or hours,maybeenergetically expensiveandinefficient, andthus AgNO 32‰ to72%in2‰(Student’s unpairedt statistically significantincreaseintotalareastainedfrom59% in individually, themostposteriorgill(i.e.4;Fig. effects model, a trendtowardsgreaterstainingin2‰versus32‰(linearfixed- change significantlywithtransfertolowsalinity, althoughtherewas previously studiedcrustaceans,overallAgNO lamellae, impliesgillswithalowersurfacearea.Incontrastto most (Lovett etal.,2006)].This,combinedwiththeirthick,plate-like and 10–16versus~300lamellaepergillcomparedwith fewer gillsandlamellaepergill[e.g.fourversuseight gills appear lesscomplexthanthoseofothereuryhalinecrustaceans, with arfeae morphologically similartothoseoftheanchialineatyid et al.,2005;Henry2012),withthegillsof transport inH.rubra,asistypicalofcrustaceansandfishes(Evans Relative expression AgNO 0.4 0.8 1.2 1.6 0.4 0.8 1.2 1.6 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 0 0 3 (Jaume andBréhier, 2005).Generally, thegillsof 01010200 150 100 50 0 01010200 150 100 50 0 01010200 150 100 50 0 E C A staining neverexceedsmorethan 30%ofthetotallamellar 3 * staining revealedthegillsasprimarysiteofion P =0.13, Fig. 2 petase)1‰tase ‰tase 45‰transfer 2‰transfer 15‰transfer 32‰ (pre-transfer) 4A). Furthermore,whenconsidered C. granulatus * * * * * * * * * Time after transfer (h) transfer after Time -test, (Genovese etal.,2000) 0.5 1.0 1.5 2.0 2.5 0.2 0.4 0.6 0.8 1.0 1.2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 C. maenas P H. rubra 0 0 0 =0.003). Incontrast, 3 * 01010200 150 100 50 0 01010200 150 100 50 0 01010200 150 100 50 0 B D F staining didnot

3B,C) showeda H. rubra 3 staining went . C. sapidus C. sapidus (Compere H. rubra being 59–71% AgNO gills developatlatezoealstages(i.e.Zoea finding suggeststhatosmoregulatorycapabilitiesin ~15 Khodabandeh etal.,2005). localized tothecentralfilamentsofeachgill(Dicksonetal., 1991; some lamellaeinallgillsshowstainingFW crayfish,itis many otherbrachyurans(CopelandandFitzjarrell,1968), while example, onlytheposteriorgillsundergo stainingin staining typicallyconfinedtojusttheosmoregulatinggills. For maximum andlarge increasesarenotpossible.As or, morelikely, thattheMRCpopulationisalreadyatornearits H. rubra (Neufeld etal.,1980).Thus,theincreasedstainingseeningill4of typically eitherrespiratoryorosmoregulatoryinnature,withAgNO as anosmoconformer. Thisisnotableasbrachyurangillsare fraction ofthetotalgillsurfaceareain euryhaline crustaceans(Lovettetal.,2006)representsalarge that the‘osmoregulatorypatch’ foundonlamellaeofother osmotic gradientrecordedinthisspecies.Furthermore,itsuggests MRCs evenin32‰,whichwouldprovideamechanismforthehigh rubra such stainingisameasureofMRCabundance,thissuggests involves thedifferent habitatsexploitedbylarvae versusadults.For proposed explanationforthis ontological shiftinosmoregulation osmoregulatory capabilitiesdevelop inlaterjuvenilestages.One 2004), whereearlyzoeal stages areosmoconformersand granulatus supported bystudiesofCrangoncrangon do notdevelopuntilthislife stageorlater. Thishypothesis is AgNO days post-hatch(CouretandWong, 1978;Iwai,2005).This * may haverelativelydenseandconstitutivepopulationsof 3 suggests eitherafairlyweakresponsetosalinitychanges staining oflarval (Charmantier etal.,2002)and The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 3 statistical analysesareasinFig. methods). Genes,symbols,asterisks,lettersand other thanthegillundercarriage(seeMaterialsand following salinitytransfers. supporting enzymesincontroltissuesofH.rubra Fig. control tissuesamples. that the3 staining inallgillswhenacclimatedto32‰and

.Relativeexpressionofiontransportersand 6. h, 15‰treatmentwasnotinvestigatedforthe H. rubra revealed thation-transporting H. rubra,evenwhenacting C. maenas (Cieluch etal.,2005), 4 Control tissuesare ), whichcorrespondsto

5, withtheexception H. rubra C. sapidus (Cieluch etal., H. rubra likely 2315 had and H. C. 3

The Journal of Experimental Biology in this upregulationissmallcompared withpreviousreports. 2.6-fold increaseinexpression 24 Support forthis hypothesiscomesfromAgNO even whenH.rubra that expressionofthesegenes is alwaysatarelativelyhighlevel, Only forNKCCwasacomparableresultseenin and AK(e.g.Luquetetal.,2005)alsofollowthisgeneraltrend. CAc (butnotCAg)(seeSerranoandHenry, 2008),NKCC,HAT vannamei 2316 these earlylifehistorystagestowardsaddressingthisquestion. developing larvae.Interestingly, larvaeof expensive osmoregulatorymechanismsarelessimportantfor et al.,2012)andoceanicsalinitytendstobeconstant,energetically crustaceans actasosmoconformersinoceanicsalinities(e.g.Henry some species(Hunte,1979a;Hunte,1979b).Becausemost export larvaetotheocean,andSW isnecessaryfordevelopmentin returning asjuveniles(Anger, 2001).Furthermore,manyatyidsalso ‘exported’ intotheoceanwheretheyundergo developmentbefore example, larvaeofmanyestuarineandriverinecrustaceansare RESEARCH ARTICLE AgNO acclimation (Lovettetal.,2006).Taken together, theresultsof ~35% to60%ofthelamellarsurfaceareaduringlowsalinity with theosmoregulatorypatchof being distributedevenlythroughoutlamellae.Thiscontrastssharply can beconsideredanosmoregulatorypatchin in abiologicalcontext.Thus,nearlytheentiresurfaceoflamellae magnitude ofthisincreasewassmall(~7%)andlikelynotsignificant increased inposteriorgillsunderhyper-regulating conditions,the osmoconforming andhyper-regulating salinities.Althoughstaining all gills/lamellaehaddenseMRCpopulationsunderboth Macrobrachium amazonicum maenas (Jayasundara etal.,2007), paramamosain and 2‰(Luquetetal.,2005),withsimilarresultsfor granulatus by Havirdetal.,2013).Forexample,NKA expressionin osmoregulatory gillsduringcomparablesalinitytransfers(reviewed expression ofthesegenesusuallyincreasesdramaticallyinthe obtained fromcontroltissue(Fig. to nochangeduringsalinitytransfer(Fig. osmoregulatory genesinthegillsof activated mechanismsofionregulation,expression posterior gillsappeartobethemostresponsivesalinitytransfers. osmoconformer; (2)allgillsparticipateinosmoregulation;and(3) level, evenatsalinitieswherethespeciesfunctionsasan osmoregulatory mechanismsconstitutivelyactivatedatthecellular the euryhalinityandosmoregulatorycapabilitiesof transporting gills,remainsunknown.Futurestudiesshouldfocuson particularly inearlylifestageslackingdevelopedandfunctionalion- osmoconformers inSW, howtheysurviveinlowersalinities, osmoregulation hypothesizedfor 2005)]. Therefore,whileitispossibletheontogeneticshiftin in CouretandWong (CouretandWong, 1978);20‰inIwai(Iwai, to lowersalinitiesinthelaboratory[e.g.15‰thisstudy;10–15‰ habitat) componentoftheanchialineecosystem(Craftetal.,2008) potentially fullstrengthSW inthehypogeal(andhypothesizedlarval successful developmentinawiderangeofsalinities,from Why arewell-characterizedosmoregulatory genesnotupregulated Vital MRCstainingwasconsistentwithAgNO In supportofthehypothesisthat H. rubra 3 and vitalMRCstainingsuggest:(1)thegillsof (Serrano andHenry, 2008;Jilletteetal.,2011), (Wang etal.,2012).Although utilizedinfewerstudies, increased 25-to55-foldaftertransferfromSW to45‰ as inothereuryhalinecrustaceans? Onehypothesisis (Chung andLin,2006),Pachygrapsusmarmoratus is functioningasanosmoconformer inSW. C. sapidus (Faleiros etal.,2010)and H. rubra

C. sapidus,whichincreasesfrom h aftertransferto2‰.However, H. rubra

H. rubra 6). Ineuryhalinecrustaceans, (Serrano etal.,2007), is duetolarvaeactingas

maintains constitutively 5), withsimilarresults H. rubra generally showedlittle H. rubra,withMRCs 3 and MRCstaining, H. rubra,witha H. rubra 3 H. rubra can undergo Litopenaeus staining, as during Scylla have C. C. maintaining anosmoticgradientof~868 one ofthestrongestosmoregulatorsdescribedamongcrustaceans, an endemicHawaiiananchialineatyidshrimp,appearstorepresent osmoregulation fromananchialinecrustacean. Serrano etal.,2007;Henry2012). salinity transfers(seeHenryandCameron,1982a;Henry, 2001; not significantlychangeinnon-osmoregulatorytissuesduring skewed theexpressionresults,asgenesunderinvestigationdo unlikely thatusingthegillundercarriageinsteadofindividualgills euryhaline atyidsoranchialinecrustaceansingeneral.Finally, itis this patternofelevatedosmoregulatoryprocessesischaracteristic fluctuations theyencounter, anditwillbeinterestingtoseewhether allow processes isanadaptationtoanchialinehabitats,suchastrategymay whether thishypothesizedchronicupregulationofosmoregulatory the gillsof which indicateelevatedcellularmechanismsofosmoregulationin H Pond; OWAI, Waianae BoatHarbor;PB,PapaBear;PUHO3A,Pu’uhonua o Keawaiki Bay;KONA,KonaCoast; MAKA3, Makalawena3;MIKE,Mike’s Cape Hanamanioa;IH,IssacHale; JP, Joe’s Pond;KBP, KalaeloaUnit; KIKI, except forPUHO3A.BB,BabyBear; BH,BlueHole;EP, Eric’s Pond; HM, used intheotherexperiments. transcriptome sequencing,andthegray starindicatestheoriginofanimals staining, graycirclesindicateoriginsof measured, blackcirclesindicateoriginsof study. Fig. 7).Fieldmeasurementsweretakenevery Hawaii, MauiandOahu(Fig. haloclines) weremeasuredfrom12anchialinehabitatsontheislandsof spatially inthewatercolumn,potentialverticalsalinitygradients(i.e. To determinethemagnitudeofsalinitychange developing afurtherunderstandingofosmoregulationingeneral. responses ofanchialineorganisms tosalinityasameansof Future studiesshouldcontinuetoexplorethephysiological of thetwoisresponsibleforthisdeviationfrompreviousmodels. is unclearwhetherselection,evolutionaryhistoryoracombination crustaceans onlyunderlowsalinityareconstitutivein Instead, osmoregulatorymechanismstypicallyactivatedinother euryhaline crustaceansdonotadequatelycharacterize Notably, previouslydescribed osmoregulatoryprocessesfor Haloclines inHawaiian anchialine habitats MATERIALS ANDMETHODS Pond; TAP, Tap’s Pond. ō 19° 20° 21° 22° In conclusion,thisreportrepresentsthefirstattemptstodescribe naunau NationalHistorical Park3A;RES1,Restoration 1;SKIP, Skippy’s

.Mapdepictinganchialinehabitatsandanimaloriginsforthis 7. Black squaresindicatehabitatswhereverticalsalinitygradientswere H. rubra The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 H. rubra Kauai 0°22 0°24 205° 204° 203° 202° 201° to copewiththerapidandcontinuoussalinity OWAI PB, TAP,RES1 KBP, MIKE,BB, regardless ofsalinity. Although itisunclear Halocaridina rubra Oahu EP JP, SKIP, BH H. rubra H. rubra Molokai lineages usedin PUHO3A

mOsm MAK3 KIKI were observedatallsites HM (/) larvae usedinAgNO H. rubra KONA Maui Halocaridina rubra, 0 kg − N might encounter 1 Hawaii H H. rubra.It 2 H. rubra. 100 km O inFW. IH 3

The Journal of Experimental Biology [K (ICP-OES; PerkinElmerOptima7000DV, Waltham, MA,USA) tomeasure were subjectedtoinductivelycoupledplasmaopticalemissionspectrometry samples withminimalintracellularfluidleakage,threehemolymph (Wescor 5100C). the experimentalmediawereconfirmedusingvaporpressureosmometer observed at56‰,where50%ofshrimpperishedwithin48 pooling strategy, with transferred to~0–56‰,followedbysamplingafter48 initial timeseries(seeResults),shrimpacclimatedto32‰werealso measured onavaporpressureosmometer(Wescor 5100C).Basedonthis in tightlysealedtubesat–80°Ctopreventevaporationuntilosmolalitywas Approximately 10 filter andpreventingcontaminationofthehemolymphsample. 14,000 rpm for5–10min,withcellulartissuedebrisbeingretainedbythe enough hemolymphforquantification.Tubes werecentrifugedat N Corning CostarSpin-X0.22 μmcentrifugefiltertube(Sigma-Aldrich),with shrimp withascalpelblade.Sixpersamplewerepooledinto the outerdorsalportionsofthoracicandabdominalcarapace using Kimwipes(Sigma-Aldrich,StLouis,MO,USA),andthenlacerating extracted byanesthetizingshrimponice,wickingoff anysurfacewater sampled at0,2,6,12,24,48and96 (reviewed byHenryetal.,2012).Shrimpwerethentransferredto15‰and induction/up-regulation instudiesinvolvingtransferstolowsalinity thus, thosevalueshavealsoservedasacontrol,startingpointforstudiesof below) areatlow, baselinelevelsincrustaceansacclimatedtohighsalinity; activity andexpressionoftransportproteinssupportingenzymes(see changes whenanimalsareexposedtoalteredsalinities.Furthermore,the osmoconformers, andthisconditionservesasthebaselineforcomparing aquaria foratleast1 developer (Eastman KodakInc.,Rochester, NY, USA)while shaking, water. Thiswasfollowedbyincubation for1 RESEARCH ARTICLE AgNO 15‰ wererinsedthreetimeswith deionizedwater, stainedwith0.05% To determinesitesofiontransport, wholeanimalschronicallyacclimatedto Prior toexperiments, continuous, year-round reproductioninthelaboratory. the aquaria.Thishusbandrytechniqueisidealfor microbial andalgalgrowthoccurringontheporousvolcanicrockpresentin no circulatingwaterandfeeding.Shrimpwereallowedtograzeonthe laboratory, shrimpwereheldat15‰with~200animalsper38 experiments, seebelow)werefromtheSouthMauilineage.In important tonotethatmostanimalsusedinthisstudy(exceptforlarval represents atleasteightdistinctgeneticlineages(Craftetal.,2008),itis of collectionduring2011. Because Individuals of five habitats,YSImeasurementswerealsorecorded~12 vapor pressureosmometer(Wescor 5100C,Logan,UT, USA).Fortheother a verticaltransectevery30 Additionally, forasubsetofsevenhabitats,watersampleswerecollectedin meter (Yellow SpringsInstruments,Yellow Springs,OH,USA). 15 previously reportedfor (120–150 (Sowers etal.,2006),butmuchlowerthantypicalintracellular [K significantly. leakage ofaspecificionshouldnotalterhemolymphosmoticconcentration Moreover, intracellularosmolalityisinequilibriumwithhemolymph;thus, using handnetsandshippedtoAuburnUniversity, AL,USA,within~2 AgNO Osmolality duringsalinitytransfer Animals =3–5 samplespertimepoint.Thispoolingschemewasnecessarytoobtain To confirmthattheabovecentrifugationmethodproducedhemolymph + cm fromthehabitats’ surfacetobottomwithahandheldYSIconductivity ]. Onaverage,[K 3 3 while shakingfor20 and MRC staining of gills and MRC stainingof mmol H. rubra

l − 1 μl ofhemolymphwasrecoveredpersampleandfrozen ). Thissuggestsintracellularleakagewasminimal. H. rubra +

were collectedfromCapeHanamanioa(HM),Maui ] was33.4±0.52 N month. Atthissalinitymarinecrustaceansare Litopenaeus vannamei =3–6 samplespersalinity. Mortalitywasonly

cm, frozen,andmeasuredforosmolalityusinga

min, andagainrinsedthreetimeswith deionized individuals wereacclimatedto32‰in4 H. rubra

h post-transfer. Hemolymphwas mmol across theHawaiianIslands

h insaturatedKodak D-76

l hemolymph (16 − 1 , whichishigherthan

H. rubra,yielding h followingrainfall.

h usingthesame

h. Salinitiesof

l aquarium,

mmol

days

l − + 1

] ) l ~7 and werecollectedfromdistinctcoloniesmaintainedinthelaboratoryfor undercarriage dissectedandrinsedina700 32‰ (N et al.,1997;Choe1999).Shrimpwereacclimatedtoeither2‰or Invitrogen, Carlsbad,CA,USA)stainingandconfocalmicroscopy(Heijden transfer, MRCdensity wasquantifiedusingDASPMI(MolecularProbes, were alsostainedwithAgNO (3) onlyspecificgillsrespondedtosalinity. (1) salinityinfluencedstaining;(2)stainingcorrelatedwithgillnumber;and per gillnumberintotal).Overall,thismodeladdressedspecificallywhether: as bothleftandrightgillsweremeasuredperindividual(20salinity interaction betweenthetwo.A randomeffect ofindividualwasalsoincluded fraction stainedwasmodeledasafunctionofsalinity, gillnumberandthe Institutes ofHealth,Bethesda,MD,USA)(Schneideretal.,2012).The fraction ofeachgillstainedwasquantifiedusingImageJv1.45s(National salinity) andthenstainedphotographedusingtheaboveprotocol.The chronically acclimatedtoeither2‰or32‰foratleast1 five oftheeightgeneticlineagesfoundacrossHawaiianIslands(Fig. quantified fromeachgillusingImageJ. each salinitytreatment,theareafractionstainedoffourcentrallamellaewas with theexcitationandemissionfiltersetforfluoresceinisothiocyanate.For laser scanningmicroscope(NikonInstrumentsInc.,Melville,NY, USA) desiccation. Vital MRCstainingwasvisualizedwithaNikonA1confocal drop ofshrimpRinger’s solutionandcoveredwithacoversliptoprevent undercarriages werethenplacedonaglassslidewithconcavityinsingle Gill with shrimpRinger’s solutionlackingDASPMI(Karnakyetal.,1984). 1 1978; Iwai,2005). based onmorphologicalfeaturesdescribedelsewhere(CouretandWong, (Promega) substituted withphenol–chloroform–isoamyl alcohol(P2069, chloroform extraction usingtheRNAgentsTotal RNA IsolationSystem experimental transferstoavoidpseudoreplication. sample werehousedinindividual ~400 experimental treatment…’ (Hurlbert,1984).Giventhis,shrimp fromasingle single tank,containingafixed number offish[orshrimp],foreach identified ascommittingpseudoreplication becausetheyoftenutilize‘…a N 3–6 shrimp(dependingonsize)werepooledforasinglesample, with tissue, exceptforthe15‰,3 musculature, digestivetract,nervoussystem)wasalsoutilizedasacontrol (Promega, Madison,WI,USA).Theremainingtissuefromeachshrimp (i.e. denaturing solutionfromtheRNAgentsTotal RNA IsolationSystem undercarriages (seeabove)dissectedintotubescontainingice-cold followed byasinglerinsewithdeionizedwater. AgNO 24 Havird etal.,2013),shrimptransferredto2‰and45‰weresampled at those resultsandpreviousstudies(Luquetetal.,2005;Serrano 2007; (i.e. at32‰)and3,8,2448 15‰ or45‰.Forthetransferto15‰,shrimpweresampledbefore chronically acclimatedto32‰for1 To quantifygeneexpressionin To determinewhethersalinityinfluencedAgNO Microscope (LeicaMicrosystems,Wetzlar, Germany)at1–8×magnification. Shiraishi, 1997).AnimalswerephotographedusinganS8APOStereo the productionofAgCl(Croghan,1958;Hollidayetal.,1990;Kikuchiand transport epithelia,whicharepermeabletosilverand/orchlorideions,through incubated inshrimpRinger’s solutioncontaining25 to bedissectedseparately. Followingrinsing,gillundercarriageswere approach wasutilizedbecauseindividualgillsweretoosmallanddelicate minimum amountofsupportingmusculature/exoskeletalmaterial.This osmoregulation). Thisgillundercarriageconsistedofthegillsanda shrimp Ringer’s solutionconsistentwith Expression of osmoregulatory genesduringsalinitytransfer osmoregulatory of Expression

=5–6 samplespertreatment.Notably, suchphysiologicalstudieshavebeen h whileshakingatroomtemperaturetolabelMRCspriorbeingrinsed To investigatetransportepitheliaduringdevelopment, To morecloselyinvestigatechangesinthegillsof Total RNA wasisolatedfrom gillandcontroltissuesbyphenol– h and7 years. Thedevelopmentalstageofeachlarva(Zoea =10 persalinity)for1 days post-transfer. Shrimpwereanesthetizedoniceandgill The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051

3 h post-transfertreatment.Foreachtreatment, H. rubra using theaboveprotocol.Larvaecamefrom

month, anesthetizedoniceandtheirgill

h and7

month andthentransferredto2‰, during salinitytransfer, shrimpwere H. rubra

mmol days post-transfer. Basedon

ml containersduringthe hemolymph duringhyper- 3

l H. rubra − staining, animalswere μmol 1 NaCl solution(i.e.a 1 –Zoea 3

staining blackens month (N l H. rubra − 1 during salinity 4 DASPMI for ) wasscored =10 per larvae 2317

7)

The Journal of Experimental Biology HAT NKCC CAg produced onlyasingle amplicon.Standardcurvesof thresholdcycle( AK CAc initial denaturingfor3 a heatingrateof0.5°Cevery5 AK). Followingthelastcycle,melting curveanalyses(from55to95°Cwith and 1 Table H 2318 co-transporter (NKCC),theH below) analyses. cDNA wascheckedbyPCRpriortobeingsubjectedqRT-PCR (see thus normalizingeachsampletototalRNA (Bustin,2002).Theresulting Superscript IIreversetranscriptasewithanoligo-dT primer(Invitrogen), Poly-A RNA in2 μgoftotalRNA persamplewasreversedtranscribedusing genomic DNA contaminationwasobservedintheseexaminedsamples. Bioanalyser 2100(AgilentTechnologies, Wilmington, DE,USA).No quality/quantity checkedforarepresentativenumberofsamplesusing (Thermo FisherScientificInc.,Waltham, MA,USA)andRNA quantified foreachsampleusingaNanoDrop2000spectrophotometer and RNase-Zap(Ambion,Austin,TX,USA).Total RNA concentrationwas tissue homogenizationsbyusingsteriletoolsrinsedwithRNase-freewater Sigma). RNase-freeconditionsweremaintainedduringdissectionsand NKA, Na NKA Target gene RESEARCH ARTICLE designed forH.rubra et al.,2005;Serrano2007;Havird2013),specificprimerswere playing rolesincrustaceanosmoregulationduringsalinitytransfer(Luquet Table These primerswerespecificallydesignedfor Homologs forthesixgenesin searchable BLAST databases(www.auburn.edu/~santosr/halo_blast.htm). and HILO)(seeCraftetal.,2008)(Fig. generated fromtheEastHawaiiandWindward Oahugeneticlineages(EP a functionoftemplate availability( membrane-associated, respiratoryisoformofCA (CAg),theNa the cytoplasmic,osmoregulatoryisoformofcarbonicanhydrase(CAc), sequences foreachgenefrom nuclease-free water, 0.3 triplicate andconsistedof12.5 (Bio-Rad Laboratories,Hercules,CA,USA).Reactions(25 Biosystems, FosterCity, CA,USA)usingtheiQSYBRGreenSupermixKit qRT-PCR onanABI7500Real-Time PCRSystemthermocycler(Applied the SouthMauigeneticlineage. template cDNA toensuregenerationofasingleampliconfrom et al.,2006).TheqRT-PCR 1)weretestedbyPCRof primers(Table in ordertoavoidamplificationofanycontaminatinggenomicDNA (Choe a conservedintron/exonboundaryknownfromothercrustaceanhomologs South Mauilineage.Lastly, primersweredesigned(whenpossible)tospan two geneticlineagestominimizeanydifferences thatmightariseinthe qRT-PCR primersweredesignedtoregionswith100%identitybetweenthe Potential openreadingframeswereextractedfromresultingmatchesand (Altschul etal.,1990)searchesagainstthetwo available onGenBank.ThesewerethenutilizedasqueriesinBLASTX + To compare For eachofthesixgenes,mRNA levelswereassessedpersamplevia -ATPase; AK,argininekinase.

min at55°C(NKA andCAc),58°C(HAT) or60°C(NKCC,CAgand 1) and1 μltemplatecDNA,usingthefollowing cycleparameters: 1. Nucleotidesequencesfor + /K + -ATPase α H. rubra targeting sixgenes:Na -subunit; CAcandCAg,carbonicanhydrasecytoplasmicmembrane-associatedisoforms;NKCC,Na

min at95°C,followedby40cycles of10 mRNA expressionofgenespreviouslyidentifiedas μl forwardandreverseprimers (25 NKCCR2020 NKCCF1826 CAgF373 HATR994 HATF800 CAgR562 CAcR613 CAcF444 AKR1001 AKF821 NKAR2854 NKAF2679 Primer C. sapidus, +

s) wereperformedtoconfirmthat reactions -ATPase (HAT) andarginine kinase(AK). H. rubra μl iQSYBRGreenmix(2×),10.9 C Halocaridina rubra t versus log

were identifiedbydownloading 7) thatarepubliclyavailableas C. maenas + H. rubra /K + -ATPase H. rubra 10 AGGCACGAATCTTATGACGAGCGA ATCGTCCACAGCTCTTGGTGCTAA TTAGGGCACTGACCAAAGGAGTGA AGCCTGGGCTCAGAACACACTATT TCAGCGAACCAGGGTAGGTGAAAT GTCAGGCCCATACGACGTTTGTTA TGGGAATCTGGGTGATGGAACCTT AGTGCGAGAAGCACGTCCTCATTA TCAAATCGGCCCACTGGACAATCA TTTGGGTGTGTTTCTGACCGTTGG ACCTGGGTACCACTGTTCGTTCTT TGGCTTCCTTCCTCCCAAACTCTT Sequence (5 cDNA volume)were and othercrustaceans using transcriptomes α -subunit (NKA), transcriptomes. μl) wererunin specific primersusedinqRT-PCR ofosmoregulatorygenes H. rubra

′ + μmol s at95°C to3′ /K + C /2Cl ) t ) as l − of 1 – ; maximized fortheinitialreaction; Serrano etal.,2007;Havird2013). among thetreatmentsbasedonpreviousstudies(Luquetetal.,2005; as thiswaspredicted generated byserialdilutionofasinglesamplefromthe2‰24 http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.103051/-/DC1 Supplementary materialavailableonline at Education (ACHE). Doctoral GraduateResearchFellowshipfromtheAlabamaCouncilonHigher Graduate StudiesforGenetics/PhysiologyfromTheCrustaceanSociety, anda Instructors (PADI) FoundationResearchAward (no.5089),aFellowshipin Excellence graduatefellowshipprogram,aProfessionalAssociationofDiving J.C.H. fromtheAuburnUniversityCellularandMolecularBiosciencesPeaks of Grant program[DEBno.1311500 toJ.C.H.].Fundingsupportwasalsoprovided to S.R.S. andEPS11-58862 toR.P.H.] anditsDoctoralDissertationImprovement Funding wasprovidedbytheNationalScienceFoundation[DEBno.0949855 to R.P.H. revisedthemanuscript. experiments, analyzedthedataandwrotemanuscript.J.C.H.,S.R.S. J.C.H., S.R.S.andR.P.H. designedtheexperiments.J.C.H.performed The authorsdeclarenocompetingfinancialinterests. material Movie environmental fielddata.We alsothankDavidWeese forprovidingsupplementary and WildlifeService,HawaiiOffice) forassistanceincollectingshrimpand We thankDavidWeese, StephanieIrvin,KileySeitzandLorenaWada (USFish (code availableonrequest)(RCoreTeam, 2013). Statistical analyseswereperformedintheRv2.12.0statisticalenvironment respectively. and MoletteBiologyLaboratoryforEnvironmentalClimateChangeStudies, contribution nos117 and21totheAuburnUniversity(AU)MarineBiologyProgram anonymous reviewerswhosecommentsimprovedthemanuscript.Thisrepresents Cannon assistedintranscriptomelibrarypreparation.We aregratefultotwo assistance withICP-OESanalyses.FranziFranke,KevinKocotandJohanna Michael Millerprovidedassistancewithmicroscopy. JonathanOliverprovided v1.2 (AppliedBiosystems). subsequent reaction.RelativeexpressionwasthenquantifiedusingSDS such thatthe lshl .F,Gs,W,Mle,W,Mes .W n imn D.J. Lipman, and K. E.W. Anger, Myers, W., Miller, W., Gish, S.F., Altschul, References Supplementary material Funding Author contributions Competing interests Acknowledgements Statistical analyses Vol. 14. local alignmentsearchtool. Lisse, TheNetherlands: A.Balkema. 20) h ilg fdcpdcutca ave In (2001). Thebiology ofdecapodcrustaceanlarvae. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051

1. StephenKempf,MariaMazzillo-Mays,ShannaHanesand R 2 of thelinearregressionstandardcurvewas a priori 194 189 194 175 180 169 Amplicon length(nucleotides) J. Mol.Biol. to havethehighestlevelsofgeneexpression 215 C t , 403-410. was thenheldconstantforeach C t was determinedforeachgene + /K + /2Cl – co-transporter; HAT, Crustacean Issues

h treatment, (1990). Basic ,

The Journal of Experimental Biology hoaiHse,S,Tils .P,Camnir .adBuaz,M. Boumaïza, and G. Charmantier, J.P., Trilles, S., Dhaouadi-Hassen, lese,M.F. Glaessner, G.N. Pellerano, and G.A. Rosa, D.A., Paz, C.M., Luquet, G., Genovese, rs,M.G. Gross, iko,J . ilmn .M,Re,R .adRy,D.B. Roye, and R.D. Roer, R.M., Dillaman, J.S., Dickson, aers .O,Glmn .H,Fril .P n caaa J.C. McNamara, and R.P. Furriel, M.H., Goldman, R.O., Faleiros, rga,P. C. Croghan, J., Kahiapo, S., Hau, Jr, T. Y., Iwai, M.N., Yamamoto, A.D., Russ, J.D., Craft, re,G. Fryer, vn,D . iraii .M n he K.P. Choe, and P. M. Piermarini, D.H., Evans, ort .L n og D. Wong, and C.L. Couret, rie .A,Okn .adMNmr,J.C. McNamara, and H. Onken, C.A., Freire, RESEARCH ARTICLE oead .E n izarl,A.T. Fitzjarrell, and D.E. Copeland, opr,P,Wno,S,Pqex . ils .adGfie,G. Goffinet, and R. Gilles, A., Pequeux, S., Wanson, P., Compere, on J.W. Born, öthr . ibr,D n ekr W. Becker, and D. Siebers, K., Böttcher, rc,R . ors .E,Zean .A n e,M.T. Lee, and D.A. Ziemann, J.E., Norris, R.E., Brock, R.E. Brock, ilc,U,Camnir . rust . hrate-ars .adAgr K. Anger, and M. Charmantier-Daures, E., and Grousset, G., M. Charmantier, U., Charmantier-Daures, Cieluch, F., Buchholz, F., Aujoulat, K., Anger, U., Cieluch, D.H. Evans, and P. Piermarini, K., Hyndman, R., Rose, J., Havird, K.P., Choe, aeo,J.N. Cameron, utn S.A. Bustin, hn,K-.adLn H.-C. Lin, and K.-F. Chung, he .P,Ewrs . orsnSelr .I,To,T n lion,J.B. Claiborne, and T. Toop, A.I., Morrison-Shetlar, S., Edwards, K.P., Choe, D. Towle, and M. Charmantier-Daures, G., K. Charmantier, Anger, and M. Charmantier-Daures, L., Gimenez, G., Charmantier, C.V. Batterton, and J.N. Cameron, Ecophysiology ofafreshwatershrimp, physiology ofthebranchiae. proliferation ofspecialisedcells. granulatus The morphometricchangesinthegills oftheestuarinecrab R of Dominica. Hall Inc. Society ofAmericaandUniversityKansas Press. Differential adjustmentingillNa of nitrogenouswaste. dominant siteofgasexchange,osmoregulation,acid-baseregulation,andexcretion Procambarus clarkii characterization ofiontransportingandrespiratoryfilamentsinthegills of Baetica. (Crustacea, Decapoda)inthreeTunisian reservoirs:preliminaryresults. 53, 675-689. etal. Y. Fujita, (Crustacean: Decapoda:Atyidae)intheHawaiianarchipelago. C., Muir, islands: thephylogeographyandevolutionof S., Ziegler-Chong, C.T., Chong, (Decapoda, Atyidae). 151A ion transportincrustaceangillsandexcretoryorgans. Zellforsch. Mikrosk.Anat. blue crab( 3905. Bull. Mar. Sci. relation totheexternalsalinity. Ultrastructural changesinthegillepitheliumofgreencrab ). branchial epitheliainthedevelopingbrownshrimp, Macrobrachium amazonicum expression duringosmoregulatoryacclimationinthecinnamonshrimp pacifica 235-244. 261. tissues ofosmoregulatingshorecrabs, water qualityinanchialinepondsoftheKona,Hawai’i,coast. (2005). Osmoregulation,immunolocalizationofNa green crab G. Charmantie, 48-57. Exp. Biol. distribution, localization,andpotentialfunctioningillsduringsalinityacclimation. (2006). COX2inaeuryhalineteleost, atlantic hagfish( J. Comp.Physiol.B blue crab,Callinectessapidus:water, electrolyte,acid–baseandammoniaexcretion. water. (RT-PCR): trendsandproblems. osmoregulatory organsof (1999). ImmunolocalizationofNa (ed. D.H.Evans),pp.165-230.BocaRaton,FL:CRCPress. regulation inaquaticarthropods.In Ser. grapsid crabChasmagnathusgranulata Ontogeny ofosmoregulation,physiologicalplasticityandlarvalexportstrategyinthe , Arthropoda 4 229, 185-194. , 272-304. J. Comp.Physiol.B (1977). Studiesonthefunctionalmorphologyandecologyofatyidprawns 15, 175-183. (Atyidae) and Physiol. Biochem.Zool. 209 Carcinus maenas (1987). StatusoftheanchialinepondsystemKona-HawaiiCoast. (2002). QuantificationofmRNA usingreal-timereversetranscriptionPCR Callinectes sapidus (1968). Osmoregulatorycapacitiesoftwocarideanshrimps. (1972). under hyper-andhyporegulationconditions arenotcausedby Philos. Trans. R.Soc.B (1958). Themechanismofosmoticregulationin , 1696-1708. 41, 633-634. (1978). NaClbalanceinbluecrabs,Callinectessapidus,fresh (1969). Decapoda.In Myxine glutinosa , Vol. 2(ed.R.C.Moore),pp.399-533.Lawrence,KS:Geological (2004). Ontogenyofosmoregulatorystructuresandfunctionsinthe . Biol.Bull. Oceanography: A View oftheEarth 123, 143-148. Crustaceana Physiol. Rev. Palaemon macrodactylus 92, 1-22. (1978). Larvaldevelopmentof 123, 127-135. (2006). OsmoregulationandNa,K-ATPase expressionin Scylla paramamosain J. Exp.Biol. (Crustacea, Decapoda). (Decapoda, Palaemonidae). Tissue Cell 180 + J. Anat. Rathbun) withnotesonmodifiedmitochondria. /K J. Mol.Endocrinol. ) gill.Comp.Biochem.Physiol. 78, 1017-1025. , 154-166. + + 34, 301-309. Osmotic andIonicRegulation:CellsAnimals 85, 97-177. /K( (1978). Antennalglandfunctioninthefreshwater - andV-ATPase activitiesandtransporter mRNA (1968). Thesaltabsorbingcellsinthegillsof 277 Treatise onInvertebratePalaeontology + )-ATPase inmitochondrion-richcellsofthe 197 Carcinus maenas 35, 234-242. , 57-129. Fundulus heteroclitus (Crustacea, Decapoda). 21, 299-318. (1990). Carbonicanhydraseinbranchial , 239-246. Atyaephyra desmarestii (2008). A structure-functionanalysis of (2005). Themultifunctionalfishgill: Halocaridina rubra . + (Palaemonidae). Comp. Biochem.Physiol. /K 29, 23-39. + Crangon crangon J. Exp.Biol. -ATPase, andultrastructureof . NewJersey, NJ:Prentice- Halocaridina rubra (2009). Osmoticandionic Comp. Biochem.Physiol. . (1987). Characteristicsof J. Exp.Biol. J. Exp.Zool. (1991). Distributionand Pac. Sci. Artemia salina (2008). Islandsunder : primarysequence, Carcinus maenas 124A Limnol. Oceanogr. 207 Mar. Ecol.Prog. Chasmagnathus Biol. Bull. Holthuis, 1963 , 161-168. , 325-336. 41, 200-208. (Millet, 1831) (Decapoda, 213 255 Syncaris Holthuis (L.): the , 3894- (2004). (2000). (2010). (1989). (2002). – , 251- 144A Zool. 134 Part Z. in J. , , aaudr,N,Twe .W,Wiruh .adSaig-iro,C. Spanings-Pierrot, and D. Weihrauch, D. W., Towle, N., Jayasundara, am,D n rhe,F. Bréhier, and D. Jaume, er,R .adCmrn J.N. Cameron, and R.P. Henry, eje,A,Vrot . yesen . i . og,S n lk G. Flik, and S. Bonga, J., Li, J., Eygensteyn, P., Verbost, A., Heijden, wi T. Y. Iwai, aid .C,Hny .P n isn A.E. Wilson, and R.P. Henry, J.C., Havird, ars .R n als,D. Bayliss, and R. Harris, lfe T. M. Iliffe, S.H. Hurlbert, er,R . uu . ne,H n ehac,D. Weihrauch, and H. Onken, C., Lucu, R.P., Henry, D. W. Towle, and D. Weihrauch, S., Gehnrich, R.P., Henry, ilte . amc,L,Lwnti,M n er,R.P. Henry, and M. Lowenstein, L., Cammack, N., Jillette, er,R .adWts S.A. Watts, and R.P. Henry, er,R .adCmrn J.N. Cameron, and R.P. Henry, R.P. Henry, R.P. Henry, er,R.P. Henry, ut,W. Hunte, W. F. Humphreys, un,D .T,Wn,T,Bye,M n hog N.T. Phuong, and M. Bayley, T., Wang, D.T. T., Huong, C.W. Holliday, otus L.B. Holthuis, L.B. Holthuis, R.D. Roer, and D.B. Roye, C.W., Holliday, er,R . arls .E,MCry .M n ol,D.W. Towle, and M. McCarty, E.E., Garrelts, R.P., Henry, ut,W. Hunte, se,P-. clnok .B n okn,T. S. Hopkins, and J.B. McClintock, P.-W., Hsueh, Gill-specific transcriptionalregulation of Na Soc. Atyidae) fromanchialinecavesonthe French Mediterraneancoast. Halocaridina Rubra Resources. Honolulu, HI:DivisionofAquaticResources,HawaiiDepartmentLandandNatural 90A distribution andcharacteristicsinrelationtoNa Subterranean Ecosystems Zool. euryhaline crab, structure. J.Exp.Biol. euryhaline shorecrab Comp. Biochem.Physiol. euryhaline greencrab, of activityandexpressionthreetransport-related proteinsinthegillsof 101 crab, Exp. Zool. of carbonicanhydraseinselectedaquaticandterrestrialdecapodcrustaceans. Exp. Biol. fresh waterorseawater:quantificationbyconfocallaserscanningmicroscopy. crustaceans. carbonic anhydraseinductioninthegillsoftwoeuryhalinespeciesdecapod gills ofeuryhalinecrustaceans. Mitochondria-rich cellsingillsoftilapia( years. Comp.Biochem.Physiol. response tosalinitytransfer:ameta-analysisof59quantitativePCRstudiesover10 ATPase andotherosmoregulatorygenesinthegillsofeuryhalineanimals 59-76. Amsterdam:ElsevierScience. experiments. rosenbergii growth andmoultingcyclesofthegiantfreshwaterprawn( 5 environments oftheNorthernGulfMexico. bioaccumulation oftoxicmetals. crustacean gill:osmotic/ionicregulation,acid-basebalance,ammoniaexcretion,and maenas carbonic anhydraseinductioninthegillsofeuryhalinegreencrab, land-locked saltwaterpools. induction ofbranchialcarbonicanhydraseandNa ornithine decarboxylaseactivity. the bluecrab, regulation. innocous Crustaceana Micratya poeyi Aust. Sinkhole, ananchialineremipedhabitatinnorth-westernAustralia. life historycharacteristicsofthebluecrabs mud fiddlercrab, 128 islands), withthedescriptionofonenewgenusandfourspecies. Indo-West Pacificlocalities (SiniaPeninsula,FunafutiAtoll,MauiandHawai‘i shrimp branchial Na,K-ATPase activityandtransepithelialpotentialdifference inthebrine , 231-242. , 255-264. , 1-48. , 303-308. 144 292 82, 89-98. Callinectes spidus Artemia salina . Comp.Biochem.Physiol. , 387-414. (1979b). Thecompletelarvaldevelopmentofthefreshwatershrimp , 595-603. (Herbst) rearedinthelaboratory(Decapoda,Atyidae). 200 (1979a). Thecompletelarvaldevelopmentofthefreshwatershrimp 221 Am. Zool. ) atdifferent salinities. (2005). Criticalsalinity, sensitivity, andcommitmentofsalinity-mediated (2005). (2000). Anchialinecaveecology. In (2001). Environmentallymediatedcarbonicanhydraseinductioninthe (1984). Theroleofcarbonicanhydraseinbloodionandacid-base The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 J. Exp.Zool. Suppl. 5 Ecol. Monogr. , 55-64. (1973). Carideanshrimpsfoundinland-lockedsaltwaterpoolsatfour (1963). Onredcolouredshrimps(Decapoda,Caridea)fromtropical (1985). SalinityinducedchangesingillNa,K-ATPase activityinthe , 309-321. Callinectes sapidus (Gufrin-Meneville) rearedinthelaboratory(Decapoda,Atyidae). (1984). Pseudoreplicationandthedesignofecologicalfield Uca pugnax Carcinus maenas (1999). Physico-chemicalprofileandenergyfixationinBundera , Captive BreedingoftheEndemicHawaiianRedShrimp, 24, 241-251. Part I:Reproduction,LarvalDevelopmentandFirstFeeding . J.Exp.Biol. , 153-166. 210 Pachygrapsus marmoratus Carcinus maenas , duringacclimationfromhightolowsalinity. (2005). A newspeciesofTyphlatya (Crustacea:Decapoda: 158A , 2070-2081. 303A (ed. H.Wilkens,D.C.CulverandW 54, 187-211. (2001). Earlycarbonicanhydraseinductioninthegillsof Zool. Meded. (1988). Gill(Na . J.Exp.Zool. , 189-193. J. Exp.Biol. , 45-56. (1982a). Thedistributionandpartialcharacterization J. Exp.Zool. Front. Physiol. 8D, 131-140. (1982b). Acid–basebalanceintheeuryhalineblue Aquacult. Res. , duringlowsalinityacclimationisindependentof 136A 151 , inresponsetolowsalinityexposure. , 279-296. , 243-258. , inresponsetohigh salinityacclimation. 16, 261-279. rohoi mossambicus Oreochromis 233 alnce similis Callinectes 204 + Mar. Ecol. 289 +K (2013). AlteredexpressionofNa + 3 , 199-208. , 991-1002. + (1990). Salinity-inducedchangesin regulation. + /K , 431. 41, e135-e143. , 350-358. )-ATPase indecapodcrustaceans: : sequencevariantsandpromoter + (1993). Populationdynamicsand Ecosystems oftheWorld.30. -ATPase alpha-subunitinthe (2012). Multiplefunctionsofthe + /K 14, 239-257. + Comp. Biochem.Physiol. (2003). Salinity-mediated (2010). Osmoregulation, ATPase activityinthe (2011). Down-regulation and Crustaceana . F. Humphreys). pp (2002). Differential C. sapidus J. R.Soc.West. Macrobrachium Zool. J.Linn. ) adaptedto J. Exp.Biol. Zool. Verh. Carcinus J. Exp. (2007). (1997). Suppl. 2319 in bay + Atya /K J. J. + - .

The Journal of Experimental Biology uut .M,Wiruh . ee,M n ol,D.W. Towle, and M. Senek, D., Weihrauch, C.M., Luquet, 2320 RESEARCH ARTICLE hdbne,S,Kti,M,Ajua,F,Camnir .adCharmantier- and G. Charmantier, F., Aujoulat, M., Kutnik, S., J.A. Khodabandeh, Zadunaisky, and L.T. Garretson, K.J., Degnan, Jr, K.J., Karnaky, uu .adFi,G. Flik, and C. Lucu, oet .L,Clla . ann .C,Le .H,Eaglso . ulr E.M. Muller, A., Evangelisto, D.H., Lee, A.C., Cannon, T., Colella, D.L., Lovett, K. Shiraishi, and S. Kikuchi, ailk .A n rc,R.E. Brock, and J.A. Maciolek, J.A. Maciolek, ne,H. Onken, J.B. Pritchard, and C.W. Holliday, G.J., Neufeld, A.G. Lima, and J.C. McNamara, S.C. Faria, and J.C. McNamara, J.A. Maciolek, rin,A,Avrz .adAcrz G. Alcaraz, and F. Alvarez, A., Ordiano, uu . Pavicić C., Lucu, in,O. Kinne, atl .H n amr L. Farmer, and L.H. Mantel, éuu,A. Péqueux, ilr . er,R,Delr .adKas D. Kraus, and J. Doeller, R., Henry, S., Piller, ola,J.W. Pohlman, Am. J.Physiol. Identification andquantificationofmitochondria-richcellsintransportingepithelia. Tissue Res. Kinau, Maui,Hawai‘i. the euryhalinecrab branchial iontransportermRNA expressionduringacclimationtosalinitychangein Biochem. Physiol. gills oftheshorecrab Na,K-ATPase inthebluecrab acclimation tosalinemedia. (Decapoda, Palaemonidae):evidencefromultrastructuralchangesinducedby across thegillepitheliumoffreshwatershrimp B gill iontransportmechanismsinthedecapodCrustacea:areview. of hyperregulating Changes inNa ars M. Daures, of thebluecrab D.W. Towle, and Part 2(ed.O.Kinne),pp.821-996.NewYork, NY: JohnWiley&Sons. Caenestheriella gifuensis types ofepithelialcellarrangedalternatelyinthegillfreshwaterbranchiopod Mantel), pp.53-161.NewYork, NY: AcademicPress. the Crustacea,vil.V, InternalAnatomyandPhysiologicalRegulation Hawaii Island. shrimps. Bull.Mar. Sci. Physiol. Chinese crabs( consumption ofthehololimneticprawn, leptodactylus salinities (Decapoda,Palaemonidae). physiology oftwoeuryhalinecrabspecies, function ofacclimationsalinity. similis possibilities. 182 , 997-1014. : energyproduction,transport-relatedenzymesandosmoregulationasa (1971). Salinity:animals.In 123A (1999). ActiveNaClabsorptionacrosssplitlamellaeofposteriorgills (1995). Osmoticregulationincrustaceans. 319 Hydrobiologia (2005). Ontogenyoftheantennalglandsincrayfish , 377-384. (1986). EnvironmentalfeaturesandbiotaofanchialinepoolsonCape (Crustacea, Decapoda):immunolocalizationofNa University ofHawaiiSeaGrantAdvisoryReportAR-74-04. + 246 (1983). DistributionandbiologyofIndo-Pacificinsularhypogeal Callinectes sapidus , 167-174. /K (2011). Thebiogeochemistryofanchialinecaves:progressand Eriocheir sinensis . Ivanković J., , + 149A Carcinus maenas (2006). Effect ofsalinityonosmoregulatorypatchepitheliaingills , R770-R775. -ATPase activity, unsaturatedfattyacidsandmetallothioneinsin Chasmagnathus granulatus (1999). Na Stygologia 33, 606-618. Carcinus aestuarii , 362-372. (Crustacea). 677 Biol. Bull. (1997). Ultrastructureandionpermeabilityofthetwo (1983). Osmoticandionicregulation.In Callinectes sapidus J. Exp.Biol. , 33-51. + -K . Pavicić D., , 2 (1974). , 119-129. + (2012). Evolutionofosmoregulatorypatternsand ) adaptedtodifferent salinities. -ATPase andNa (1997). Therouteofionandwatermovements . Biol.Bull. . Am.J.Physiol. Marine Ecology:EnvironmentalFactors Crustaceana 192 Zoomorphology Aquatic SurveyfortheKonaCoastPonds, , 321-331. after diluteseawateracclimation. 198 Macrobrachium tuxtlaense (2005). Osmoregulationandoxygen Hmr .adNje,M. Najdek, and D. -Hamer, 210 Callinectes sapidus , 349-358. . J.Exp.Biol. . J.Exp.Zool. (1995). A comparisonofthegill , 132-139. 78, 1013-1022. + (1980). Salinityadaptationofgill /Ca 276 J. Crustac.Biol. 117 2+ , R490-R499. exchange activitiesingills , 53-62. Macrobrachium olfersii 208 211, 215-224. (2005). Inductionof , 3627-3636. + J. Comp.Physiol. ,K Comp. Biochem. and + The Biologyof -ATPase. 15, 1-60. Callinectes at varying (ed. L.H. Astacus , Vol. 1, (1984). (2008). Comp. Cell ol,D .adKy,W. T. Kays, and D.W. Towle, ors . hrate-ars . hflt .adAgr K. Anger, and S. Chifflet, M., Charmantier-Daures, G., Torres, E.W. Taylor, and H. Taylor, o,L . ai,D . au,I .adHny R.P. Henry, and I.P. Saoud, D.A., Davis, L.A., Roy, adr,I.P. Zanders, kt B. Sket, R.P. Henry, and K.M. Halanych, L., Serrano, oes .D,Yug .P,Goel . rwy .L n oas,J.R. Tomasso, and C.L. Browdy, M., Grosell, S.P., Young, A.D., Sowers, W. D. Williams, and M.J. Smith, K.W. Eliceiri, and W. S. Rasband, C.A., Schneider, aa,K,Tra,M n ag S.L. Yang, and M. Türkay, K., Sakai, J.A. Riegel, R CoreTeam ol,D . amr .adHri,J. Harris, and G. Palmer, D.W., Towle, ern,L n er,R.P. Henry, and L. Serrano, hal,M .adHny R.P. Henry, and M.G. Wheatly, A.-L. Wang, and W. J.-Z. W. Warner, Li, D.-X., Cai, Y., Liu, W.-N., Wang, L., Wang, T. Iliffe, B.R., Kuhajda, B., Stelbrink, K., Roe, Y., Cai, T. J., Page, K., von Rintelen, long-term exposuretodifferent salinitiesonthelocationandactivityofNa Physiol. Harrisson andA.G.Humes),pp.203-293.NewYork, NY: JohnWiley&Sons. ions. InMicroscopicAnatomyofInvertebrates,Decapoda,Crustacea, Comp. Biochem.Physiol. exposure toartificialseasaltoramixed-ionsolution:relationshippotassiumflux. Hemolymph osmolalityandcationconcentrationsin 565 complex (Crustacea:Decapoda:Brachyura). Litopenaeus vannamei anhydrase activityandninhydrinpositivesubstancesinthePacificwhiteshrimp, pressure intheurineofcrayfishes. ATPase inacclimationofbluecrabs( Biochem. Physiol. euryhaline crayfish dependent ATPase andcarbonicanhydraseactivityduringsalinityacclimationofthe ATPase inthegillsofjuvenilemittencrab, J. Exp.Zool. epithelium oftwoosmoregulatingcrabs, Callinectes sapidus expression andactivityoftwocarbonicanhydraseisoformsinthebluecrab biogeographical problem. (Edmondson) (Crustacea,Decapoda,Atyidae)intheSolomonIslands:anintriguing 25 yearsofimageanalysis. Physiol. Foundation forStatisticalComputing.Available at:http://www.R-project.org Zool. maenas carbonic anhydraseisoformsinthegillsofeuryhalinegreencrab, Bay. Boston,MA:Little,BrownandCo. to environmentalstress. types ofATPases fromthePacificwhiteshrimp, Phylogenet. Evol. frequent caveinvasionsandchallengescurrenttaxonomichypotheses. T. vonRintelen, and phylogeny offreshwatershrimps(Crustacea:Decapoda:Caridea:Atyidae)reveals J. Hughes, M., , 1-76. 196 (1996). Theecologryofanchihalinecaves. , inresponsetolowsalinity. 147 147A , 315-321. (1968). Analysisofthedistributionsodium,potassiumandosmotic , 460-465. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103051 (2013). 239 , 404-411. (1980). Regulationofbloodionsin (1976). , 311-318. 63, 82-96. 65A Pacifastacus leniusculus . J.Exp.Biol. R: A LanguageandEnvironmentforStatisticalComputing Beautiful Swimmers:Watermen, CrabsandtheChesapeake , 97-108. , acclimatedtolowandhighsalinities. Mol. Biol.Rep. 145A Hydrobiologia Nat. Methods (1986). BasolaterallocalizationofNa (2008). Differential expressionandinductionoftwo , 176-180. (1992). Gillsandlungs:theexchangeofgases 210 (1981). Theoccurrenceof J. Exp.Biol. (1987). BranchialandantennalglandNa Comp. Biochem.Physiol. , 2320-2332. (2006). Revisionofthe (2012). Drawntothedarkside:amolecular Callinectes sapidus 39, 6427-6438. 85, 49-58. alnce sapidus Callinectes 9 , 671-675. . J.Exp.Biol. (1976). RoleofgillNa Abh. Senckenberg.Naturforsch.Ges. (2007). Salinity-stimulatedchangesin Eriocheir sinensis 48, 587-596. ioeau vannamei Litopenaeus Trends Ecol.Evol. Carcinus maenas (2012). NIHImagetoImageJ: Litopenaeus vannamei (2007). Branchialcarbonic 133 ) tolowsalinity. Helice , 73-86. and 3D, 186-193. Antecaridina lauensis . + Carcinus maenas /K (2007). Effects of Comp. Biochem. Comp. Biochem. / Chasmagnathus 11 + + - ATPase ingill +K o.1 (ed.F. Vol. 10 , 221-225. + (2012). Two (L.). in response -dependent Carcinus J. Exp. (2006). Comp. during + + Mol. -K /K . R + + - - .

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