Application of Physiological Tests to Determine Specific

Application of Physiological Tests to Determine Specific Monovalent and Divalent Ion Supplementation for Culture of Marine Species

CalvinFISHER＊1,CharlotteBODINIER＊2,AdamKUHL＊3,andChristopherC.GREEN＊1

Abstract:Thecultureofmarineandeuryhalinefishesinlowsalinityoriondeficientwatershasbeenanareaof interest formanyaquaculturistsdueto theexpenseofmarinesaltmixes.TheGulfkillifish(Fundulus grandis)isaeuryhalineteleostfoundabundantlyincoastalmarshesalongtheGulfofMexico.Theabilitytoinvestigatethemolecularunderpinningsofspecificionsinthismodelspeciescouldhaveanumberofimplicationsforothercommerciallyimportantmarinefinfishspecies.Althoughstudieshaveexaminedtheinfluenceofsalinityonadultsandjuveniles,fewhaveinvestigatedtheroleofsalinityorspecificionconcentrationsinlarvae.Theseinvestigationsutilizeamodelteleosttodeterminetheroleofspecificmonovalentanddivalentionsatbiochemicalandmolecularlevels.　SeparatefourweektrialswereconductedexposingnewlyhatchedGulfkillifishtoconcentrationgradients ofpotassium (K+), calcium (Ca2+), andmagnesium (Mg2+).TheK+ supplementationconsistedof0.3, 1.3,and2.9mM.TreatmentgroupsforCa2+consistedof0.2, 1.1, 1.5,and2.1mMCa2+,while trialsusingMg2+consistedof0.1, 2.7, 5.1,and10.4mMMg2+.All treatmentsweremaintainedatasalinityof9.5-10‰usingcrystalsalt (99.6%NaCl).Each investigationconsistedof four50-Laquariumsstockedat7 larvaeper liter foreachconcentration.Fishweresampledat0, 1, 3, 7, 10, 14,and28dposthatch(DPH).Uponeachsamplingthestandpipewasadjustedtomaintainaconstantdensity foreach treatment.Collectedsampleswereanalyzed forwholebody ionconcentrations (K+,Na+,Mg2+,Cl－),Na+/K+-ATPase (NKA)activity,dryweight, andexpression/localizationof ion transportproteins (NKA,Na+/K+/2Cl－cotransporter (NKCC)andcysticfibrosistransmembraneconductanceregulator(CFTR)).　MortalityandgrowthwassignificantlyinfluencedbyK+concentration(P< 0.05).NodifferenceswereobservedamongtreatmentgroupsforNKAenzymeactivity,howeverat28-dposthatch(dph)thereweresignificantdifferencesindryweightamongK+treatment.AtsevendphdifferencesinintestinalNKAandCFTRstainingwereobserved,andNKAmRNAexpressionwasalso foundtobehigherinthe0.3mM[K+]groupthaninothertreatmentgroups.SurvivalwassignificantlyinfluencedbybothMg2+andCa2+concentration (P≤ 0.05).Highestsurvival (71.1%) intheCa2+trialwasnoted inthe0.2mM[Ca2+] treatment. IntheMg2+trial,highestsurvivalwasnoted inthe2.7mM[Mg2+]treatment(82.9%).NKAenzymeactivitywasreducedanddelayedpeakswereobserved inwholebody ioncomposition in theMg2+ treatments. Inaddition,decreasedCFTRintensitywasobservedatthegillandintestineepitheliumforthe0.05mM[Mg2+]treatmentat1dph,indicatingthatMg2+deficiencyhasapossibleeffectonlarvalosmoregulatorycapability.Theroleofintestinalepitheliuminionuptakenotonlyallowsthepotentialforuptakeandmaintenanceofionbalancefromdietarysources,butalsodemonstratesthepotentialtomodulateconcentrationsofspecificionsinpreparedwatersforeuryhalineandmarineteleosts.

Key words:Gulfkillifish,osmoregulation,potassium,magnesium,euryhaline2015年 1 月30日受理（ReceivedonJanuary30, 2015）＊1 AquacultureResearchStation,LouisianaStateUniversityAgriculturalCenter,BatonRouge,LA,USA＊2 RosenstielSchoolofMarine&AtmosphericScience,Miami,FL,USA＊3 HuntsmanCorporation,Houston,TX,USA Correspondingauthor:ChristopherC.GREENE-mail:[email protected]

水研センター研報，第40号，97－107，平成27年Bull.Fish.Res.Agen.No. 40，97－107，2015

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Calvin FISHER, Charlotte BODINIER, Adam KUHL, and Christopher C. GREEN

　Themajor ions in seawater are; chloride (Cl－),sodium (Na+),magnesium (Mg2+), Calcium (Ca2+),sulfate (SO4－), andpotassium (K+),withavarietyof other ionspresent in lowconcentrations.Thethree ionsCa2+,Mg2+, andK+ are necessary formaintainingelectrolyteandacid-basehomeostasis,and the regulationof osmolality and intracellularfluids(Fielderet al., 2001).Thehighcostofsyntheticmarine salt mixes has facil itated interest inalternative low-costsaltsourcesandsupplementingphysiologicallyimportantions.Potassiumisinvolvedin ionregulationof intracellular fluidsandcanbeaddeddirectly to thewater, usually in the formofKCl, or supplemented in thediet (Wilson andElNaggar, 1992).Calcium andmagnesium bothinfluence the permeability of osmoregulatorymembranesmaking them critical ions in teleostionic regulation (Silva et al., 2005).Magnesium isresponsiblefortheactivationofnumerousenzymesmaking it a necessary cofactor to the transferof phosphategroups and the activation ofATP-dependent ion pumps such asNKA (Bijvelds et al., 1998). In freshwater fish,Mg2+ deficiency isassociatedwithincreasedNa+andCa2+levelsaswellaslowK+levels(Bijveldset al., 1997). 　Ionregulationiscontrolledbyionocytesinwhichthere are several proteins involved in ion andwaterexchange (Evanset al., 2005;Lorin-Nebel et al., 2006).Previous investigationshave focusedonNa+/K+-ATPase (NKA),Na+/K+/2Cl－cotransporter(NKCC), and the cystic fibrosis transmembraneconductanceregulator(CFTR)asthethreeprimaryproteins involved in ionoregulatory processes(Hirose et al., 2003).The ionoregulatory processis initiatedbyabasolaterally locatedNKAwhichcouplestwoextracellularK+withthreeintracellularNa+generating an electrochemical gradient thatdrives the ions according to expression, location,andabundanceofNKCCandCFTR(Bodinieret al., 2009;Kanget al., 2008).NKAisresponsible fortheloweringofintracellularNa+concentrationsallowingthebasolateralNKCCto importNa+,K+, andCl－,theexcessCl－isthensecretedthroughthechloridechannelCFTR.AdditionalwaterexchangeandCl－

ionregulationoccurs intestinally (Christensenet al., 2012)andithasalsobeensuggestedthatsubstantialMg2+ uptake occurs intestinally (Marshall and

Grosell, 2005).The apically locatedNKCC2mostlikelyplays an active role in the reabsorption ofions(Lorin-Nebelet al., 2006).Theexactmechanismby which Mg2+ uptake occurs has yet to bedescribed indetail. It is suggested thatsignificantdifferences inMg2+regulationexistamongspecies,knowledgeofMg2+ transportacrossepitheliaandcellmembranesisstill limited(Bijveldset al., 1998).Byexposingeuryhalineteleoststovaryingsalinitiesor individual ionconcentrations, itmaybepossibleto immunolocalizespecificosmoregulatoryproteinsin thegill filaments and intestinal epithelium tobetter understand the physiological adaptationsthesespeciesundergo tocopewithenvironmentalchallenges.　TheGulfkillifish,F. grandis,inhabitstheestuarinewatersof theGulfofMexicoandAtlanticcoastofFloridawhere it iscommonlyusedasabaitfishbyanglers for speckled trout,Cynoscion nebulosus,flounder,Paralichthys lethostigma, and reddrum,Sciaenops ocellatus. FromTexas toFlorida it isreferred to by many regional names includingbutnot limited to:mudminnow,mudfish, cocahoeminnow, andbullminnow.F. grandis are closelyrelated to themummichog,F. heteroclitus,whichinhabitstheAtlanticcoastandissimilarlyutilizedbyanglersasalivebait.Bothspeciesarecharacterizedasa“hardy”baittolerantofwideswingsinsalinity,temperature,andotherconditionsencounteredbylivemarinebait.F. grandisoccupycoastalmarsheswheresalinitycanchangedramaticallyoverashorttimeperiod.These fishhave theability to live insalinitiesthatrangefromfreshwater(0 ‰ )toneardouble the concentration of seawater (up to 70ppt) for severaldays.Exploitation of this salinitytolerance in F. grandis allows investigators theabilitytoillicitphysiologicalresponsesacrossawiderangeofionconcentrations.　Severalstudieshave investigatedtheuseof lowsalinity inland seawater orwater frombrackishaquifersasasource formarineaquaculture.Muchoftheseinlandsalinewaterswerefoundtohaveiondeficiencies,wherespecificmanipulationofthemajorionsinsaltwaterhavesignificantlyincreasedgrowthandsurvival(Doroudiet al., 2006;Fielderet al., 2001;Fotedaret al., 2008).Manystudieshaveinvestigatedtheeffectsof lowsalinitywaterson juvenileand

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Ion Supplementation for Culture of Marine Species

adulteuryhalinespecies(Coulonet al., 2012;Doroudiet al., 2006;Fielderet al., 2001;Fotedaret al., 2008;Pattersonet al., 2012;Royet al., 2007).Fewstudieshave attempted to explore the importance ofspecific ion supplementation ineuryhaline teleostlarviculture.Theobjectives of thisworkwere todemonstrate theuseofphysiologicalexaminationsatbiochemical andmolecular levels todeterminespecific supplementation ofmonovalent (K+) anddivalent ions (Ca2+ andMg2+) as replacements forsaltwaterinthecultureofF. grandislarvae.

Materials and Methods

　Three separate four-week experiments wereconductedto investigatethe impactofexternalK+,Ca2+andMg2+concentrationson larvalF. grandis.

Newlyhatched (< 4hold)F. grandis larvaewerestocked, in triplicate, at adensityof 7 larvaeperL into 50-L aquaria maintained on a commonrecirculating systemutilizingbiological andultravioletfilters.Larvaewere fedadietofArtemia sp.nauplii (30Artemiafish－1d－1) forthefirst7datarateof threetimesperday followedbyOtohimeTMB1 (ReedMaricultureInc.,Campbell,CA,USA)dietfed toapparent satiation4 timesperday for theremainderofthestudy.After4weeks,survivalwascalculatedbydrainingallaquariaandcountingeachindividual.　Treatmentswereestablishedbyaddingcrystalsalt(DiamondCrystalSolarSalt, 99.6%NaCl)to10‰withionsupplementation.Potassiumsupplementationconsistedof0.3, 1.3, 2.0and2.9mM(Table1).Thisinvestigationrepresents [K+] fromvaluessimilar to

Table 1.Meansalinityandionconcentrations(±SEM)givenforeachK+ ,Ca2+,andMg2+Concentrationtreatmentgroup. Superscriptlettersdenotestatisticallysignificantdifferences inspecific ionconcentrationsamongtreatmentgroups(P< 0.05)

Table 1. Mean salinity and ion concentrations (± SEM) given for each K+ ,Ca2+, and Mg2+ Concentration treatment group. Superscript letters denote statistically significant differences in specific ion concentrations among treatment groups (P < 0.05)

K+ Treatment group Parameter 1 2 3 4** K+ (mM) 0.33 ± 0.05A 1.31 ± 0.04B 2.06 ± 0.04C 2.96 ± 0.04D

Na+ (mM) 170.19 ± 3.40A 157.52 ± 6.08AB 151.56 ± 7.03AB 138.01 ± 0.83B

Mg2+ (mM) 0.04 ± 0.00A 0.12 ± .01A 0.11 ± 0.01A 11.55 ± 0.10B

Ca2+ (mM) 0.31 ±0.00A 0.43 ± 0.05A 0.30 ± 0.03A 2.25 ± 0.02B

Salinity(‰) 9.8 ± 0.28 9.4 ± 0.4 9.4 ± 0.4 9.6 ± 0.1 pH 8.56 ± 0.05 8.27 ± 0.10 8.24 ± 0.09 8.07 ± 0.11 TAN (mg/L) 0.03 ± 0.01 0.02 ± 0.00 0.01 ± 0.00 0.04 ± 0.03 NO2 (mg/L) 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 Hardness (mg/L)* 37.25 ± 1.55 39.17 ± 2.06 33.83 ± 2.20 1442.67 ± 11.04Alkalinity (mg/L)* 184.00 ± 25.16 186.00 ± 6.55 182.50 ± 6.98 209.83 ± 3.06*Reported as CaCO3 ** Represents reference salt group

Ca2 Treatment groupsParameter 1 2 3 4 K+ (mM) 2.37 ± 0.03 2.32 ± 0.02 2.33 ± 0.02 2.31 ± 0.05 Na+ (mM) 165.31 ± 3.07 166.72 ± 4.83 166.72 ± 4.83 165.84 ± 2.97 Mg2+ (mM) 0.11 ± 0.01 0.16 ± 0.02 0.15 ± 0.01 0.12 ± 0.01 Ca2+ (mM) 0.20 ± 0.02A 1.08 ± 0.03B 1.50 ± 0.04C 2.07 ± 0.05D

Salinity(‰) 10.3 ± 0.03 10.1 ± 0.07 10.1 ± 0.06 10.0 ± 0.09 TAN (mg/L) 0.03 ± 0.02 0.03 ± 0.02 0.01 ± 0.01 0.02 ± 0.02 NO2 (mg/L) 0.05 ± 0.02 0.03 ± 0.02 0.02 ± 0.01 0.01 ± 0.00 Hardness (mg/L)* 193 ± 32.76A 1090 ± 15.81B 1433 ± 32.24C 1740 ± 31.52D

Alkalinity (mg/L)* 178 ± 17.97 263 ± 57.64 140 ± 4.08 170 ± 7.07 *Reported as CaCO3

Mg2+ Treatment groupsParameter 1 2 3 4 K+ (mM) 2.37 ± 0.03 2.40 ± 0.04 2.29 ± 0.05 2.41 ± 0.02 Na+ (mM) 168.48 ± 3.64 169.96 ± 4.11 162. 64 ± 4.83 170.55 ± 4.97 Mg2+ (mM) 0.05 ± 0.01A 2.88 ± 0.08B 5.76 ± 0.16C 11.52 ± 0.19D

Ca2+ (mM) 0.19 ± 0.02 0.24 ± 0.02 0.15 ± 0.01 0.26 ± 0.02 Salinity(‰) 9.9 ± 0.04 10.2 ± 0.03 10.1 ± 0.03 10.0 ± 0.05 TAN (mg/L) 0.02 ± 0.01 0.01 ± 0.02 0.02 ± 0.02 0.03 ± 0.02 NO2 (mg/L) 0.01 ± 0.03 0.03 ± 0.02 0.00 ± 0.00 0.02 ± 0.01 Hardness (mg/L)* 102 ± 25.82A 320 ± 44.82B 530 ± 29.35C 1040 ± 36.21D

Alkalinity (mg/L)* 165 ± 19.53 160 ± 4.71 150 ± 10. 62 185 ± 21.83 *Reported as CaCO3

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freshwater sources (0.33mM) toconcentrationsof2.96mM, foundsalinewatersat10 ‰.Potassiumconcentration in [Ca2+] and [Mg2+] experimentsweremaintainedat2.35mM,asdeterminedbyaprevious study (Fisher et al., 2013).Calciumwassupplemented togive final concentrationsof 0.20, 1.08, 1.50, and2.07mM (Table1),whileMg2+wassupplemented togiveconcentrationsof 0.05, 2.88, 5.76, and 11.52mM.The [Mg2+] experiment alsoreceivedCa2+ supplementation to thewater inthe formofCaCO2,asdetermined in thepreviousexperiment (Table1).Weeklywatersampleswerecollected to quantify ion concentrations via ICPanalysisbytheLSUAgricultureCenter;AgricultureChemistryDepartment(BatonRouge,LA,USA).　Fishweresampledat0, 1, 3, 7, 10, 14,and28dposthatch(DPH).Uponeachsamplingthestandpipewas adjusted tomaintain a constantdensity foreach treatment.Threereplicatesof5 larvaewereused toestablishanaveragedesiccateddrymassduetotheinabilitytoaccuratelymeasureindividualdrymass of larvae less than 7dph.Wholebodyionconcentration (Na+,K+andMg2+)wasanalyzedusing themethodsdescribedby (VanGenderen, 2003;Fisheret al., 2013).Threereplicatesof5larvaewerecollectedateachtimepointtodetermineNa+/K+-ATPaseactivityusing theprotocoldescribed inMcCormick (1993)andexpressedasµmolADPmgprotein－1h－1.　Six larvaewere collectedper treatmentgroupat1, 3,and7dphandpreserved inZ-fix (Anatech,LTD,BattleCreek,MI,USA).Immunocytochemistryreactionswereperformed according tomethodsdescribed inBodinier et al. (2010) andFisher et al. (2013) forNKA,CFTR, andNKCC1. IonocytelengthmeasurementsweretakenusingImageTool®version 3.0 (University ofTexasHealth ScienceCenter,SanAntonio,TX,USA).Aminimumof50cellsweremeasuredpersamplingperiod foreachtreatment.

Results

Growth and survival:The0.3mM [K+] treatmentresulted in 100%mortalitywithin 24h of hatch.No significant differenceswere seen in survivalbetweenthe1.3and2.0mM[K+]groups(survival≤

5%);howeverthe2.9mM[K+]treatmentresultedinsignificantlygreatersurvivalafter4weeks(survival~60%).Finaldrymassofthe2.9mM[K+]referencegroupwassignificantlyhigherthanboththe1.3and2.0mM[K+]treatments(P≤ 0.05).The2.9mM[K+]referencegrouphadafinaldrymassof4.93 ± 0.54mgwhile the1.3and2.0mM [K+] treatmentshaddrymassof2.39 ± 0.19mgand2.21 ± 1.55mg,respectively.　Survival and dry mass were signif icantlyinfluencedbyboth [Ca2+] and [Mg2+] experiments.Supplementation of Ca2+ showed a decrease inmeanfinalmassandmeansurvivalwith increasingconcentrations of Ca2+ in the absence of Mg2+ (Fig. 1A). Supplementation ofMg2+ resulted inapproximatelya5-foldincreaseinsurvivalwithMg2+supplementationascomparedtonosupplementation,nodifferenceswereobservedamongsupplementedtreatmentgroups.Finalmasswasnotsignificantly

Fig. 1.FinalmassandsurvivalofGulfkillifishlarvaeafter4weeksin(A) [Ca2+]treatmentsor(B) [Mg2+]treatments.Lettersrepresentsignificantdifferencesamongtreatmentgroups.Lettersdenotesignificantdifferencesamongtreatments.

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influenceduntilaconcentrationof11.52mMMg2+wasreached(Fig. 1B).

Whole-body ion concentrations & Na+/K+-ATPase activity : No differenceswere found inNa+/K+-ATPase activity among [K+] treatment groupsor among time (P ≥ 0.05) . Whole body Mg2+concentration showednodifference for treatmentortreatment×dphinteraction;howeversignificantdifferencewasdeterminedfordph(P≤ 0.05).Therewasasignificant increase in [Mg2+]withinthefirstweekfollowedbyarapiddecrease forall three[K+]treatmentgroups.Whole bodyK+ concentrationhadno significantdifference in treatment×dphinteraction, although significantdifferenceswereobservedamongtreatmentsandfordph(P≤ 0.05).　Nodifferenceswereobservedamongtreatmentsforwholebody ioncomposition in the [Ca2+]study.The [Mg2+] experiment showeddelayedpeaks inwholebody [Na+], [Ca2+],and [K+] for the0.05mM[Mg2+] treatment group,with ion concentrationsreaching their highest point 7 days later thanother treatments.Asexpected, theMg2+deficienttreatmentgroups (0.05 and 2.70mM [Mg2+]) alsoshowedasignificantdecreaseinwholebody[Mg2+].　Na+/K+ -ATPase activity in the [Ca2+] studywas significantly lower at 0 and 1 dph followed

by a significant increase in activity throughoutthe experiment, however no treatment specificdi f ferences in act ivity were determined atany specific sample dates. The [Mg2+] studydemonstrated significant differences inNa+/K+-ATPaseactivitybetweenMg2+deficienttreatmentsandthosewithsufficientsupplyat0dphfollowedbyminor,butnotsignificant, increasesthroughouttheexperiment(Fig. 2).

Immunocytochemistry & cell volume:Allnegativeslides, with no primary antibody, showed noimmunostaining as expected (not illustrated). In[K+] treatmentsat7dph,nosignificantdifferenceswere determined among treatments for gi l lionocyte length.Gill ionocytes from the 1.3mM[K+] treatmentwere found tohavea significantlylargerarea than those in the2.0and2.9mM [K+]treatments.IntestinalCFTRandNKAwerepresentat7dph inallsurviving [K+] treatmentgroups.Nochangeswere observed inNKAorCFTR in thegill ionocytes. IntestinalNKAandCFTRstainingappeared to show a decrease in intensity asK+concentration increased (Fig. 3).Very littlestainingwasobservedforNKCCbothintestinallyandatthegillepithelium.　Nodifferencesamongtreatmentswereobserved

Fig. 2.WholebodyNKAactivityforlarvaestockedin[Mg2+]treatmentsafter0, 1, 3,and7dph.Lettersrepresentsignificantdifferencesamongtreatmentgroupsatthatday.

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forNKAorNKCCstaining intensity inthegillsorintestine,norwerethereanysignificantdifferencesamong treatments for gill ionocyte length orquantityat1, 3,and7dph forboththe [Ca2+]and[Mg2+] experiments. In the [Mg2+] experiment, adecrease inCFTRstaining intensitywasobservedat 1dph inboth the intestine (Fig. 4) andgill inthe0.05mM[Mg2+] treatment,nootherchanges inCFTRstainingintensitywereobserved.

Discussion

　ThechemicalgradientgeneratedbytheactivationofNKAisoneoftheprimarydrivingforcesbehindion regulation, thus theability tomeasurewholebodyNKAactivitymayallowfortheevaluationoflarval fish osmoregulation capacity inhyper andhypo-osmoticenvironments. It ispossible thatK+isa limiting factor inkillifish larvalosmoregulationandadeficiencycanrestrict theabilityofNKAtofunction.Thecurrentmodel forchloridesecretionasdescribedbyMcCormicket al. (2003)suggestsabasolateralNKAandNKCCcoupledwithanapicallylocatedCFTR-likeprotein.BecauseK+ isalsoused

by the chloride cotransporter,NKCC, to assistin the transport of chloride ions, anextracellularK+ shortagecould reducea larval fish’s ability toregulatesodiumandchloride ionconcentrationsatcriticalbiologicalmembranes.Despite the fact thattherewerenostatisticaldifferencesinNKAactivitybetweenanytreatmentgroups,itisstillconceivablethatsufficientK+wasnotavailableforthefunctionofNKCC. If so then the inabilityof the larvae toregulateeitherNa+orCl－ ionsmaypossiblyhavebeenafactorinthelowsurvivalofthe0.3, 1.3and2.0mM [K+]groups.Thereferencegroup (marinemixsalt)was theonlygroup thatdidnotdisplaypoorsurvivalanditispossiblethatthepresenceofotheressential ionssuchasMg2+andCa2+playedasignificant role in thesurvivalof this treatmentgroup.　Potassiumsupplementation across thegradientexaminedinthecurrentstudyataconstantsalinitynotonlyaltered survival,butaffecteddifferencesamong molecular components o f intest ina lepithelium. Although potassium treatments inthe current studydidnot appear to alterwholebodyNa/KATPaseactivity, immunofluorescence

Fig. 3.Representativehistological sections from larval intestinesafteroneweek in [K+] treatmentgroupscut transverselyat5µm intervals. ImmunofluorescenceofNKA (red)andCFTR (green).The2.9mmolK+grouprepresentsa reference saltusedandcontainsother ionswhichmaybeessential for the functionofosmoregulatoryproteins.L:lumen;v:villi

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showed increased density of NKA and CFTRproteinsasK+concentrationdecreased.WholebodyK+concentrationwasgreater in the referenceascomparedtotreatmentswith1.3and2.0mMofK+bothofwhichremainedlowerthroughoutthestudyasexpectedduetotheinabilitytoselectivelyretainK+ ions. It is important tonote that thereferencegroupcontaining thehighest concentrationofK+was also comprised of greater concentrations ofMg2+andCa2+,whichcouldhavemodulatedNa+andCl－absorption inthe intestinerelativeto lowerK+treatmentsinthecurrentstudythatwererelativelylow in thesedivalent ions (Marshall andSinger, 2002;O’Grady, 1989).Growthandsurvivalmetricsfor thereference treatmentmayreflectcombinedeffectsofa full topartialcomplementofother ionsinadditiontotheK+gradientexamined.Theroleofintestinalepithelium in ionuptakenotonlyallows

thepotential for uptake andmaintenance of ionbalancefromdietarysourcesandthusimportantinaquaculture,butalsodemonstrates thepotential tomodulateconcentrationsofspecificionsinpreparedwatersforeuryhalineandmarineteleosts.　CalciumandMagnesiumare critical in teleostion regulation,varyingconcentrationsofCa2+andMg2+canimpactthepermeabilityofosmoregulatoryepitheliatobothionsandwater(Silvaet al., 2005).Intheseawateradaptedeel,removalofCa2+fromtheexternalmediumdiminishedtheactiveexcretionofsodiumbyhalf, excretionreturnedtonormal levelafterthereintroductionofCa2+tothemedium(IsaiaandMasoni, 1976). Properdevelopment ofmanyeuryhalineteleosts isrelatedtoenvironmentalCa2+concentration, for example red drum (Sciaenops ocellatus) frydisplayedadrop inbloodosmolaritywhen transferred from salt to freshwater, this

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decline inosmolaritywasreducedby theadditionof Ca2+(Wurts and Stickney, 1989) . PreviousstudieshavedeterminedthatMg2+ iscritical intheactivationofATP-dependent ionpumps (Bijveldset al., 1998)making itcritical for theactivationofNKA.Inthisstudy,boththe0.05and2.88mM[Mg2+]treatmentgroupsdisplayed significantly reducedNKAactivityathatch.　In the current study both Ca2+ and Mg2+had effects on survival, growth, andmolecularcomponentsatboththegillfilamentsand intestinalepithelium.IntheabsenceofMg2+supplementationlarvalF. grandishadreducedsurvival,decreasedNa+/K+ -ATPase activity, and adelayedpeak inwholebodycompositionsforseveralionsessentialforlarvaldevelopment.Adecrease inCFTR intensitywasalsoobservedinthegillsandintestineofMg2+deficienttreatmentasseenbyimmunocytochemistrystainingat1dph.ThisisanindicationthatMg2+isanecessity for larvalF. grandisgrowth, survival,and ion regulation. However, the Mg2+ of 10‰seawater is approximately 11.52 mM and thegreatestgrowthandsurvivalwereachievedinthisstudyataconcentrationof 2.88mMMg2+.Theseresultsdemonstratethatthepotentialforeuryhalineteleostculture inpreparedwatermayonlyrequirea fractionof the ionconcentrations found in10‰seawater.Alsotheroleofintestinalepitheliuminionuptakeallows forthepotential for ionmaintenancethroughdietarysources.

Acknowledgements

　Thisworkwassupported,inpart,throughgrantsfrom theSouthernRegionalAquacultureCenterandLouisianaSeaGrant.The authorswould liketoacknowledge the faculty, studentsandsupportstaff thatassistedwith these investigations:PaigeO’Malley, EmilyMcArdle, ChrisMariani,MikeCoulon,JoshPatterson,CharlieBrown.

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Annotated Bibliography

FisherC.,BodinierC.,KuhlA.,andGreenC., 2013:Effects of potassium ion supplementation onsurvival and ion regulation in Gulf killifishFundulus grandis larvaereared in iondeficientsalinewaters.Comp. Biochem. Physiol., Part A., 164, 572-578.

　This study encompasses results pertaining totheK+ ionmanipulationportions of the abstractpresentedabovefortheUJNRScientificSymposium.Thisinvestigationincreased[K+]fromvaluessimilarto freshwatersources (0.33mM) toconcentrationsof 2.96 mM, found in saline waters at 10‰. Anumberofbiochemical andmolecular techniques

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were performed to examine the effect of thisK+ iongradient,which included:wholebody ioncomposition,Na+/K+ -ATPase (NKA)activity, gillionocytemorphometrics, relativegeneexpression(NKA, NKCC, and CFTR), and correspondingimmunocytochemistry at the gill and intestinalepithelium.Resultsmore tangible toaquaculturist,suchasgrowthandsurvival,indicatedthepresenceofathresholdwithinthegradientexaminedwherebysurvivalwasnotdifferentbetween1.3 to2.9mM[K+].Utilizing immunocytochemistry,thedifferencesbetweentheseseeminglysimilar treatmentgroupsindicatedthatthetreatmentsgroupsbetween1.3to2.9mM[K+]wasdifferent in termsofgill ionocytearea,NKAandCTFRlocalization.

OstrowskiA.D.,WatanabeW.O.,MontgomeryF.P.,RezekT.C., ShaferT.H.,Morris Jr J.A., 2011:Effectsofsalinityandtemperatureonthegrowth, survival,wholebodyosmolality, andexpressionofNa+/K+ATPasemRNA in redporgy (Pagrus pagrus) larvae.Aquacult., 314, 193-201.

　These authors invest igated a number ofphysiologicalparameterspertainingtosalinityandtemperature in embryosand larvaeof redporgy(Pagrus pagrus),which isviewedasahigh-marketvaluemarine specieswith good potential as anaquaculturespecies.Embryosandresulting larvaewererearedatfourtemperatures(17, 19, 21, 23℃)andtwosalinities(24and34‰).Larvae(16dph)weretransferred fromtheirrespectivesalinities to44‰to represent a sublethal hyperosmotic challenge.TheseauthorsdemonstratedsignificantincreasesinNKAmRNAexpressioninindividualsacclimatedto24 ‰at24haftertransferredto increasedsalinity,while individuals from34‰exhibitednosignificantchanges inNKAexpression.Temperaturewasnotobserved to influence expression ofNKA,whilemetabolic parameters related to growth wereinfluencedby the temperature gradient in theirstudy. The authors utilized traditional growthmetrics includingmolecular tools to anticipateoptimum salinity (24‰) and temperature (23℃)conditionsthatareoptimumforlarvalrearingofthisspecies.

BodinierC.,SucréE.,Lecurieux-BelfondL.,Blondeau-BidetE.,CharmantierG., 2010:Ontogeny ofosmoregulation and salinity tolerance in thegilthead sea bream Sparus aurata. Comp.Biochem.Physiol. -PartA:Mol. IntegrativePhysiol., 157, 220-228.

　The gilthead sea bream (Sparus aurata) is acommercially importantaquaculturespecies,whichspawnsintheoceanandwhoseresultinglarvaeandjuvenilesmigrate to lower salinity estuaries andlagoons.Theseauthorsinvestigatedthedevelopmentof salinity tolerance ingiltheadseabreamfrom3, 30, 75, 96,and300dposthatch(dph)bychallengingthemwith9salinitiesrangingbetween freshwaterand45.1‰.Utilizing immunohistochemistry, theseauthors localized the NKA throughout thesechallenges todocument location of ion regulationwithinrespecttothisionoregulatoryprotein.Initially,immunopositiveNKAionocyteswere laocted intheintegumentalong theyolksacand integumentaryfoldsrepresenting thebranchial slits.A functionalshift from integument togillswasdemonstrated30and70dph,whenboththe integumentandgillswere observed to locally expressNKA,wherebyfrom70 to300dph thegills remain themainsiteof osmoregulation. Increases in osmoregulatorycapacity for thisspeciesat the intervalsexaminedwithinthisstudyrelatedtotheshiftsandpatternsobservedthroughimmunohistochemistry.

ChristensenA.K.,HiroiJ.,SchultzE.T.,McCormickS.D., 2012:Branchialionocyteorganizationandion-transport protein expression in juvenilealewivesacclimatedto freshwaterorseawater. J. Exp. Biol., 215, 642-652.

　Ingill ionocytes, ion transport is activatedbythe basolaterally locatedNKAwhich generatesan electrochemical gradient by coupling twoextracellular K+ with three intracellular Na+,driving the ionsaccording toexpression, location,and abundance of other proteins such asNKCCandCFTR.Christensenet al. investigatedchangesin alewife physiology and branchial epitheliumas individualswere acclimated to freshwater orsaltwaterandrepresentsthefirststudyof itskindto characterizemultiple ion-transportproteins ina non-salmonid anadromous fish. Corresponding

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increasesinNKA,NKCC1,andCFTRabundanceatthegillepitheliumwithincreasingsalinitywasusedto establish agillmodel forhypo-osmoregulation.Ingill ionocytes,NKA is responsible for loweringintracellularNa+allowingthebasolateralNKCC1toimportNa+,K+,andtwoCl－ions.ExcessintracellularCl－ is then secreted through theCFTRchloridechannel.NKCC1,thesecretoryisoformisexpressedbasolaterally in thegill ionocytes,whileNKCC2 isidentifiedasanabsorptiveisoformandisexpressedapically along the intestinal andurinarybladderepitheliumofsaltwaterandeuryhalineteleosts.Theauthorsdetermined thekeydifferencesbetweenfreshwaterandseawateracclimatedalewivesinthecontextof ion transportersat thegill epithelium.The implicationsof these investigationhasassistedinincreasingtheinformationmulticellularcomplexesofmature ionocytes and the role of salinity andspecificioninthemaintenanceofhomeostasis.

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