Posted on Authorea 17 Aug 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.159769275.58126994 | This a preprint and has not been peer reviewed. Data may be preliminary. aiiycoet rhge hnsaae Camnire l,20;Lgo n hrate,21) Such 2015). Charmantier, and Lignot in 2009; in regulation al., osmoregulation isosmotic et Typical (Charmantier and seawater salinity extent. than low some higher in to or hyperregulation to changes with close salinity and hyper-isosmotic, salinity osmotic ambient is hemolymph to regulate crustaceans can response estuarine they in unclear. ions osmoregulators; are the minor concentrations are However, organisms Therefore, estuaries ionic marine crustaceans. inhabiting 2007). of euryhaline species al., survival some crustacean et the for Most Roy affect least 2006; ions at invertebrates, 2005, minor Fotedar, marine which and of by survival mechanisms Prangnell the 2005; for al., required et are Davis et 2004; (Cawthorne al., salinity same et K the of of seawater presence vannamei artificial the Litopenaeus diluted addition, in prawn In survive euryhaline 1983). can Na the al., it by However, although replacement invertebrates. their solution, marine and NaCl essential of 0.17% be life not the inverte- should on marine ions effect minor of fluids, fluids body body of of lality SO osmolality as the such maintaining ions in minor role crucial Na a brates. play seawater in ions Dissolved ambient to susceptive less is INTRODUCTION cations, dehaani 1| minor In C. ambient that osmolality–dependent. by japonicus. indicated are affected M. strongly cations and hardly results minor tridens were These ambient H. rate to of species. survival compared effects this and cations the of minor composition that habitat concentration ionic ambient concluded K+ the hemolymph hemolymph thus conditions, reflecting the the is hypo-osmotic of probably increase dehaani, under It an C. contrast, and japonicus. In in japonicus, M. M. contrast, on exist. and inhibitors effect not tridens lethal exchanger did a H. Na+/H+ crabs; cations in and on of minor effects anhydrase different ambient composition cytoplas- ATPase, carbonic had if ionic cations Na+/K+ of even minor as and Administration such rate osmolality uptake, survival exchanger. Na+ the the for Na+/H+ affected required increased and gills also japonicus the anhydrase, cations M. in carbonic and genes minor tridens mic specific Macroph- ambient H. of tridens, The expressions of Helice survival regulating crabs: by for seawater. hemolymph euryhaline required with cations) were three minor conditions cations of (ambient minor isosmotic gills K+ Ambient and the under but Ca2+, dehaani. in the SO42–, Mg2+, Chiromantes expression affect and of and gene significantly K+, effects japonicus, and Ca2+, the They thalmus Mg2+, composition, analyzed ions. study as ionic such This total hemolymph ions survival, of unclear. minor on are ~85% contains organisms constituting also marine Seawater seawater, on invertebrates. in effects marine their ions of dissolved fluids abundant body most of the osmolality are Cl– and Na+ Abstract 2020 17, August 2 1 Yamaguchi Masahiro manner minor condition-dependent ambient osmotic by to controlled according are cations thus crabs and euryhaline gills, of the survival in the expressions gene composition, Hemolymph sk iyUiest rdaeSho fSineFclyo Science of Faculty Science of School Graduate University City College Osaka Suzuka Technology, of Institute National + n Cl and – r h otaudn osi ewtr osiuig~5 fttlin,i diinto addition in ions, total of ~85% constituting seawater, in ions abundant most the are 4 2– and 1 Mg , n oih Soga Kouichi and eietslatisulcatus Melicertus 2+ Ca , 2+ n K and , + Mg , + 2+ fdsovdin r eurdol o anann h osmo- the maintaining for only required are ions dissolved If . 2 n SO and , frel called (formerly 1 4 2– nrae h uvvlrt ferhln prawns euryhaline of rate survival the increases .latisulcatus) P. eau mondon Penaeus + Sode l,20;Zhu 2003; al., et (Saoud n Cl and antsriein survive cannot – hudhv no have should Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. yeH type Na catalyzes which the (CAc), of anhydrase carbonic H activation cytoplasmic of is cells et formation chloride (Freire in the regulation enzyme key ionic Na Another and and express low. environment, osmotic cells relatively exposed hemolymph Chloride the Na in crabs 2012). to transports role al., euryhaline which compared important et which an range higher Henry by play wider 2008; osmolality gills mechanisms al., a hemolymph posterior the tolerate in their investigated and cells maintain have chloride euryhaline water studies be fresh/brackish to Many to estuaries salinity. in ambient species of crustacean allows pattern osmoregulatory a erdi 1. mlLNC otiigol MO h uvvlrt a acltd2 after h 24 calculated was rate K survival The triplicate. DMSO. Na in Hemolymph only least group 2.3| at containing control mmol/L performed 513.3 The NaCl were in In hydrate. mmol/L reared experiments hydrochloride solution. were The amiloride 513.3 stock crabs transfer. of the mmol/L) in mmol/L) of (187.9 (0.9 individuals reared mg/mL four mg/mL was survivals, 50 0.25 on containing a Amiloride amiloride reared solution DMSO. of prepare also solution NaCl with effect were to only NaCl the crabs supplemented DMSO assess 3% the solution of to in of the NaCl order dissolved effect number 3% to was same the the transferred to the hydrate then assess transferred group, hydrochloride then and control to and the experiments seawater order As natural to mg/mL In acetazolamide. in 1 prior containing of solution. seawater days mg/mL natural stock 2 1 in containing acetazolamide as reared mol/L) were used of (1.11 crabs were mmol/L) the dimethyl mg/mL 2008), of (5.6 in individuals 200 al., dissolved four et was a survivals, Tresguerres studies Acetazolamide on prepare respectively. 2005; acetazolamide crustacean NHE, al., to in the et (DMSO) and Genovese used (CA) sulfoxide 2003; been anhydrase al., carbonic already et for have Henry inhibitors 1990; which Towle, hydrate, and hydrochloride otherwise (Burnett unless amiloride presented and and Acetazolamide calculated Na was and 16 h triplicate. anhydrase a 48 in in Carbonic least at incubator 2.2| at rate an performed survival in were The kept experiments were point. The tanks time noted. The each 1. at Table removed 21 in were reared at summarized cycle were were light/dark study species h this each h/8 in of the used individuals examine media four to bathing rate, order survival In on Japan. 177 media Prefecture, bathing Mie of 210 City, Yokkaichi in composition in ionic estuary of river effects Suzuka the media from bathing collected various in or tridens rates habitats survival H. of their evaluation of and microenvironment Animals the 2.1| in METHODS difference AND MATERIALS the 2| to crabs: due euryhaline be three could of phylogeny. species gills euryhaline the in between gills. expression the in gene japonicus expressions and Macrophthalmus gene are composition, the water of hemolymph ionic environmental Mg changes to of hemolymph in induce contribute effects factor(s) factor(s) the also what analyzed what might study and present This salinity, genes in at ambient these unknown changes of and is signal by 2013), key it modified 2017). However, as al., Griffith, used are et regulation. 2012; levels Lv ionic al., 2011; expression and et al., osmotic whose Henry et 2009; genes (Towle al., numerous salinity et ambient identified Charmantier has 2008; al., analysis et Transcriptome (Freire hemolymph of composition + n soi concentrations osmotic and , × 195 + × TaeadNa and ATPase 300 × and 0 mpatctnswt 0 Lo ahn ei (for media bathing of mL 500 with tanks plastic mm 100 × 5m lsi ak ih10 Lo ahn ei (for media bathing of mL 1000 with tanks plastic mm 95 .japonicus M. + + + n HCO and /H rmtectpamt h eoyp n anan nrclua Na intracellular maintains and hemolymph the to cytoplasm the from + + + , /K xhne NE oae nteaia ebae te oeue,sc sV- as such molecules, Other membrane. apical the in located (NHE) exchanger ° and , .Tesria aeo rb a sesda 6 4 0 n 8h n carcasses and h, 48 and 40, 24, 16, at assessed was crabs of rate survival The C. + eecletdfo h aaarvretayi s City. Tsu in estuary river Tanaka the from collected were /2Cl 3 – hrmne dehaani. Chiromantes rmH from – ornpre NC) r loivle nrgltn aiiyadionic and salinity regulating in involved also are (NKCC), cotransporter 2+ + Ca , / H 2 n CO and O + 2+ n K and , xhne inhibitors exchanger + 2 2 n h eie H derived the and , hratrcle abetmnrctos)o survival, on cations”) minor “ambient called (hereafter + /K h ieetrsos oabetmnrcations minor ambient to response different The + Tae(K)i h aoaea membrane, basolateral the in (NKA) ATPase .japonicus M. .tridens H. + uprsNa supports ) and . h opstosof compositions The .dehaani C. + .dehaani C. + eietridens Helice paethrough uptake concentration ,o in or ), was , Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. h rb eeaeteie n isce,ad~300 4 and at dissected, and japonicus M. anesthetized were crabs The eemaue ihavprpesr soee Mdl50,Wso,Lgn T USA). Να UT, concentrations ανηψδρασε, Logan, νις osmotic Wescor, 5600, The and (Model respectively. C-122) osmometer 2.4 Japan), pressure (model Kyoto, vapor sodium (HORIBA, a using with C-131) measured measured were (model were respectively, meters solutions, ion sample potassium undiluted and diluted 1:10 ojgtdat-ioiei nioy(oh)a iuino :00 inl eedtce sn ir blue nitro using detected digoxigenin-labeled were with Signals phosphatase– 1:2000. hybridized alkaline of with then dilution treated a and and at blocked, (tRNA) (Roche) washed, sodium antibody was RNA 0.5% anti-digoxigenin membrane conjugated transfer the solution, Finally, Denhardt’s 0.005% overnight. 5x using probes and formamide, prehybridized to RNA 50% (SDS), was gel (SSC), membrane the sulfate citrate resultant Isogen from saline-sodium dodecyl The using transferred 5x overnight. was containing gills (Roche) solution RNA of membrane a Next, pair nylon mix charged formalin. posterior labeling positively 5.5% most RNA a and the DIG (MOPS) from 10x acid extracted 8.5 the -morpholino)propanesulfonic and was for cDNA Gene), RNA instructions (Nippon using Total manufacturer’s reagent Briefly, Switzerland). the 2000). to Basel, al., according (Roche, et synthesized (Yamaguchi were described probes NKA previously encoding genes as for performed, fragments was blotting Northern 2008). Henry, and NKA Serrano 2.5 for 1997; AY035548 al., and et U09274, (Towle respectively) EU273943, NKCC, AY03550, are numbers Accession tridens for dehaani LC572289 and LC572288, in products gene the of sequence acid were primers amino Degenerate the was resul- of nas amplification. and RNA (PCR) basis Japan), reaction the total Osaka, instructions. chain on (TOYOBO, and polymerase manufacturer’s Ace designed for ReverTra crabs, the templates and from as to primer served isolated (dT) according cDNAs oligo tant was Japan) an Tokyo, gills using Gene, synthesized of were (Nippon pair cDNAs reagent posterior Isogen most using the extracted Briefly, NKA 2001). encoding Wakahara, (cDNAs) and DNAs complementary in of NKCC isolation and Amplification h eune fioae DA eedpstdt eBn Acsinnmeso NKA determined. of for LC572287 numbers were and (Accession sequences LC572286, GenBank nucleotide LC375964, to LC214855, their deposited are finally, NKCC were and and cDNAs 94 NHE, (TOYOBO), isolated at vector of s sequences cloning 30 The pTA2 for a cycles into 40 inserted follows: for- 72 as at CAc: 43 were s PCR 30 follows: and for as ture, conditions were case, PCR every nested the In for from used products forward, the sequences NKCC: of 5’-ARYTCCATNACYTGRAARCT-3’. primer part reverse, 5’-CANGGNGGNGTNGTNA-3’; 5’-ATHTGGGGNGTNATGYT-3’; The using reverse, PCR template. 5’-CAYTGGGGNAARACNAAYGA-3’; nested performed a ward, we as cDNA, amplification NKCC and first CAc -TAYGTNCTNGARCARTTYCA- of amplification 5’- For 5’ forward, 5’- or NHE: forward, 5’-GCRAARCANCCNGCRTADAT-3’. 5’-TAYGTNTTRGARCARTTYCA-3’ 5’-CKRAANGCRTCNAGYTG-3’; AAYATHTGGGGNGTNATG-3’; reverse, NKCC: 5’-TGYTCNACRTARTTYTTCAT-3’; or forward, reverse, 5’-CKRAANGCRTCYAAYTG-3’ AARATHGGNTTYCAYATGAC-3’; reverse, 3’; CAc: 3’; α uui:frad ’AGCGNCCYTTG3;rvre 5’-GGRTGRTCNCCNGTNACCAT- reverse, 5’-ATGACNGTNGCNCAYATGTGG-3’; forward, subunit: ° ,45 C, | | ° , n h uentn a olce ob sda h apeslto.Na solution. sample the as used be to collected was supernatant the and C otnstrituberculatus Portunus otenblotting Northern μλφςτο ν σλτο φΝα οφ ισολατιον ανδ Αμπλιφιςατιον , ° .japonicus M. .I diin mn cdsqecswr rdce rmtecN rgetsqecsof sequences fragment cDNA the from predicted were sequences acid amino addition, In ). ,ad45 and C, .tridens H. fhmlmhwscletdfo ahca.Te eecnrfgda 2390 at centrifuged were They crab. each from collected was hemolymph of ) ° ,rsetvl.Apie C rdcswr lcrpoee n xrce,adthen and extracted, and electrophoresed were products PCR Amplified respectively. C, ° .TeanaigtmeauefrNKA for temperature annealing The C. , and , .japonicus M. + /Η .dehaani C. + and , ξηνε,αδΝα ανδ εξςηανγερ, ,japonicus M, μ fttlRAwseetohrsdi %aaoeglcnann x3-( 1x containing gel agarose 1% in electrophoresed was RNA total of g α .vannamei. L. uui,Cc H,adNC stmlts ioiei-aee RNA digoxigenin-labeled templates, as NKCC and NHE, CAc, subunit, and , n oprdwt hs ftesoecrab shore the of those with compared and .dehaani C. n C720 C721 C722 n C723for LC572293 and LC572292, LC572291, LC572290, and , + h rmrsqecsue o C eea olw:NKA follows: as were PCR for used sequences primer The /Κ 3 + eepromd speiul ecie (Yamaguchi described previously as performed, were + μ /Κ (for l Ταεασβντ ψολσι ςαρβο- ςψτοπλασμις συβυνιτ, α ΑΤΠασε + /2῝λ α .tridens H. uui,Cc H,adNC a 50 was NKCC and NHE, CAc, subunit, – ςοτρανσπορτερ alnce aiu,Criu mae- Carcinus sapidus, Callinectes .tridens H. and ° ,3 tanaigtempera- annealing at s 30 C, .dehaani C. α + α n K and uui,Cc H,and NHE, CAc, subunit, uui,Cc H,and NHE, CAc, subunit, C186 LC375965, LC214856, , .maenas C. + ocnrtosin concentrations r˜150 or ) α × uui,CAc, subunit, o min 5 for g (GenBank μ (for l ° H. C. C, N Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. nrae n ece 0%i ohseis(i.2,B,idctn ht533mo/ NaCl+MMCK mmol/L 513.3 that indicating of B), 2A, composition (Fig. ionic species the replicate both in to 100% solution) reached NaCl+MMCK and mmol/L increased 513.3 as and seawater, to artificial referred was solution MgSO mmol/L 27.4 with Na besides ions minor tridens ambient H. some of administration whether determine To that indicating movements, LC slowed as determine such damages, of signs dehaani few a lethal dehaani observed median we C. NaCl that although mmol/L 1C), revealed (Fig. 513.3 analysis solution that Profit contrast, showed In crabs. respectively. results in mmol/L, These few damage The severe (LC moribund. B). caused were concentrations 1A, cations solution minor (Fig. NaCl seawater without of mmol/L solution that 513.3 to in comparable survived was salinity its although ftreseisws10 nntrlsaae n eaieyhg n80mo/ alslto Fg 1), However, (Fig. salinity. solution low of NaCl in (most mmol/L hyperregulators crabs 8.0 are all of and in of in majority euryhaline high rate decreased the relatively strongly survival significantly The are and rate seawater. seawater species survival natural natural the three and in all 2.0) 100% that of was indicating factor species a with three crabs, diluted of euryhaline geometrically of was solution survivals NaCl on cations minor dehaani of effect investigate To conditions isosmotic under crabs 3.1 3 Student’s by show of the performed figures analysis if were using the significant groups in performed two statistically were bars between groups Error comparisons Student’s three values Na group. test, than Mean and each Williams’ more in by triplicate, among followed crabs in (ANOVA), comparisons variance three least values least Mean at at performed errors. in were standard measured rates were survival concentrations evaluate osmotic to experiments rearing All as 5-bromo-4-chloro-3-indolyl- determined were and levels tetrazolium expression relative blue 2.7 the nitro and using quantified was blotting. at detected signals Northern (Roche) were the in was of antibody Signals RNA membrane intensity RNA anti-digoxigenin the single-stranded The Subsequently, Finally, phosphatase–conjugated 1:2000. digest phosphate. overnight. alkaline formalin. of (Roche) to with RNA membrane 5.5% dilution T1 treated nylon digoxigenin-labeled and a then charged RNase with MOPS positively and and 1x a hybridized blocked A to containing was and gel RNase gel gills the total with agarose Briefly, from of treated 1% transferred 2001). pair was in al., posterior RNA electrophoresed et (Sugiyama and Next, most al. the overnight. et from Sugiyama probes extracted to according was performed RNA was assay (Fig. protection solution RNase NaCl mmol/N 4.3 or level 5) expression 2.6 (Fig. relative NaCl+MCK The mmol/L USA). image 513.3 1.0. using Maryland, in was quantified expression Health, 9) were the of which as of Institute calculated both (National was RNA, ImageJ ribosomal program, electrophoresed blotting of processing Northern amount of signals with detected normalized of was intensity The 5-bromo-4-chloro-3-indolyl-phosphate. and tetrazolium | RESULTS | | | ttsia analysis Statistical assay protection RNase ffcso min io ain nsria n eoyp opsto feuryhaline of composition hemolymph and survival on cations minor ambient of Effects eerae n80 60 21 42 2.,267 n 1. mlLNC ouin(.. 1. mmol/L 513.3 (i.e., solution NaCl mmol/L 513.3 and 256.7, 128.3, 64.2, 32.1, 16.0, 8.0, in reared were hwdhg uvvlrtsee nhg ocnrtoso alsltos rfi nlsscudnot could analysis Profit solutions, NaCl of concentrations high in even rates survival high showed and epnst min ouin ieetycmae to compared differently solutions ambient to responds 50 .japonicus M. nti species. this in 50 for ) .tridens H. 4 52mo/ MgCl mmol/L 25.2 , .tridens H. n533mo/ alslto,533mo/ alslto a supplemented was solution NaCl mmol/L 513.3 solution, NaCl mmol/L 513.3 in P au a esta 0.05. than less was value .tridens H. and .dehaani C. .japonicus M. and .tridens H. n l of all and .japonicus M. a ihsria ae( rate survival high a had 2 . mlLCaCl mmol/L 9.9 , eerae nti ouin h uvvlrt significantly rate survival The solution. this in reared were 4 and .japonicus) M. t ee431.57 were .japonicus M. ts ihBnern orcin rTkystest. Tukey’s or correction, Bonferroni with -test .tridens H. 2 n 07mo/ C teresultant (the KCl mmol/L 10.7 and , idi 1. mlLNC solution, NaCl mmol/L 513.3 in died ± > .tridens H. thge alcnetain,and concentrations, NaCl higher at 0) vni 1. mlLNaCl mmol/L 513.3 in even 80%), 71mo/ n 164.37 and mmol/L 37.1 + t n Cl and ts.Rslswr considered were Results -test. and .tridens H. .japonicus. M. , – .japonicus M. etr uvvlrt of rate survival restore niiul who individuals + Because K , and , ± + and , 22.3 C. C. Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. n533mo/ alslto oprdt 1. mlLNC+C ouina n86mo/ NaCl mmol/L 8.6 in as solution NaCl+MCK mmol/L 513.3 to in compared gills solution posterior NaCl most in mmol/L the gene 513.3 in this in genes of all expression in of the CAc expression detect the to that out japonicas revealed carried was analyses assay These protection RNase therefore, alMK n 1. mlLNC ouin o eeaaye yNrhr ltig oee,the However, blotting. Northern in by mmol/L 513.3 high analyzed NaCl, sufficiently were mmol/L not h 8.6 was the 6 gene in for CAc held of solutions crabs levels of NaCl gills expression mmol/L posterior 513.3 most the and in NaCl+MCK, genes these of expression The of that to respectively, similarity, maenas; C. of te rbseis h eue mn cdsqecso h NKA in the those to of similar sequences highly acid were amino genes deduced and these The of , sequences species. fragment crab cDNA the other that showed results Sequencing DAfamnsfrteNKA the for fragments cDNA oaayetepsiiiyta hne nteepeso fgnsivle nNa frag- in cDNA involved of experiments, gills genes rearing posterior in of most crabs of the expression death in the the analyzed to NKA in led of changes ultimately ments and that composition possibility ionic hemolymph the analyze To Να ανδ ανηψδρασε, ςαρβονις ςψτοπλασμις Na 3.2 hemolymph change not 2). Table did F; media in bathing 3C, not of in but compositions concentrations solution, ionic NaCl+MCK osmotic in mmol/L differences contrast, 513.3 In in 2). that and in to solution concentration compared NaCl+MCK higher osmotic mmol/L significantly 513.3 hemolymph to K K Moreover, compared hemolymph hemolymph E). less the the significantly to addition, was comparable solution In was NaCl B). Na mmol/L 3A, hemolymph 513.3 (Fig. in The solution incubation. NaCl+MCK after mmol/L h of Na 6 concentration Hemolymph at solution. osmotic tridens NaCl determined and mmol/L were 513.3 composition and concentrations ionic solution), MgCl NaCl+MCK hemolymph mmol/L mmol/L the 25.5 513.3 examine with supplemented to K NaCl was both of study crabs, presence the these the of that step indicating Next D), 2C, (Fig. species Mg that (either both suggesting of D), rate 2C, MgCl survival (Fig. mmol/L solution NaCl+K+25.5 mmol/L NaCl+K 513.3 mmol/L Both 513.3 MgCl in K mmol/L species 25.5 that both specific indicating K in the of recover B), determine addition fully To 2A, not NaCl. (Fig. did mmol/L rate KCl 513.3 and solution), of lacking NaCl+K presence survival mmol/L medium the 513.3 The bathing in salts. species the K additional of both four in role of the survival species mmol/L of for 513.3 both one NaCl, indispensable any in mmol/L 513.3 mmol/L minus decreased follows; 513.3 NaCl+MMCK as rate of condition mmol/L survival, isosmotic presence 513.3 for of the and media required in bathing NaCl+MMCK, are of survive kinds ions to several minor species in ambient reared both which for investigate ions To minor NaCl. ambient sufficient contains solution .tridens H. | σλτο ν ξρσινααψι φγνςεςδν Να ενςοδινγ γενες οφ αναλψσις εξπρεσσιον ανδ Ισολατιον .dehaani C. .japonicus M. and + h 1. mlLNC ouinwssplmne nywt 07mo/ C rfre oa the as to (referred KCl mmol/L 10.7 with only supplemented was solution NaCl mmol/L 513.3 the , a ihri . mlLNC ouincmae o533mo/ alMKslto except solution NaCl+MCK mmol/L 513.3 to compared solution NaCl mmol/L 8.6 in higher was 2+ .japonicus M. n hs fteNEof NHE the of those and .tridens H. + rCa or , ln sisffiin.Tu h 1. mlLNC+ ouinwssplmne iheither with supplemented was solution NaCl+K mmol/L 513.3 the Thus insufficient. is alone .japonicus M. α uui,Cc n H eeapie yPRadioae,adgn xrsinwas expression gene and isolated, and PCR by amplified were NHE and CAc, subunit, a 5,9% n 4 iiaiy epciey ota of that to respectively, similarity, 94% and 95%, 95%, had 2 2+ r99mo/ CaCl mmol/L 9.9 or sncsayadsffiin o h uvvlof survival the for sufficient and necessary is ) utemr,teepeso falgnsi h otpseirglswsas higher also was gills posterior most the in genes all of expression the Furthermore, . , .japonicus M. edi 1. mlLNC ouinwssgicnl ihrcmae o513.3 to compared higher significantly was solution NaCl mmol/L 513.3 in held .dehaani C. and , α .maenas C. uui,Cc n H ee ee70 0,ad80b,respectively. bp, 800 and 300, 700, were genes NHE and CAc, subunit, .dehaani C. + hc a ihsria aei 1. mlLNC ouin(Fig. solution NaCl mmol/L 513.3 in rate survival high a had which , .tridens H. .tridens H. and , .tridens H. ocnrto ncashl n86mo/ alslto Fg 3D, (Fig. solution NaCl mmol/L 8.6 in held crabs in concentration 2 and .dehaani C. Fg AC Twee l,19;SraoadHny 2008). Henry, and Serrano 1997; al., et (Towle 4A–C) (Fig. 2 . mlLCaCl mmol/L 9.9 , .tridens H. a 6,7% n 5 iiaiy epciey ota of that to respectively, similarity, 75% and 78%, 76%, had and 2 , , n 1. mlLNC++. mlLCaCl mmol/L NaCl+K+9.9 mmol/L 513.3 and .japonicus M. .japonicus M. + .tridens H. 5 .japonicus M. /Η nuae nte86mo/ al 1. mmol/L 513.3 NaCl, mmol/L 8.6 the in incubated .japonicus M. + and εξςηανγερ .japonicus M. edi 1. mlLNC ouinwas solution NaCl mmol/L 513.3 in held and , and , 2 eerae nti ouin h survival The solution. this in reared were n 07mo/ C rfre oas to (referred KCl mmol/L 10.7 and , .tridens H. α uui of subunit ob eetdb otenblotting; Northern by detected be to .dehaani. C. .dehaani C. .tridens H. + eerae nteesolutions. these in reared were + /Κ + and ocnrto ncasheld crabs in concentration .maenas C. + .tridens H. ocnrto nboth in concentration a 4,9% n 92% and 93%, 94%, had + Ταεασυβυνιτ, α ΑΤΠασε .japonicus M. h egh fisolated of lengths The and n iaetcation divalent a and + .japonicus M. + .tridens H. .japonicus M. K , paedisturbed uptake hs fCAc of those ; + .japonicus M. , n osmotic and , .japonicus M. + 2 K , . restored and + (Table and , were + M. H. is . Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. naltreseis(i.8–) ncnrs,tehmlmhK hemolymph the contrast, In 8A–C). (Fig. hemolymph species the in three differences all significant Na no in hemolymph were and There solutions, incubation. MCK after Na NaCl+1.2 h composition mmol/L 6 4.3 ionic determined and were hemolymph concentrations NaCl on mmol/L conditions 4.3 F). the 7D, hypo-osmotic in in under (Fig. examined were rearing cations concentration of minor osmotic initiation and ambient after of h effects 48 Possible all K at NaCl of effect mmol/L effect lethal 4.3 lethal the of the block supplementation K attenuated contrast, that In cations indicating divalent solution. 7E), these MgCl MCK (Fig. 1.2 mmol/L recover NaCl+ the 30.5 mmol/L of 4.3 hardly with one-fourth 4.3 in not (almost in that did mmol/L lethality as 2.7 rate the to level survival for KCl comparable the diluting a after seawater), Even to in 7D). decreased concentration significantly (Fig. solution rate MCK survival NaCl+1.2 the mmol/L solution), K NaCl+1.2 mmol/L on japonicus effect lethal have CaCl cations minor which in examine salts To additional of concentrations in mortality higher high solution. that caused MCK indicated concentration NaCl results lower These a with 7B). combination few (Fig. A solution rate MCK most survival 7). NaCl+1.2 contrast, The In (Fig. solution. moribund. MCK solution were NaCl+1.2 MCK mmol/L NaCl+1.2 17.1 mmol/L and solution), MCK 4.3 NaCl+1.2 NaCl, of mmol/L mmol/L in 4.3 4.3 MgCl using as decreased mmol/L conducted to 30.5 considerably were with experiments rate supplemented some further NaCl survival Because solution, mmol/L NaCl+MCK the shown). mmol/L contrast, of not 8.6 in (data In survival intact Additional solution the NaCl+MCK salts. shown). on mmol/L additional not 8.6 without effect (data solution any species NaCl have mmol/L both not 17.1 and did solution, salts NaCl mmol/L salinity, 8.6 ambient low solution), at MgCl crabs euryhaline mmol/L of survival 25.2 the on cations tridens minor H. ambient of effects the investigate To conditions significant hypo-osmotic statistically under not crabs were results the 3.4 but solution, NaCl 6A). mmol/L 513.3 (Fig. to in compared higher also acetazolamide was rate survival of of The and rate D). CA rate survival of survival the inhibitor the an increased increased amiloride, acetazolamide or significantly acetazolamide Amiloride with supplemented respectively. solution NHE, NaCl mmol/L 513.3 in reared japonicus M. nrae xrsino ee novdi Na in involved genes of expression Increased eewsehne n533mo/ alslto u o n86mo/ alslto Fg 5). NHE (Fig. of solution NaCl expression mmol/L the 8.6 contrast, in In not solution. but in NaCl solution different mmol/L NaCl 3.3 mmol/L 8.6 was 513.3 in genes in was these enhanced but was solution of gene NaCl expression mmol/L the 513.3 in hand, to other NKA compared the encoding drastic On genes of 5). expression (Fig. solution .tridens H. + | | ocnrto ewe rb nuae n43mo/ alad43mo/ al12MKsolutions MCK NaCl+1.2 mmol/L 4.3 and NaCl mmol/L 4.3 in incubated crabs between concentration ffcso abncahdaeadNa and anhydrase carbonic of Effects 2 ffcso min io ain nsria n eoyp opsto feuryhaline of composition hemolymph and survival on cations minor ambient of Effects 12C n 34mo/ C 12)wr de o43mo/ alslto eaaey In separately. solution NaCl mmol/L 4.3 to added were (1.2K) KCl mmol/L 13.4 and (1.2MC) erdi . mlLNC ouinsplmne ih1. mlLKl(eerdt s4.3 as to (referred KCl mmol/L 13.4 with supplemented solution NaCl mmol/L 4.3 in reared , .japonicus M. n533mo/ alslto.T xmn hspossibility, this examine To solution. NaCl mmol/L 513.3 in and .dehaani C. 2 . mlLCaCl mmol/L 9.9 , .tridens H. 2 n 17mo/ CaCl mmol/L 11.7 and and , a 0% u htof that but 100%, was and .dehaani C. .japonicus M. .japonicus. M. α uui a nacdi 1. mlLNC ouinbtwsless was but solution NaCl mmol/L 513.3 in enhanced was subunit .tridens H. 2 n 07mo/ C rfre oa . mlLNaCl+MCK mmol/L 8.6 as to (referred KCl mmol/L 10.7 and , + .tridens H. .japonicus M. eerae n86mo/ alslto upeetdwith supplemented solution NaCl mmol/L 8.6 in reared were paecudb osbecueo h et of death the of cause possible a be could uptake + niiul uvvdi . mlLNC n 71mmol/L 17.1 and NaCl mmol/L 4.3 in survived individuals /H erdi 1. mlLNC ouinsplmne with supplemented solution NaCl mmol/L 513.3 in reared nadto,teepeso fCcgn a o induced not was gene CAc of expression the addition, In .tridens H. 2 i o aealta ffc on effect lethal a have not did 2 + 6 .japonicus M. .japonicus M. 17mo/ CaCl mmol/L 11.7 , + , .japonicus M. xhne inhibitors exchanger .japonicus M. 5hatriiito fraig lhuhi i not did it although rearing, of initiation after h 15 erdi 1. mlLNC ouin(i.6B– (Fig. solution NaCl mmol/L 513.3 in reared and + niiul uvvdi hsslto but solution this in survived individuals .dehaani C. ocnrto ncasicbtdi 4.3 in incubated crabs in concentration 05mo/ MgCl mmol/L 30.5 , .japonicus M. and , .japonicus M. infiatydcesdt 7 n4.3 in 17% to decreased significantly .tridens H. .tridens H. 2 .dehaani. C. n 34mo/ C (referred KCl mmol/L 13.4 and , hwn 0%sria in survival 100% showing , niiul tl appeared still individuals n43mo/ NaCl+1.2 mmol/L 4.3 in and .japonicus M. and hywr incubated were They + 2 .japonicus M. .japonicas M. K , .dehaani. C. n 17mmol/L 11.7 and .japonicus M. + .tridens H. n osmotic and , + instead, ; accounts and , were The and M. in Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. Mg uvv nNC ouinwt iia aiiy(21mo/,crepnigt .9) hs aaso that show data These 0.19%). to both corresponding 1983), mmol/L, al., (32.1 salinity et similar (Cawthorne with solution solution NaCl NaCl 0.17% in in survive h 48 survive cannot K (2) 2003), al., crabs, euryhaline of species of three Habitats study, this In ne ssoi odto.Frcmlt eoeyo h uvvlrt n533mo/ alslto,both solution, SO NaCl (1) mmol/L 513.3 examined: in both rate for survival ions the of recovery complete K For Na besides condition. of ions isosmotic mortality minor under high ambient The some indicates solution. solution NaCl NaCl mmol/L 513.3 in both not but that revealed study 2006a). This al., et (Lovett survival crab its shore Ca for the either to of removal damage Furthermore, lethal Na seawater 2007). besides artificial al., ions, et diluted Roy minor in K ambient low of some vannamei presence remains L. the that it that indicating shows although 1983), prawns solution, of al., NaCl et 0.17% (Cawthorne in Cl salinity affects h same significantly environment 48 the prawn the euryhaline first of of the of the composition mortality ionic the during also example, For 100% but salinity crustaceans. euryhaline only the of not survival in that the differences reported been the has to It due possibly are species 4.1 phylogeny. these or of habitats water their environmental of to microenvironment 2000). Kobayashi, responses 1993; ( different Varunidae Kijima, The and families: (Irawan different water ( three fresh Sesarmidae to to belong adaptive is species and These regions upstream more invaded In 9). (Fig. low relatively was expression the compared 4 although decreased h cations, In in 6 minor expression species. ambient for NKCC of three solution contrast, presence MCK all the NaCl+1.2 in in mmol/L solution decreased 4.3 NaCl in mmol/L incubated 4.3 crabs to of gills and posterior it most of of expression that K gene of to NKCC and similarity in of isolated, sequence involved and acid PCR amino genes by deduced amplified of was expression NKCC of NKA the fragment cDNA in solution, changes MCK that K possibility hemolymph the analyze Να To ανδ γενε τηε οφ ναλψσις 3.5 K MgCl of of role in specific solution know japonicus. to M. NaCl conducted mmol/L were experiments 4.3 in Additional to significantly compared concentration higher osmotic significantly was solution tridens MCK NaCl+1.2 mmol/L | + – DISCUSSION 2+ r seta o t uvvl nadto,aayi fincpolso nadwl ae o cultivation for water well inland of profiles ionic of analysis addition, In survival. its for essential are , | | n iaetctos ihrMg either cations, divalent and ffc fabetmnrctosi ewtro h uvvlo uyaiecrabs euryhaline of survival the on seawater in cations minor ambient of Effect σλτο φτεγν νοιγΝα ενςοδινγ γενε τηε οφ Ισολατιον α n Ca and and uui a nlzdi h otpseirglsof gills posterior most the in analyzed was subunit 2 CaCl , .tridens H. .japonicus M. .dehaani C. and .tridens H. 2+ diitaino C nrae h eoyp K hemolymph the increased KCl of Administration + 2 + u o K not but , n C Fg 8H). (Fig. KCl and , ocnrto n liaeyldt h et of death the to led ultimately and concentration 4 n ihrMg either and .latisulcatus M. 2– .maenas C. sntnee o uvvlof survival for needed not is and and ,although ), u o in not but .japonicus M. + .japonicus M. r eurdfor required are , .dehaani C. Fg D.Nrhr ltigrvae htNKA that revealed blotting Northern 4D). (Fig. .tridens H. 2+ .maenas C. Sode l,20;Dvse l,20;PageladFtdr 05 2006; 2005, Fotedar, and Prangnell 2005; al., et Davis 2003; al., et (Saoud 2+ rCa or + .dehaani C. .tridens H. rCa or /Κ .japonicus M. + 2+ Mg , + r etitdwti sure,but estuaries, within restricted are .tridens H. r ieetcmae oohrerhln rsaen previously crustaceans euryhaline other to compared different are a esaetdb min io ain Fg 9). (Fig. cations minor ambient by affected less was and 2+ r seta o both for essential are Ταεασβντυδρηπ-σοι ςονδιτιονς ηψπο-οσμοτις υνδερ συβυνιτ α ΑΤΠασε hwn htbt iaetctos u o K not but cations, divalent both that showing , r eurd(i.2.I diin h eurdabetminor ambient required the addition, In 2). (Fig. required are , 2+ and .japonicus M. .maenas C. n SO and , Fg EG.Mroe,adto fmnrctosincreased cations minor of addition Moreover, 8E–G). (Fig. .tridens H. .tridens H. + .dehaani C. , 2+ /Κ 7 u o in not but , .japonicus M. .tridens H. rMg or 4 + 2– /2῝λ .tridens H. Lvt ta. 06) n 3 while (3) and 2006a), al., et (Lovett + nrae h uvvlrt ferhln prawns euryhaline of rate survival the increases and a uvv vni . mlLNC solution NaCl mmol/L 8.6 in even survive can .tridens H. 2+ n Cl and .tridens H. , .japonicus M. eogt h aespraiy Grapsoidea. superfamily, same the to belong – .tridens H. + .japonicus M. ορνπρε ν ξρσινα- εξπρεσσιον ανδ ςοτρανσπορτερ rbt,btntK not but both, or and + ocnrto scmie administration combined as concentration .tridens H. .japonicus M. and , ntehmlmhinccmoiinin composition ionic hemolymph the on – .japonicus M. , r eurdfrteecast survive to crabs these for required are .japonicus M. ,Oyoia ( Ocypodidae ), .dehaani C. and and .tridens H. .dehaani C. .japonicus M. and , and unlike .japonicus M. α n43mo/ NaCl+1.2 mmol/L 4.3 in uui xrsini the in expression subunit .japonicus M. KCepeso also expression NKCC , + a 1,7% n 77% and 79%, 81%, had .dehaani C. + .vannamei L. and , rmsaae causes seawater from , and rnpr disturbed transport .japonicus M. .mondon P. saseiswhich species a is .japonicus M. n533mmol/L 513.3 in .dehaani. C. lhuhboth although , + r required are , .mondon P. eeused. were Tbe3). (Table Sodet (Saoud reaches + ,and ), The and can H. Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. ihpeiu tde.I diin h xrsino hs ee npseirglsas nrae nboth ambient that in indicating increased enhanced strongly Na The also increased seawater. solution, gills the with NaCl for in conditions posterior account K isosmotic mmol/L could enhanced in under genes 8.6 genes also these genes these in of was of these expression as expression genes of the solution these decrease expression al., NaCl cations of et the minor mmol/L Liu expression 513.3 addition, 2006b; the in In al., that species et hypo-osmotic revealed studies. Lovett under study 2003; previous Towle, increases This with and crabs Lucu euryhaline 2016). 2003; in al., al., tridens NHE et et addition, Henry and In Pan 1999; CAc, 2017). 2015; Flik, Griffith, NKA, 2012; and al., encoding (Lucu et genes conditions Henry 2009; of al., et expression Charmantier the 2008; al., transports et (Freire which membrane cells, apical chloride of Na H membrane Na of from basolateral incorporate force the to driving in required generating are distributed hemolymph, of cells is the regulation chloride NKA to ionic Na in the compared hemolymph. in expressed higher role to NHE osmolality prominent water and a hemolymph play Na CAc, their gills maintain mainly NKA, posterior to the and ions, in gills cells ambient the Chloride in incorporate environment. cells crustaceans chloride through euryhaline actively conditions, hypo-osmotic Under 4.2 solution. NaCl mmol/L hand, other and the turn, On in phylogeny. tridens might, and/or difference H. habitats This their respectively. of condition differ, dehaani microenvironments osmotic also C. the the functioning between and are difference crustaceans, ions the euryhaline minor reflect of ambient species the among which differ in survival itself for ions minor required the ) hssget htteltaiyi h 1. mlLNC ouinwsntbcueo nihbto of inhibition an of because not was solution NaCl mmol/L 513.3 the in lethality the that suggests This Na hemolymph 3). however, study, in this both In increased 1984). al., et Haves and most the in of NHE death and CAc, of NKA, cause encoding genes the the be of expression might increased gills the Thus posterior 2006). al., et (Sowers Na between nadto,we min Hi o,dcesdabetCa et ambient decreased (McDonald low, fish is Ca in pH Na ambient ambient epithelium of when concentration gill addition, the In the Mg Lowering intracellular ambient across of 1998). by permeability Milligan, tightness modulated Na and Ca ion the is McDonald Moreover, controlling increasing crab 1983; of thereby membranes, al., 2017). gills biological and al., in stabilizing junctions, NKA et in tight of Antunes particularly activity 2009; processes, fact, 2005, In logical al., 2017). euryhaline et al., in euryha- (Masui Mg cations et of minor (Apell survival ambient understood. the NKA these for completely of cluding indispensable role not are ecological seawater is and in physiological crustaceans cations the minor However, ambient crustaceans. the that line reported been has It NKA of of rate to expression 3). compared survival the rate the of survival increase increased high limited a respectively, showing NHE, solution, and NaCl mmol/L CA of inhibitors tridens amiloride, and acetazolamide Na of increased an through solution + + + + | ocnrto,icesdNa increased concentration, .middendorffiana D. .japonicus M. ocnrto,irsetv fevrnetlp ncrayfish in pH environmental of irrespective concentration, oeo min io ain ne ssoi odtoswt seawater with conditions isosmotic under cations minor ambient of Role ocnrto ntlotspecies, teleost in concentration rmtectpamt h eoyp n ep h nrclua Na intracellular the keeps and hemolymph the to cytoplasm the from 2 and and n CO and O and + a infiatyhg uvvlrt,ee n533mo/ alslto,i tiigcnrs to contrast striking in solution, NaCl mmol/L 513.3 in even rate, survival high significantly a has .japonicus M. .japonicus M. n K and .japonicas M. .tridens H. Iaa n iia 93,wihwudacutfrterbsns fti pce n513.3 in species this of robustness the for account would which 1993), Kijima, and (Irawan 2 + n h eie H derived the and , ocnrtosi eoyp a as nices ntemraiyrt ncrustaceans in rate mortality the in increase an cause can hemolymph in concentrations n ed oicesdmraiy(ouaadAah,17;MDnl ta. 1980; al., et McDonald 1979; Adachi, and (Jozuka mortality increased to leads and , n533mo/ alslto Fg ) n 2 nuainof incubation (2) and 6), (Fig. solution NaCl mmol/L 513.3 in ne yoomtccniin 86mo/ alslto)(i.5,consistent 5), (Fig. solution) NaCl mmol/L (8.6 conditions hypo-osmotic under and Fg 1) (Fig. + + .japonicus M. pae A nclrd el aaye h omto fH of formation the catalyzes cells chloride in CAc uptake. + /K /K + am gairdneri Salmo .dehaani C. . + ai nhmlmhi 1. mlLNC ouin nimbalance An solution. NaCl mmol/L 513.3 in hemolymph in ratio + ai.Tefloigfidnsspotti iw 1 administration (1) view: this support findings following The ratio. α uprsNa supports uui n A n osntices h Na the increase not does and CAc and subunit oprdt hti 1. mlLNC+C ouin(Fig. solution NaCl+MCK mmol/L 513.3 in that to compared 2+ .tridens H. smr dpiet oe aiiycmae to compared salinity lower to adaptive more is sivle nrglto fmr hn30ezms in- enzymes, 300 than more of regulation in involved is 8 and + paetruhatvto fNElctdi the in located NHE of activation through uptake rza latipes Oryzias + + and 2+ ocnrto,adcnoiatwt decreased with concomitant and concentration, ocnrtosi 1. mlLNC solution NaCl mmol/L 513.3 in concentrations .japonicus M. ocnrto eue lsao hemolymph or plasma reduces concentration hrxdestructor Cherax 2+ .tridens H. ly nipratrl nvrosbio- various in role important an plays n crustaceans, and , and erdi 1. mlLNaCl mmol/L 513.3 in reared + ocnrto eaieylow, relatively concentration .japonicus, M. ElsadMri,1995). Morris, and (Ellis 2+ + eue hemolymph reduces rmenvironmental from .dehaani C. + ahi magna Daphnia /K + nue only induced + n HCO and + ai (Fig. ratio .tridens H. n Cl and n513.3 in 2+ H. H. 3 – – , Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. h rsneo min io ain ne yoomtccniin.Tefc htomtcconcentration osmotic that fact The conditions. hypo-osmotic in under only cations occurs minor ambient damage of unidentified K presence some the the that is affect possibility may Another cations minor ambient that possible tridens is 15.3 H. it is Therefore, MCK 7A). NaCl+1.2 K mmol/L (Fig. 513.3 mortality in no caused they to although 8E) tridens H. (Fig. condition osmotic K accelerate to in cations Interestingly, minor hypo- ambient under by activated cations are minor transport ambient of K presence hemolymph the K in hemolymph both decreases increased in also expression conditions gene osmotic NKCC addition, In K dehaani hemolymph C. increased the NKA decreased for This 9). K (Fig. K conditions decrease hypo-osmotic might under cations K minor NKA ambient transports that NKA showed blotting 2017). Na K incorporates Griffith, to and cells 2012; direction chloride al., opposite et the Na Henry decreased in a 2009; that al., assume et to possible in K is lethality It in the solution. 8F). MCK for (Fig. NaCl+1.2 accounts solution mmol/L NaCl 4.3 mmol/L 4.3 in to decreases compared Na solution hemolymph MCK isosmotic NaCl+1.2 the mmol/L Because to 4.3 in contrast bathed were K striking crabs hemolymph The a K ambient manner. showing condition–dependent that osmotic 7), K appears it Ambient Thus (Fig. 2B). condition. solution) NaCl (Fig. mmol/L conditions K (4.3 especially cations, conditions minor hypo-osmotic ambient that revealed study This that important genes an the also ambient of is cations these identification 4.3 minor comprehensive ambient whether by A regulated know is gills. expression to adapt whose the to addressed and challenge. in transport concentrations be expression ion cation in gene involved should minor are affect studies ambient directly Future of cations euryhaline changes thereby minor for gills, necessary fluctuation. subtle the be sense salinity might in It genes to environmental composition. specific estuaries ionic of hemolymph inhabiting expression and gills crustaceans the the regulate across cations affected transport minor ion be ambient affecting species. also that would these above, suggests gills in described in study also formation those This solution junction from NaCl cell species and mmol/L these integrity 513.3 membrane on the cell roles by that different possible have is cations it minor although that suggest strongly results Ca of K addition of the removal by caused lethality the Mg japonicus of removal by NKA + + .japonicus. M. ocnrto.Hwvr ti nla hte hsdwrglto fNKA of downregulation this whether unclear is it However, concentration. | ocnrto nbtigmda(34mo/) lhuhteaeaehmlmhK hemolymph average the although mmol/L), (13.4 media bathing in concentration + oeo min io ain ne yoomtcconditions hypo-osmotic under cations minor ambient of Role rnpr nclrd el nteglso uyaiecutcas(riee l,20;Charmantier 2008; al., et (Freire crustaceans euryhaline of gills the in cells chloride in transport a usiual yMg by substitutable was smr oeatt lvtdK elevated to tolerant more is s11.5 is nwihabetmnrctosddntaettehmlmhK hemolymph the affect not did cations minor ambient which in + ocnrto.I spsil htuietfidtasotr n/rcanl novdi K in involved channels and/or transporters unidentified that possible is It concentration. .tridens H. + ti oeotyta h vrg eoyp K hemolymph average the that noteworthy is It 2+ ± rnpr rmhmlmht hoiecls osbyrsligi nrae hemolymph increased in resulting possibly cells, chloride to hemolymph from transport .9mo/,sills hntenmnlK nominal the than less still mmol/L, 1.79 nrae hi uvvlrt nlwp odtos(ouaadAah,17) These 1979). Adachi, and (Jozuka conditions pH low in rate survival their increases 2+ + nadto,w hwdta h oeo Ca of role the that showed we addition, In . ocnrto,bcuedcesdepeso fti eesol edt decreased to lead should gene this of expression decreased because concentration, + min io ain nrae h eoyp K hemolymph the increased cations minor ambient , .tridens H. α .japonicus. M. ocnrto stesm nbt ouin,teNa the solutions, both in same the is concentration uui xrsinwsatnae naltreseisi h rsneof presence the in species three all in attenuated was expression subunit + + + nteohrhn,NC sdsrbtdi h pclmmrn of membrane apical the in distributed is NKCC hand, other the On . ocnrto in concentration rmteevrnetit el,cnoiatwt Na with concomitant cells, into environment the from 2+ + + Fg ) utemr,bt hs iaetctoscudntovercome not could cations divalent these both Furthermore, 2). (Fig. a seta niomtccniin,bthrfli hypo-osmotic in harmful but conditions, isosmotic in essential was Fg ) hc sasrkn otatto contrast striking a is which 2), (Fig. and + + ± + ocnrtosadlwNa low and concentrations .japonicus M. rnpr ytmdffrnl in differently system transport a nopst nuneo h uvvlof survival the on influence opposite an has K n KChv enietfida oeue involved molecules as identified been have NKCC and NKA .8mo/,wihi infiatyhge hntenominal the than higher significantly is which mmol/L, 1.28 .japonicus M. 9 + niaigta KCde o otiueto contribute not does NKCC that indicating , ocnrto in concentration + asdlta aaeto damage lethal caused , + ic hsgn a loatnae in attenuated also was gene this since , + + paeudrhp-soi conditions. hypo-osmotic under uptake ocnrto nbtigmda Thus media. bathing in concentration .japonicus M. ocnrto in concentration + + 2+ /K rmhmlmht h cytoplasm the to hemolymph from o uvvlof survival for + .japonicus M. .tridens H. ai nhmlmhcompared hemolymph in ratio rza latipes Oryzias + + ocnrto Fg 8G). (Fig. concentration + α /K u o in not but ocnrto nhypo- in concentration uui eeaccounts gene subunit + .japonicus M. α + ai nhemolymph in ratio .japonicus M. .japonicus M. nrae hnthe when increased and uui expression subunit .tridens H. n Cl and + ocnrto in concentration nwihonly which in , .japonicus. M. .tridens H. – + Northern . /K and + bathed under nan in ratio M. in + Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. ai A odC,RueD,SodI 20)Eet fptsim ansu n g ngot and growth on age and magnesium potassium, of Effects (2005) IP 165-230. Saoud pp Raton, DB, of Boca Rouse Press, survival CRC CE, Evans, Boyd DH by DA, arthropods. Ed Davis aquatic Animals” in and regulation Cells ionic Regulation: and Ionic 165-174. and Osmotic “Osmotic (2009) 32, In D Aquaculture, Towle salinity. M, sapidus: low Charmantier-Daures G, of Callinectes Charmantier juvenile waters of crab sea Responses blue artificial (1983) and the JF Wickins natural of J, to 293-305. Davenport gills 149, T, perfused Biol, Beard DF, Exp by Biochem, Cawthorne J uptake ions. amiloride. ion magnesium and Sodium ouabain by of (1990) Na,K-ATPase Effects DW the Towle of LE, Modulation Burnett (2017) G Schreiber T, 56,1005-1016. Hitzler HJ, Apell alterations crab, salinity-induced 380-397. hermit 327, Low Physiol, thinstripe (2017) the JC of McNamara gills FA, Na Leone ultrastructure, Gar¸con DP, epithelial MN, in Lucena CD, minor Antunes ambient to difference. phylogenetic expression or gene habitats References their of of susceptibility microenvironment and the less in transport of difference ion because in likely involved cations composition, genes ionic of specific survival hemolymph the of ultimately, in and, patterns contrast, composition In expression ionic hemolymph the the affect regulate thereby cations minor Ambient o:aueadcrnccagsi amlmhoye ees xgncnupinadmtblclvl.J levels. metabolic and destruc- consumption Cherax oxygen ’yabby’ levels, Australian oxygen the of haemolymph physiology in the changes on chronic pH extreme and of acute Effects tor: (1995) S Morris BA, Ellis 403-406. 36, Soc, Aquac World water. fresh to adaptation and 5 cations minor fresh ambient other using to studies sensitivity Comparative between species. these as of in such difference lost crabs, phylogenetic water–adaptive cations to and minor due species ambient is such cations to minor for Susceptibility ambient 2000). adaptive Kobayashi, less 1993; be Kijima, would and (Irawan ambient regions upstream Furthermore, more 5). that Na (Fig. indicate hemolymph dehaani strongly solution C. and to results NaCl 7C) compared These cations (Fig. mmol/L G). minor rate ambient 513.3 8C, survival (Fig. in the conditions activated affect hypo-osmotic not not to did compared was (Fig. drastic cations expression less solutions minor but NaCl+MCK CAc enhanced mmol/L was and 513.3 expression and , NKA NaCl addition, mmol/L In 513.3 F). 3C, in bathed crabs of between rate concentrations survival of 1) decrease (Fig. the drastic conditions, isosmotic under First, of response The 4.4 to future. mortality the cause Na in cations than investigated minor other ambient ions which some by of in cations transport minor that of presence the in significantly increased | CONCLUSION | itntrsos of response Distinct . h ieetrsos oabetmnrctosbtenerhln pce ol edet the to due be could species euryhaline between cations minor ambient to response different The saatv ofehwtradhbtt fti pce sntrsrce ihnetaisbtextends but estuaries within restricted not is species this of habitats and water fresh to adaptive is ioeau vannamei Litopenaeus . .dehaani C. .dehaani C. ossetwt hsrsl,teewsn infiatdffrnei eoyp Na hemolymph in difference significant no was there result, this with Consistent hc ie nmr ptemrgo,abetmnrctoshrl ffc the affect hardly cations minor ambient region, upstream more in lives which , oabetmnrctosi ieetfo htof that from different is cations minor ambient to .dehaani C. rohi japonica Eriocheir + /K otlra erdi nadlwslnt elwtr nws lbm.J Alabama. west in waters well salinity low inland in reared post-larvae + lbnru vittatus Clibanarius .tridens H. APs muooaiainadezm iei hrceitc nthe in characteristics kinetic enzyme and immunolocalization -ATPase oabetmnrcations minor ambient to + n K and and and .japonicus. M. .dehaani. C. .japonicus M. + 10 .sinensis E. a ffce nyin only affected was .japonicus M. Aoua ignde.JEpZo clIntegr Ecol A Zool Exp J Diogenidae). (Anomura, lentvl,tedffrneo estvt to sensitivity of difference the Alternatively, oprdto Compared Vrnde,wl lcdt h relationship the elucidate will (Varunidae), .dehaani C. ne yoomtccniin hudbe should conditions hypo-osmotic under u o in not but .dehaani C. .japonicus M. + n533mo/ alwsless was NaCl mmol/L 513.3 in .tridens H. .tridens H. n K and .tridens H. .tridens H. eau monodon Penaeus .tridens H. + ocnrtosunder concentrations sls ucpieto susceptive less is and and h mechanism The . and and Tbe3 suggest 3) (Table .japonicus M. .japonicus. M. .japonicus. M. .japonicus M. + Fabricius n K and + , Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. vJ i ,Wn ,GoB hnP iJ(03 rncitm nlssof e82155. analysis 8, Transcriptome One, (2013) PLoS J osmoregulation. of Li basis P, molecular Chen the into B, insights provides Gao stress Y, salinity Wang to P, response Liu J, Lv 195-214. 135, Physiol, Integr Lucu maenas. nus Lucu 58-65. 211, Bull, oetD,VriM,Bret E anrC,GosiK e J ol W(06)Epeso profiles Expression (2006b) DW Towle JJ, Lee K, Glomski CA, Tanner Na JE, of Burgents and MP, crab composition Verzi seawater green DL, 67-77. of Lovett the 143, effect in Physiol, The farnesoate Integr (2006a) methyl Mol DW A of Borst white Physiol levels in TM, genes hemolymph Ricart anhydrase K, on carbonic Glomski osmolality two CA, of Tanner analysis DL, expression Lovett and 249-284. Cloning pp York, (2015) L New the Pan Press, of Y, University History shrimp Hu Oxford S, (Natural Thiel, Liu “Physiology M M, In Chang, Liu ES excretion. English by and with Ed Osmoregulation Japanese 4)” (2015) Vol (in Crustacea, G 113-130. Charmantier 3, J-H, Eng, their Lignot Civil environment: Ecol riverine condition. the in present crabs and brachyuran abstract) ecosystem of of the ecology properties in and Blood pattern significance II: Distribution teleost. (2000) of S Kobayashi tolerance pH species, the crab on physiology estuarine Eevironmental medaka, (1979) three H the Adachi K, among Jozuka requirements salinity of Difference 39-47. (1993) 3, dehaani Physiol, A Chiromantes Front osmotic/ionic Kijima metals. gill: B, toxic crustacean Irawan the of of bioaccumulation functions and Multiple excretion, (2012) ammonia 1-33. D Weihrauch balance, H, acid-base Onken regulation, C, Lucu RP, Henry in induction anhydrase crab, carbonic green Salinity-mediated euryhaline 243-258. (2003) the DW of Towle gills of D, Weihrauch the species two S, in Gehnrich regulation RP, sodium Henry 1965-1970. on pH 62, low Zool, of J Effect Can (1984) GE . 36, Likens Chem, TC, Toxicol euryhaline Hutchinson Environ M, Havas Mollusca. the V-H(+)- and major insects, of of anhydrase, aquatic physiology crustacea, gills ionoregulation carbonic fish, and 576-600. teleost the osmoregulation of animals: the freshwater across role on by ions perspective transport Possible Toxicological (2017) ion (2005) MB Griffith electrogenic CM Luquet in exchanger MR, crab Urcola Cl(-)/HCO3- 272-304. N, and 151, gills ATPase, crustacean Physiol, Ortiz in Integr G, transport Mol ion A Genovese of Physiol analysis Biochem structure-function Comp A organs. (2008) excretory JC and McNamara H, Onken CA, Freire 395-407. 198. Biol, Exp hsantu granulatus Chasmagnathus + ,Fi 19)Na (1999) G Flik C, ,TweD 20)Na (2003) DW Towle C, ˇ ˇ ,K ioeau vannamei Litopenaeus rza latipes Oryzias + APs uigaueadcrnchp-soi tesi h lecrab blue the in stress hypo-osmotic chronic and acute during -ATPase mJPyil 7,R490-499. 276, Physiol, J Am , eietridens Helice xoe olwp niomn.AntZo p,5,107-113. 52, Jpn, Zool Annot environment. pH low to exposed , + -K + nue yp n aiiysrse.Auclue 4,391-400. 448, Aquaculture, stresses. salinity and pH by induced , opBohmPyilAMlItg hso,12 362-369. 142, Physiol, Integr Mol A Physiol Biochem Comp . + APs n Na and -ATPase +K + APsi il fautccutca opBohmPyilAMol A Physiol Biochem Comp crustacea. aquatic of gills -ATPasein acnsmaenas Carcinus and .japonica H. + /Ca 11 2+ opBohmPyilAMlItg hso,136, Physiol, Integr Mol A Physiol Biochem Comp . Bahua rpia) oouJArcl e,44, Res, Agricult J Tohoku Grapsidae). (Brachyura: xhneatvte nglso hyperregulating of gills in activities exchange acnsmaenas Carcinus otnstrituberculatus Portunus alnce sapidus Callinectes opBiochem Comp . Daphnia Carci- Biol . in Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. rsure ,PrsS,Sbtn E osG,Lqe M(08 euaino o rnpr yp and pH by transport ion of Regulation R1033-1043. (2008) 294, CM Physiol, Luquet GG, Goss SE, [HCO Sabatini SK, Parks M, Tresguerres green of gills 115-125. in 6, expression Proteomics, gene in changes Microarray-detected ( (2011) crabs NB Terwilliger RP, 1003-1014. Henry 200, WS DW, Wu Towle Biol, DW, Exp Shearer crab J MK, Jordan euryhaline in distribution. A, the tissue expression Amstutz and in MJ, gene Rose antiporter aquaporin-3 JJ, Sodium/proton Magnani up-regulates D, (1997) stress Heidysch ME, Osmotic 82-88. Rushton 1522, (2001) DW, Towle Acta, S Biophys Inoue Biochim M, 176-180. keratinocytes. Hara 145, human Physiol, cultured Y, Integr Ota Mol Y, A Physiol Sugiyama Biochem Comp concen- flux. cation and potassium osmolality to Hemolymph (2006) JR Tomasso in CL, Browdy trations M, Grosell SP, Young the AD, Sowers in isoforms anhydrase 186-93. carbonic 3, crab, two Proteomics, of green Genomics induction euryhaline and the for expression of Differential waters (2008) gills well RP inland Henry of L, Serrano studies Suitability 373-383. (2003) 217, DB Aquaculture, Rouse culture. DA, Davis IP, Saoud magnesium and shrimp, 461-469. potassium white 262, aqueous Pacific of Aquaculture, the levels varying of waters. of respiration Effects and (2007) growth, RP survival, Henry on IP, Saoud 449-457. DA, Davis 145, LA, on Physiol, Roy water. Integr change saline Mol inland salinity potassium-fortified A and sudden Physiol 19-34. water Biochem saline 17, of inland Comp Aquac, in Effect Appl condition (2006) and J ionoregulation R 1896. moregulation, Kishinouye, Fotedar Growth latisulcatus the DI, on Penaeus Water Shrimp, Prangnell Saline King Inland Western in Concentration the Potassium of of Survival Effect anhydrase and The carbonic (2005) two R Fotedar of DI, analysis Prangnell sequence and 1606-1613. cloning 45, Molecular Sci, (2016) 347-357. Aquat S 576, Fish Liu J crab Y, swimming Can Hu the M, aluminum. in trout, Liu of D, brook absence Hu and the L, presence Pan in the transport in Sodium exposure pH (1988). low prolonged CL on pH Milligan low DG, and McDonald calcium environmental the of of interaction 141-155. trout, responses The 102, rainbow physiological (1983) the of the PRH physiology Wilkes, on the RL, calcium Walker of DG, influence McDonald The (1980) CM Wood trout, 141-148. H, rainbow gill 153, Hobe in Physiol, DG, Integr activity McDonald K+-ATPase Mol A Na+, Physiol (2009) Biochem FA Comp Leone RP, excretion. crab, Furriel blue JC, the McNamara from microsomes FL, Mantelatto 2521-2535. DC, 37, Masui Biol, Leone Cell CF, Fontes Biochem HM, crab J Scofano blue Int H, the Barrabin sites. from (Na+,K+)-ATPase JC, microsomal McNamara Gill FL, Mantelatto (2005) FA EC, Silva RP, Furriel DC, Masui 3 - acnsmaenas Carcinus nioae il ftecrab the of gills isolated in ] ioeau vannamei Litopenaeus am gairdneri Salmo otnstrituberculatus Portunus pndlto fevrnetlslnt.Cm ice hso atDGenomics D Part Physiol Biochem Comp salinity. environmental of dilution upon ) alnce danae Callinectes olwevrnetlp.JEpBo,8,109-131. 88, Biol, Exp J pH. environmental low to , acnsmaenas Carcinus uigepsr oatfiilsasl ramxdinslto:relationship solution: mixed-ion a or salt sea artificial to exposure during Neohelice am gairdneri Salmo ( n t xrsini epnet aiiyadp tes Gene, stress. pH and salinity to response in expression its and Chasmagnathus clmtdt o aiiy oe esetvso ammonia on perspectives novel salinity: low to acclimated , nrsos olwslnt.Cm ice hso D Physiol Biochem Comp salinity. low to response in , I rnha ooeuaoymcaim.JEpBiol, Exp J mechanisms. ionoregulatory Branchial II. : 12 acnsmaenas Carcinus ) granulata ioeau vannamei Litopenaeus alnce danae Callinectes eau latisulcatus Penaeus mJPyilRglItg Comp Integr Regul Physiol J Am . avlnsfontinalis Salvelinus oeua lnn,expression cloning, molecular : neatosa cationic at Interactions : ioeau vannamei Litopenaeus erdi o salinity low in reared , ihnueos- Kishinouye ffcsof effects : Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. n ftefu diinlsls ausrpeetmaso he xeiet,aderrbr represent bars error and experiments, both three in media of bathing means of < compositions represent different Values among tridens observed any salts. H. were minus differences additional solution Significant NaCl+MMCK errors. four mmol/L standard the 513.3 and of solution, NaCl+MMCK one mmol/L 513.3 same solution, NaCl the within species 2 three FIGURE among values bars different error ( significantly concentration and indicate NaCl seawater. letters experiments, and Different (ANOVA, three solutions test). concentrations NaCl species Williams’ NaCl of ambient three mmol/L different means among 513.3 all observed represent and were in Values differences 256.7, Significant logarithmically. 128.3, errors. standard scaled 64.2, represent 32.1, are 16.0, axes 8.0, Horizontal in determined was rate dehaani IUE1 FIGURE LEGENDS FIGURE are-controlled-by-ambient-minor-cations-according-to-osmotic-condition-dependent-manner composition-gene-expressions-in-the-gills-and-thus-the-survival-of-euryhaline-crabs- image3.emf file Hosted cnl ieetvle ihnec pce ( species each within (n=3) values means different represent ficantly Values solutions. MCK NaCl+1.2 3. TABLE are-controlled-by-ambient-minor-cations-according-to-osmotic-condition-dependent-manner composition-gene-expressions-in-the-gills-and-thus-the-survival-of-euryhaline-crabs- image2.emf file ( Hosted species each within values different significantly in media bathing different (ANOVA, (n=3) among means observed represent Values were differences solutions. NaCl mmol/L 513.3 and NaCl+MCK, 2. TABLE are-controlled-by-ambient-minor-cations-according-to-osmotic-condition-dependent-manner is composition-gene-expressions-in-the-gills-and-thus-the-survival-of-euryhaline-crabs- NaCl mmol/L 513.3 image1.emf mOsm/kg. determined. by not given file means are D. Hosted N. concentration budget NaCl. osmotic energy 3% measured and to growth and corresponds on mmol/L seawater by in shown ratio Na/K of 1. Effects TABLE (2004) G Huang F, juvenile Wang of S, definitive and Dong primitive C, to Zhu mesoderm dorsal salamander and the ventral of in Contribution erythropoiesis (2001) M Wakahara M, globin Yamaguchi of pattern expression salamander unexpected the and in Erythropoiesis (2000) genes M Wakahara H, Takahashi M, Yamaguchi .5 ue’ et.(,D uvvlrtso (C) of rates Survival D) (C, test). Tukey’s 0.05; tvrosNC ocnrtosadi aua ewtr4 fe ntaino ern.Tesurvival The rearing. of initiation after h 48 seawater natural in and concentrations NaCl various at P ioeau vannamei. Litopenaeus and soi ocnrtos(Omk)o eoyp ahdi . mlLNC n . mmol/L 4.3 and NaCl mmol/L 4.3 in bathed hemolymph of (mOsm/kg) concentrations Osmotic < soi ocnrtos(Omk)o eoyp ahdi . mlLNC,533mmol/L 513.3 NaCl, mmol/L 8.6 in bathed hemolymph of (mOsm/kg) concentrations Osmotic A )Sria ae f(A) of rates Survival B) (A, h opsto fbtigmdaue nti td.Cnetain fdsovdslsare salts dissolved of Concentrations study. this in used media bathing of composition The uvvlrtso (A) of rates Survival .5 u o bevdin observed not but 0.05) .japonicus M. vial at available vial at available at available P < yoisretardatus Hynobius .5 ue’ et.(W,seawater. (SW), test). Tukey’s 0.05; (ANOVA, P yoisretardatus Hynobius < qautr,24 485-496. 234, Aquaculture, https://authorea.com/users/351272/articles/475958-hemolymph- https://authorea.com/users/351272/articles/475958-hemolymph- https://authorea.com/users/351272/articles/475958-hemolymph- eietridens Helice .5.*infiatydffrn auscmae osaae ( seawater to compared values different *Significantly 0.05). P hrmne dehaani Chiromantes eietridens Helice < e ee vl 1,180-189. 210, Evol, Genes Dev . .5.Dffrn etr niaesgicnl ieetvle ( values different significantly indicate letters Different 0.05). P < P .5 Student’s 0.05; .tridens H. (B) , < 13 e il 3,204-216. 230, Biol, Dev . .5 Student’s 0.05; n (B) and arptamsjaponicus Macrophthalmus ± eietridens Helice tnaderr.Dffrn etr niaesigni- indicate letters Different errors. standard n (D) and (ANOVA, arptamsjaponicus Macrophthalmus t -test). t .japonicus M. ts ihBnern correction). Bonferroni with -test P > and .5.Dffrn etr indicate letters Different 0.05). ± arptamsjaponicus Macrophthalmus tnaderr.Significant errors. standard n (C) and , n533mo/ NaCl, mmol/L 513.3 in n533mmol/L 513.3 in Chiromantes P < 0.05; P , Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. IUE6 FIGURE 34mo/ C;(. C,3. mlLMgCl mmol/L indicate 30.5 letters MC), Different (1.2 KCl; Values errors. mmol/L experiments. 13.4 standard ( of represent ( initiation point point after bars beginning time h error the each 48 and in and values 39, experiments, different 24, significantly three 15, of solutions *Signifi- means (squares) ( K represent errors. NaCl+1.2 KCl. standard mmol/L without represent 4.3 solution bars and NaCl error mmol/L of and 4.3 rate in experiments, Survival that four from of values means different cantly represent Values rearing. (ANOVA,of media bathing errors. ( standard of values icus represent different compositions bars significantly different error indicate and letters among Different experiments, observed three were of means differences represent Significant MgCl Values mmol/L K). (1.2 30.5 KCl with mmol/L supplemented solutions of NaCl rate mmol/L Survival (D) compositions correction). different among observed were > in differences media Significant bathing errors. of standard represent bars (for error four and of means represent Values three haani of means represent 7 Values FIGURE hydrochloride rearing. amiloride of or initiation B), ( after Na and values h different (A 24 significantly CA indicate D), letters of and (C inhibitor NHE an (for of acetazolamide, inhibitor without an or hydrate, 513.3 with in solution expression NaCl the as band each Na in (NKA), shown was 1.0. Na level was is (NHE), expression expression solution in gene relative CAc NaCl+MCK All The of mmol/L solutions. expression assay. NaCl the protection mmol/L except RNase 8.6 blotting, and Northern NaCl+MCK, using mmol/L detected 513.3 NaCl, mmol/L 513.3 dens MgCl 4 mmol/L FIGURE 25.2 (MCK), bars correction). Bonferroni error with and -test bar), each in above Na media dehaani shown bathing both of crabs of compositions analyzed differences different Significant of errors. (number standard means represent represent Values solution. NaCl K (D–F) japonicus 3 FIGURE MgCl mmol/L both were 25.2 in differences MgSO4, media Significant mmol/L errors. bathing standard of represent compositions P bars MgCl different error with and among supplemented experiments, solutions observed three NaCl+K of mmol/L means 513.3 represent and NaCl+K, mmol/L 513.3 eietridens Helice 5 FIGURE crab shore the of those with .5.Dffrn etr niaesgicnl ieetvle ( values different significantly indicate letters Different 0.05). < + .tridens H. /H n43mo/ alslto upeetdwt ,27 .,ad1. mlLKl2 fe initiation after h 24 KCl mmol/L 10.7 and 5.4, 2.7, 0, with supplemented solution NaCl mmol/L 4.3 in , .5.Dffrn etr niaesgicnl ieetvle ( values different significantly indicate letters Different 0.05). erdi . mlLNC,43mo/ al12MK n 71mo/ al12MKsolutions. MCK NaCl+1.2 mmol/L 17.1 and MCK, NaCl+1.2 mmol/L 4.3 NaCl, mmol/L 4.3 in reared arptamsjaponicus Macrophthalmus + + exchanger. (ANOVA, n C F) (C, and , ocnrtosi rb edi . mlLNC,533mo/ alMK n 1. mmol/L 513.3 and NaCl+MCK, mmol/L 513.3 NaCl, mmol/L 8.6 in held crabs in concentrations + /H mn cdsqecso A NKA (A) of sequences acid Amino uvvlrtso A C) (A, of rates Survival uvvlrtso (A) of rates Survival xrsino ee noigNKA encoding genes of Expression eoyp Na Hemolymph , rfu (for four or ) + arptamsjaponicus Macrophthalmus .japonicus M. xhne;(C) 55mo/ MgCl mmol/L 25.5 (MCK), exchanger; P .japonicus M. > P hrmne dehaani Chiromantes < .5.Dffrn etr niaesgicnl ieetvle ( values different significantly indicate letters Different 0.05). .5 ilas et.(. C) 05mo/ MgCl mmol/L 30.5 MCK), (1.2 test). Williams’ 0.05; .japonicus M. .maenas. C. + erdi . mlLNC crls,43mo/ al12MK(triangles), MCK NaCl+1.2 mmol/L 4.3 (circles), NaCl mmol/L 4.3 in reared n K and .japonicus M. and , eietridens Helice (ANOVA, .tridens H. 2 eietridens Helice . mlLCaCl mmol/L 9.9 , + hrmne dehaani Chiromantes ocnrtosi A D) (A, in concentrations xeiet,aderrbr ersn tnaderr.Different errors. standard represent bars error and experiments, ) and , .tridens H. P fe ntaino nuain eoyp AC Na (A–C) Hemolymph incubation. of initiation after h 6 P < n43mo/ al . mlLNC+. C,ad4.3 and MCK, NaCl+1.2 mmol/L 4.3 NaCl, mmol/L 4.3 in and P < α 2 (B) , hrmne dehaani Chiromantes .5 tdn’ -et.(A,croi nyrs;(NHE), anhydrase; carbonic (CA), t-test). Student’s 0.05; uui,()Cc C H,ad()NC of NKCC (D) and NHE, (C) CAc, (B) subunit, α + < .5,btntin not but 0.05), n 17mo/ CaCl mmol/L 11.7 and n B D) (B, and /K .japonicas M. uui,Cc n H ntems otro il of gills posterior most the in NHE and CAc, subunit, 14 .5 ue’ et.*infiatydffrn ausfrom values different *Significantly test). Tukey’s 0.05; arptamsjaponicus Macrophthalmus P and + < 2 2 . mlLCaCl mmol/L 9.9 , Tae Cc,ctpamccroi anhydrase; carbonic cytoplasmic (CAc), ATPase; .5 ue’ et.()Sria aeof rate Survival (E) test). Tukey’s 0.05; n 07mo/ C;() 07mo/ KCl. mmol/L 10.7 (K), KCl; mmol/L 10.7 and , .japonicus M. + eue rmcN rget n alignments and fragments cDNA from deduced 2 n K and . mlLCaCl mmol/L 9.9 , arptamsjaponicus Macrophthalmus 2 P n 17mo/ CaCl mmol/L 11.7 and rtre(for three or ) .japonicus M. P eietridens Helice < .tridens H. .tridens H. + .5 tdn’ -etwt Bonferroni with t-test Student’s 0.05; < ocnrtoswr bevdamong observed were concentrations fe ntaino nuainin incubation of initiation after h 6 .5 ue’ et.(MK,27.4 (MMCK), test). Tukey’s 0.05; (ANOVA, 2 12K,1. mlLKCl. mmol/L 13.4 K), (1.2 ; 2 P n 07mo/ KCl. mmol/L 10.7 and , and and < 2 hc a eetdusing detected was which , 17mo/ CaCl mmol/L 11.7 , .dehaani C. n (C) and , B E) (B, , 2 .5 ilas et.(F) test). Williams’ 0.05; n 07mo/ KCl. mmol/L 10.7 and , .dehaani C. P .japonicus M. < P .5,btntin not but 0.05), < 2 2 12M)o 13.4 or MC) (1.2 rCaCl or n533mmol/L 513.3 in .5 Student’s 0.05; hrmne de- Chiromantes Macrophthalmus experiments, ) (ANOVA, P eietri- Helice (ANOVA, .japon- M. 2 < Values . 2 + 0.05). and , and C. P t Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. ng n r .Ihd o nls agaerve.Ti okwsspotdi atb h rn from grant the by part in also supported thank was College. to work Suzuka like This NIT, would of review. I and president language supports, advice. and English technical constructive (18-1-66), for for and Foundation College Ishida Okasan-Kato manuscript Suzuka K. the the NIT, of in Dr. crabs reading and of critical Enago group for research Wakahara of M. members Dr. thank to like would I ACKNOWLEDEMENTS (lead). Editing & Writing-Review (lead), Soga: Preparation Draft Writing-Original (lead), Investigation Yamaguchi: Masahiro interests. of conflicts CONTRIBUTIONS no AUTHOUR are there that declare authors The INTERESTS OF CONFLICT 10.6084/m9.figshare.12788132). (DOI: figshare on available dehaani LC214855, C. numbers: japonicus (accession GenBank M. in registered were of study this in of cDNAs NKA isolated of sequences The STATEMENT ACCESSIBILITY DATA MgCl ltig h eaieepeso ee a hw nec ada h xrsini . mlLNaCl mmol/L 4.3 in expression the as Northern band using each detected in were shown expressions Na was (NKA), All level 1.0. expression solutions. is relative MCK solution NaCl+1.2 The mmol/L 4.3 blotting. and NaCl mmol/L tridens 9 MgCl FIGURE mmol/L 30.5 MCK), MgCl (1.2 mmol/L KCl. H). 25.2 and (MCK), (ANOVA, KCl; D H) mmol/L for of (D, correction (number were in Bonferroni differences means ( Significant media with represent values bathing errors. Values different and standard of significantly NaCl+K, represent incubation. mmol/L indicate compositions bars 4.3 of error different NaCl, and initiation among mmol/L bar), after observed each 4.3 above h H) shown (D, 6 crabs in solutions analyzed or NaCl+MCK solutions MCK mmol/L NaCl+1.2 4.3 mmol/L 4.3 and NaCl H) IUE8 FIGURE .tridens H. arptamsjaponicus Macrophthalmus 2 17mo/ CaCl mmol/L 11.7 , netgto spotn) rtn-eiw&Eiig(supporting). Editing & Writing-Review (supporting), Investigation , .tridens H. arptamsjaponicus Macrophthalmus C722 H of NHE LC572292, ; C186 K of NKA LC214856, ; xrsino ee noigNKA encoding genes of Expression eoyp AD Na (A–D) Hemolymph C729 KCof NKCC LC572289, ; C794 A of CAc LC375964, ; 2 ocpulzto la) omlAayi la) udn custo (lead), Acquisition Funding (lead), Analysis Formal (lead), Conceptualization + n 34mo/ KCl. mmol/L 13.4 and , /K n C G) (C, and , + Tae NC) Na (NKCC), ATPase; .dehaani C. .japonicus M. + and , .japonicus M. P n EH K (E–H) and < .tridens H. 2 . mlLCaCl mmol/L 9.9 , .5 Student’s 0.05; hrmne dehaani Chiromantes hrmne dehaani Chiromantes C723 KCof NKCC LC572293, ; α C795 A of CAc LC375965, ; uui n KCi h otpseirglsof gills posterior most the in NKCC and subunit C720 K of NKA LC572290, ; 15 + C726 H of NHE LC572286, ; + ocnrtosi A E) (A, in concentrations /K t + /2Cl ts o – n –,adStudent’s and E–G, and A–C for -test 2 n 07mo/ C;() 07mmol/L 10.7 (K), KCl; mmol/L 10.7 and , – fe ntaino nuaini 4.3 in incubation of initiation after h 6 ornpre;(. C) 05mmol/L 30.5 MCK), (1.2 cotransporter; nuae n(–,EG . mmol/L 4.3 E–G) (A–C, in incubated .dehaani C. .japonicus M. 2 .dehaani C. .tridens H. 17mo/ CaCl mmol/L 11.7 , P eietridens Helice < .Rwdt r publicly are data Raw ). .5.Dffrn letters Different 0.05). C728 H of NHE LC572288, ; C721 A of CAc LC572291, ; C727 NKCC LC572287, ; 2 B ,F, D, (B, , n 13.4 and , Kouichi t Helice -test Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. 16 Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. 17 Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. 18 Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. 19 Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. 20 Posted on Authorea 17 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159769275.58126994 — This a preprint and has not been peer reviewed. Data may be preliminary. 21