The Journl of Experimentl iology 8, 469-478 Pulished y The ompny of iologists 5 doi:.4/je.87 469 Ionoregultory chnges in different popultions of mturing sockeye slmon Oncorhynchus nerk during ocen nd river migrtion J. M. Shrimpton, *, D.. Ptterson, J. G. Richrds, S. J. ooke 4, P. M. Schulte, S. G. Hinch 4 nd. P. Frrell,5 Ecosystem Science nd Mngement Progrm, University of Northern ritish olumi, Prince George,, VN 4Z9, nd, Fisheries nd Ocens nd, Science rnch, Pcific Region, oopertive Resource Mngement Institute, School of Resource nd Environmentl Mngement, Simon Frser University, urny,, V5 S6, nd, Deprtment of Zoology, 4 entre for pplied onservtion Reserch, Deprtment of Forest Sciences nd 5 Fculty of griculturl Sciences, University of ritish olumi, Vncouver,, V6T Z4, nd We present the first dt on chnges in ionoregultory physiology of mturing, migrtory dult sockeye slmon Oncorhynchus nerk. Frser River sockeye were intercepted in the ocen s fr wy s the Queen hrlotte Islnds (~85 km from the Frser River) nd during freshwter migrtion to the spwning grounds; for some popultions this ws distnce of over 7 km. Sockeye migrting in sewter towrd the mouth of the Frser River nd upriver to spwning grounds showed decline in gill N +,K + -TPse ctivity. s result, gill N +,K + -TPse ctivity of fish rriving t the spwning grounds ws significntly lower thn vlues otined from fish cptured efore entry into freshwter. Plsm osmollity nd chloride levels lso showed significnt decreses from sewter vlues during the freshwter migrtion to spwning res. Movement from sewter to *uthor for correspondence (e-mil: shrimptm@unc.c) ccepted 5 Septemer 5 Summry freshwter incresed mrn expression of freshwterspecific N +,K + -TPse isoform ( ) while hving no effect on the sewter-specific isoform ( ). In ddition, gill N +,K + -TPse ctivity generlly incresed in ctive spwners compred with unspwned fish on the spwning grounds nd this ws ssocited with mrked increse in N +,K + -TPse mrn. Increses in gill N +,K + - TPse ctivities oserved in spwners suggests tht the fish my e ttempting to compenste for the osmotic perturtion ssocited with the decline in plsm chloride concentrtion nd osmollity. Key words: sockeye slmon, Oncorhynchus nerk, ionoregultion, migrtion, slinity, spwning. Introduction The migrtions of ndromous fish etween freshwter nd sewter represent remrkle trnsformtions in the mechnisms tht control ionoregultion. Fish ctively tke up ions in freshwter nd must excrete excess ions in sewter (Hor, 988). dditionlly, migrtion is n energeticlly demnding nd physiologiclly chllenging phse of life history (McKeown, 984; Hinch et l., 5) nd the trnsition etween freshwter nd sewter is often time of incresed mortlity (Fisher nd Percy, 988; S. J. ooke, S. G. Hinch, G. T. rossin, D.. Ptterson, K. K. English, J. M. Shrimpton, G. Vn Der Krk nd. P. Frrell, mnuscript sumitted for puliction). While we know much regrding the morphologicl, ehviourl nd physiologicl chnges tht occur during the downstrem migrtion of juvenile slmon smolts s they prepre for entry into sewter (Mcormick nd Sunders, 987), comprtively little is known regrding migrtion of dults from sewter ck to the freshwter spwning grounds. Increses in gill N +,K + -TPse ctivity occur in fish in freshwter nd re preprtory for the incresed ionic concentrtion of sewter s juvenile slmon migrte to the mrine environment (Mcormick nd Sunders, 987). In fct, the elevtion in enzyme ctivity hs een proposed s predictive of the entry dte into sewter (Nielsen et l., 4). Recently, Richrds et l. () demonstrted differentil mrn expression of N +,K + -TPse -suunit isoforms in response to sewter trnsfer nd speculted tht isoform switching etween freshwter-responsive N +,K + -TPse isoform ( ) nd sewter-responsive N +,K + -TPse isoform ( ) is importnt for ionoregultory competence during slinity trnsfer. Isoform switching potentilly offers highly sensitive indictor of osmoregultory sttus. The elevted gill N +,K + -TPse ctivity limits ionic perturtion in plsm of fish following movement into sewter. Fish trnsferred to sewter tht re not prepred for the incresed slinity showed lrge perturtions in plsm ions, decresed survivl (Shrimpton et l., 994) nd reduced swimming performnce (runer et l., 99). While similr preprtory physiologicl chnges re expected to occur in THE JOURNL OF EXPERIMENTL IOLOGY
47 J. M. Shrimpton nd others preprtion for mturing dult slmon returning to freshwter, their nture nd their consequences on plsm osmollity nd ion levels re unknown. Whtever these chnges re, they re unlikely to exctly mirror the prr smolt trnsformtion. Juvenile slmon tht re prevented from migrting into the ocen survive in freshwter (Shrimpton et l., ); in contrst, there is evidence tht mturing dult slmon cnnot remin in sewter, ut must move into freshwter. Hirno et l. (978) showed tht mturing chum slmon O. ket did not survive trnsfer from freshwter into full strength sewter, nd the physiologicl chnges in ionoregultory ility tht ccompny mturtion pper to e irreversile. In the present study, we ssessed gill N +,K + -TPse ctivity, plsm osmollity nd chloride concentrtion, nd the mrn levels of two isoforms of the N +,K + -TPse suunit, to evlute physiologicl nd moleculr chnges tht occur in nturlly migrting slmon. To exmine the dynmics of ionregultion during migrtion, we intercepted wild dult sockeye slmon homing to the Frser River wtershed, ritish olumi, nd nd collected gill tissue nd plsm smples from these mturing fish migrting in sewter nd in-river, nd on the spwning grounds. We cptured fish from s fr wy s ~85 km to ~5 km from the mouth of the Frser River to exmine physiologicl chnges tht occur during migrtion in sewter nd whether physiologicl chnges necessry for ionoregultion in freshwter were completed efore moving into the river. Sockeye migrting upriver nd on the spwning grounds were lso smpled to ssess the effect of migrtion nd finl mturtion on ionoregultion. For this nlysis, we focussed our smpling efforts on four geogrphiclly isolted spwning ggregtes, ech comprising multiple spwning popultions of sockeye slmon: Hrrison, Lte Shuswp, hilko nd Quesnel. Most popultions of sockeye tht spwn in the Frser River wtershed (hilko nd Quesnel) enter the river within week of rriving t the mouth of the estury. In contrst, lte-run sockeye slmon (Hrrison nd Lte Shuswp), nmed for their lte summer rrivl, normlly congregte in the Frser River estury for 6 weeks prior to entering freshwter nd inititing their upriver migrtion to ntl spwning grounds (ooke et l., 4). The stocks we selected lso vried in their migrtion distnces in the freshwter of the Frser River wtershed, rnging from less thn km (Hrrison) to over 7 km (Quesnel). Mterils nd methods Fish cpture nd smpling The four stocks of sockeye slmon Oncorhynchus nerk Wlum tht we trgeted were intercepted in the costl wters of ritish olumi nd t vrious points during their upstrem migrtion through the Frser River system (Fig. ). Migrting sockeye were smpled while still in sewter off the Queen hrlotte Islnds (QI, ~85 km; the mouth of the Frser River is km), off Port Renfrew on Vncouver Islnd (PR, ~ km), in Johnstone Strit (JS, ~ km), nd in Georgi Strit (GS, ~5 km). Slinities off the north cost of ritish olumi re greter thn. In Johnstone Strit, slinities show sesonl fluctution due to river run-off in the spring, ut generlly exceed 7 in ugust when the sockeye migrte through this region. For the mjority of Georgi Strit the se surfce slinity is lso greter thn 7 during ugust nd Septemer when sockeye were migrting through this re. In Georgi Strit close to the mouth of the Frser River, however, surfce wter slinities re lower nd fluctute with the tide. In this re sockeye seem to prefer to km N Study re nd Frser River wtershed Fig.. Mp of Frser River wtershed nd costl ritish olumi showing loctions where fish were intercepted during migrtion. Smples were collected off the Queen hrlotte Islnds (QI), Port Renfrew (PR), Johnstone Strit (JS), nd Georgi Strit (GS) in sewter. Smple loctions within the Frser River were ottonwood (), Whonnock (W), Hells Gte (HG), ner hurn reek (c), nd on the Thompson River (T) ner shcroft. Spwning sockeye from Hrrison stock (H) were smpled from Wever reek; Lte Shuswp stock (Sh) from the dms River; hilko stock (h) from the hilko River; nd Quesnel stock (Q) from the Horsefly River. QI Pcific Ocen PR JS GS h H Q c T HG W Sh ritish olumi US THE JOURNL OF EXPERIMENTL IOLOGY
Ionoregultory chnges in mturing slmon 47 e elow the hlocline s they migrte nd were cught with deeper fishing ger. During their upstrem migrtion, sockeye were cught in the lower Frser River t ottonwood (, km), which is esturine nd t Whonnock (W, 5 km), which is eyond the sltwter intrusion. Lower Frser sockeye of the Hrrison stock were smpled on the spwning grounds in Wever reek (H, 7 km). Mid-Frser sockeye of the Lte Shuswp stock were smpled in the Thompson River ner shcroft (T, 7 km), nd on the spwning grounds in the dms River (Sh, 484 km). Two stocks of Upper Frser sockeye were smpled, hilko nd Quesnel. hilko stock ws smpled smpled t Hells Gte (HG, km) nd on the spwning grounds in the hilko River (h, 56 km). Quesnel stock ws smpled t Hells Gte, ner hurn reek on the Frser River (c, 44 km) nd on the spwning grounds in the Horsefly River (Q, 748 km). Fish were collected using vriety of methods specific to ech loction. The following methods were employed: trolling off the west cost of the Queen hrlotte Islnds nd in Georgi Strit; purse seine off the cost of Vncouver Islnd ner Port Renfrew nd in Johnstone Strit; gill net ner the mouth of the Frser River t ottonwood nd t Whonnock; long dip nets t Hells Gte; ech seines t shcroft nd hurn reek; ech seines to cpture sockeye rriving t the spwning grounds; nd smll dip nets to cpture ctively spwning fish. ll groups were smpled on the spwning grounds t lest twice in ech yer nd fish were ssessed for stge of mturtion. There is considerle knowledge of run timing for ech of the stocks smpled; therefore, smpling ws timed to intercept the stocks of interest. Stock identifiction for ech fish smpled ws determined y vrition in genetic mrkers, s outlined y echm et l. (995). Genetic determintion, however, ment tht stock identifiction ws determined posteriori. s result, smple size vried considerly for ech stock exmined, mong loctions, nd lso etween yers. The fish cught during migrtion were grouped sed on spwning ggregte nd represent multiple spwning popultions within wtershed. In contrst, the fish smpled on the spwning grounds were from single spwning popultion in tht ggregte. In ll cses, however, the single spwning popultions chosen were the lrgest spwning popultion for tht ggregte in oth smpling yers. Tissue smpling In most cses these fish were smpled s prt of norml test fishery or stock ssessment opertions nd specific tretment of the fish ws dependnt on the ger type. Every effort ws mde to minimize the time from cpture to tissue smpling. In those cses involving seine nets, fish remined in the ocen constrined y the seine net until they were individully dipnetted out for smpling. Fish cught using troll line or dip net were lnded nd smpled within minutes. Where possile the sok time for gill nets ws reduced to less thn 5 min nd only fish tht were still vigorous t cpture were smpled. Fish were killed with single low to the hed nd immeditely smpled for lood nd gill tissue. ml of lood ws collected from the cudl vsculture using vcutiner syringe (.5, guge) for ssessing plsm chemistry, nd the tips of 6 8 filments from the first gill rch were removed for nlysis of N +,K + -TPse ctivity. Gill tissue nd centrifuged plsm smples were stored on dry ice for severl dys efore trnsfer to 8 freezer where they were held until nlysis. Fork length (FL, cm) ws mesured nd n dipose fin clip ws removed for DN stock identifiction. nlysis of gill N +,K + -TPse ctivity Gill N +,K + -TPse ctivity ws mesured ccording to the microssy protocol of Mcormick (99). Gill filments were homogenized in SEI uffer (5 mmol l sucrose, mmol l N EDT, 5 mmol l imidzole, ph 7.) contining.% sodium deoxycholte. Following centrifugtion ( g for.5 min), the superntnt ws used to determine N +,K + - TPse ctivity y linking TP hydrolysis to the oxidtion of nicotinmide denine dinucleotide (NDH), mesured t 4 nm for min t 5, in the presence nd sence of.5 mmol l ouin. Protein content in the gill homogente ws mesured using icinchoninic cid () protein ssy (Pierce, Rockford, IL, US). Specific ctivities were expressed s mol DP mg protein h. Determintion of plsm osmollity nd chloride Plsm smples were thwed, vortexed nd centrifuged for 5 min immeditely efore nlysis. Osmollity ws mesured in duplicte on l smples using model 55 Wescor Vpour Pressure meter (Logn, UT, US). Plsm chloride concentrtions were mesured in duplicte using model 445 Hke uchler digitl chloridometer (Knss ity, MO, US); vlues were checked ginst chloride stndrd ( mmol l Nl) efore nd fter pproximtely every duplictes. Gill N +,K + -TPse nd isoform expression n nlysis of N +,K + -TPse -suunit isoforms expression, s descried y Richrds et l. (), ws performed on suset of gill smples from Upper Frser stocks cught (i) in the Georgi Strit, (ii) t Whonnock, (iii) during migrtion t Hell s Gte (river km), (iv) while the fish were holding on the spwning grounds efore spwning (green), nd (v) fully mture fish (spwners). Fish cptured on the spwning grounds were smpled from the Horsefly River, Quesnel Wtershed. riefly, totl RN ws extrcted from gill tissue using TriPure Isoltion Regent (Roche Dignostics, Montrel, Q, nd) nd quntified spectrophotometriclly. First-strnd cdn ws synthesized from 5 g of totl RN using oligo(dt 5 ) primer nd Revertid TM H-Minus M-MuLV reverse trnscripse (MI Ferments Inc., urlington, ON, nd). The expression of N +,K + -TPse - nd - isoforms ws estimted using quntittive rel-time PR (qrt-pr; I Prism 7 sequence nlysis system; pplied iosystems Inc., Foster ity,, US). PR THE JOURNL OF EXPERIMENTL IOLOGY
47 J. M. Shrimpton nd others rections contined l of cdn, 4 pmoles of ech isoformspecific primer nd Universl SYR green mster mix (pplied iosystems Inc.) in totl volume of l. Primers were designed from trout N +,K + -TPse (Gennk ccession No. Y99), N +,K + -TPse (ccession No. Y99) nd elongtion fctor (ccession No. F498). Primer sequences were s follows: N +,K + - TPse - forwrd, 5 GG GG G GT T ; N +,K + -TPse - reverse, 5 GG G TG T GG T T ; N +,K + -TPse - forwrd, 5 TG T T T T T ; N +,K + -TPse - reverse, 5 G T GT GTT T TGG T ; nd elongtion fctor- forwrd, 5 GG T TG GTT G G G ; elongtion fctor- reverse, 5 G GG T T TG G. ll qrt-pr rections were performed s follows: min t 5, min t 95, followed y 4 cycles of 95 for 5 s nd 6 for min. Melt curve nlysis ws performed following ech rection to confirm the presence of only single product of the rection. The melting temperture of the mplicon otined with cdn from sockeye slmon ws identicl to tht of the trout mplicon, strongly indicting tht the primers developed for trout lso mplify the expected trget in slmon. Negtive control rections were performed for selection of smples using RN tht hd not een reverse trnscried to control for the possile presence of genomic contmintion. Genomic DN contmintion never constituted more thn :496 strting copies for ny gene exmined. One rndomly selected smple ws used to develop stndrd curve relting threshold cycle to cdn mount for ech primer set nd ll results re expressed reltive to these stndrd curves. mrn mounts re normlized reltive to elongtion fctor nd expressed reltive to the gill smples collected from fish cught in Georgi Strit. Freshwter migrtion did not ffect the expression of elongtion fctor when expressed reltive to totl mrn reverse trnscried; therefore, ny chnges in gene expression re due to chnges in N +,K + -TPse nd not due to chnges in the control. Sttisticl nlysis Sockeye collected t the pek of the run were compred for gill N +,K + -TPse ctivity, plsm chloride nd osmollity using one-wy nlysis of vrince (NOV) to determine whether loction hd significnt effect on these vriles for the nd smples. In, our smpling ws more extensive in freshwter thn sewter. In, we smpled migrting sockeye more extensively in sewter thn freshwter. s consequence, fish were not cptured from the sme loctions in oth yers. We could not, therefore, compre the dt directly etween the yers for ll loctions. However, comprisons etween yers nd mong stocks were mde for fish cptured t the common smpling sites of Whonnock nd the spwning grounds using two-wy NOV. For ech yer, fish smpled on the spwning grounds were seprted into those tht were not fully mture (green) nd those tht were fully mture fish redy to spwn (spwners). When fctors were found to e sttisticlly significnt, Tukey s test ws used to determine differences etween the smples collected from the different loctions. Sttisticl significnce ws tken t level of P<.5. ll vlues re expressed s mens ± S.E.M. Specific ctivity of gill N +,K + -TPse ctivity, mrn gene expression nd mrn gene expression were exmined for differences y loction using one-wy NOV, followed y Tukey s test if significnt differences were found. Results Physiologicl chnges were oserved in dult sockeye slmon returning from the ocen to spwning res. We found Gill N +,K + -TPse ctivity (μmol DP mg protein h ) Plsm osmollity (mosm) Plsm [chloride] (mmol l ) 4 5 6 4 Hrrison,,D D QI JS PR GS H W S 5 5 Sewter Freshwter Distnce (km) Fig.. () Gill N +,K + -TPse ctivity ( mol DP mg protein h ), () plsm osmollity (mosm), nd () plsm [chloride] (mmol l ) levels for Hrrison sockeye slmon cptured in sewter nd freshwter during migrtion to the spwning grounds (distnce, km). The mouth of the Frser River is defined s km. Smpling loctions s identified in Fig.. Spwners were smpled in Wever reek, mjor spwning triutry in this system. The lst two smples were collected on the spwning grounds (7 km; H, holding, S, spwners), ut sexully mture fish re offset to prevent overlp of the dt points. Smple size rnged from 54 (JS, ) to (holding, ; W, ). Vlues re mens ± S.E.M. Symols with common letter do not differ significntly. THE JOURNL OF EXPERIMENTL IOLOGY
Ionoregultory chnges in mturing slmon 47 significnt chnges in gill N +,K + -TPse ctivity etween sewter nd freshwter loctions in oth yers of our study nd for ll popultions of sockeye exmined. For the Hrrison stock, gill N +,K + -TPse ctivity showed significnt chnge with loction in (F,45 =8.6, P<.) nd (F 7,56 =., P.) (Fig. ). Plsm osmollity (, F,46 =7.4, P.;, F 7,6 =46.5, P.) nd [chloride] (, F,46 =.9, P.;, F 7,64 =69., P.) lso showed highly significnt reltionships with smpling loction. For the Shuswp stock, gill N +,K + -TPse ctivity similrly showed significnt chnge with loction in (F 4,75 =9.98, P<.) nd (F 8,7 =.7, P.) (Fig. ), s did plsm osmollity (, F 4,86 =48.85, P.;, F 8,75 =4.7, P.) nd [chloride] (, F 4,87 =7., P.;, F 8,75 =9.86, Gill N +,K + -TPse ctivity (μmol DP mg protein h ) Plsm osmollity (mosm) Plsm [chloride] (mmol l ) 4 5 6 4 Shuswp Fig.. () Gill N +,K + -TPse ctivity ( mol DP mg protein h ), () plsm osmollity (mosm), nd () plsm [chloride] (mmol l ) levels for Shuswp sockeye slmon cptured in sewter nd freshwter during migrtion to the spwning grounds (distnce, km). The mouth of the Frser River is defined s km. Smpling loctions s identified in Fig.. Spwners were smpled in dms River, mjor spwning triutry in this system. The lst two smples were collected on the spwning grounds (48 km; H, holding, S, spwners), ut sexully mture fish re offset to prevent overlp of the dt points. Smple size rnged from 74 (GS, ) to 6 (QI, ). Vlues re mens ± S.E.M. Symols with common letter do not differ significntly.,, c D, c QI GS W H JS T S 5 5 Sewter Freshwter Distnce (km) P.). Likewise, for the hilko stock, gill N +,K + -TPse ctivity showed significnt chnge with loction in (F,4 =7.48, P.) nd (F 6,6 =6.58, P.) (Fig. 4), s did plsm osmollity (, F,4 =5.57, P.;, F 6,9 =47., P.) nd [chloride] in (F 6,9 =5.95, P.), ut not in (F,4 =., P=.4). For the Quesnel stock, gill N +,K + -TPse ctivity showed significnt chnge with loction in (F 5,9 =4.95, P<.) nd (F 6,86 =6., P.) (Fig. 5), s did plsm osmollity (, F 5,96 =5.4, P.;, F 6,9 =.65, P.) nd [chloride] (, F 5,95 =9., P.;, F 6,9 =6.74, P.). Gill N +,K + -TPse ctivity (μmol DP mg protein h ) Plsm osmollity (mosm) Plsm [chloride] (mmol l ) 4 5 6 4, hilko,,,, Fig. 4. () Gill N +,K + -TPse ctivity ( mol DP mg protein h ), () plsm osmollity (mosm), nd () plsm [chloride] (mmol l ) levels for hilko River sockeye slmon cptured in sewter nd freshwter during migrtion to the spwning grounds (distnce, km). Smpling loctions s identified in Fig.. The mouth of the Frser River is defined s km. Spwners were smpled in Horsefly River, mjor spwning triutry in this system. The lst two smples were collected on the spwning grounds (56 km; H, holding, S, spwners), ut sexully mture fish re offset to prevent overlp of the dt points. Smple size rnged from (W, ) to (HG, ). Vlues re mens ± S.E.M. Symols with common letter do not differ significntly. c QI JS PR W H HG S 5 5 Sewter Freshwter Distnce (km) THE JOURNL OF EXPERIMENTL IOLOGY
474 J. M. Shrimpton nd others Ionoregultory chnges in sewter Fish were smpled in the se t four loctions, one ~85 km, second km, third km, nd fourth <5 km from the mouth of the Frser River. Depending on stock, there were significnt decreses in gill N +,K + -TPse ctivity, plsm osmollity nd chloride levels s the sockeye slmon moved etween these three sltwter loctions (Figs 5); enzyme ctivity declined significntly for Shuswp sockeye etween the Queen hrlotte Islnds nd Georgi Strit. The pttern ws similr for the other stocks, ut smll smple size nd no Quesnel nd hilko sockeye cught in Georgi Strit for limited sttisticl power nd smple comprisons. Plsm osmollity nd [chloride] vlues declined significntly for Gill N +,K + -TPse ctivity (μmol DP mg protein h ) Plsm osmollity (mosm) Plsm [chloride] (mmol l ) 4 5 6 4 Quesnel Fig. 5. () Gill N +,K + -TPse ctivity ( mol DP mg protein h ), () plsm osmollity (mosm), nd () plsm [chloride] (mmol l ) levels for Quesnel River sockeye slmon cptured in sewter nd freshwter during migrtion to the spwning grounds (distnce, km). Smpling loctions s identified in Fig.. The mouth of the Frser River is defined s km. Spwners were smpled in Horsefly River, mjor spwning triutry in this system. The lst two smples were collected on the spwning grounds (749 km; 749; H, holding, S, spwners), ut sexully mture fish re offset to prevent overlp of the dt points. Smple size rnged from 5 (W, ) to (QI, ). Vlues re mens ± S.E.M. Symols with common letter do not differ significntly.,,,,,,,,,c c QI PR GSW HG c S H 5 5 Sewter Freshwter Shuswp nd Hrrison stocks, respectively, etween Queen hrlotte Islnd nd Georgi Strit; ut not hilko nd Quesnel. These dt provide evidence for pre-freshwter ionoregultory preprtions occurring efore fish encountered nd entered the Frser River. Ionoregultory chnges with freshwter entry The ottonwood smple site (river, km) is esturine. For the Shuswp nd Hrrison stocks intercepted t ottonwood, oth plsm osmollity nd chloride hd declined significntly compred with Strit of Georgi smples (Figs nd ). t the Whonnock smple site (river, 5 km) fish re in freshwter. Here gill N +,K + -TPse ctivity hd significntly declined in three of the four stocks compred with the ottonwood nd Georgi Strit smples. Two-wy NOV indicted tht there were no significnt differences mong stocks (P=.97) or etween yers (P=.78) for gill N +,K + - TPse ctivity. Except for plsm osmollity in the Shuswp stock, which showed significnt increse, there ws no significnt chnge in plsm osmollity nd [chloride] etween the ottonwood nd Whonnock smples (Fig. ). Gill N +,K + - TPse ctivity differed significntly etween Georgi Strit nd Whonnock for Shuswp () nd Hrrison nd Shuswp stocks in. Ionoregultory chnges during freshwter migrtion For ll stocks exmined, gill N +,K + -TPse ctivity, s well s plsm osmollity nd [chloride] vlues, of fish rriving t the spwning grounds were significntly lower thn vlues from the Georgi Strit. Thus, from the Queen hrlotte Islnds to the spwning res, there ws firly consistent decline in gill N +,K + -TPse ctivity, with the exception of vlues from fish cptured shortly fter entry into freshwter. ecuse the different fish stocks migrted different distnces in freshwter, it ws possile to exmine the effects of freshwter migrtion time nd distnce on the ionoregultory chnges. The loss of N +,K + -TPse ctivity per unit distnce ws highest for the fish stock tht migrted the shortest distnce (Hrrison stock), nd lest for the fish stock migrting the longer distnces (over 75 km in freshwter for the Quesnel stock; Figs 5). Regrdless, ll stocks rrived t the spwning grounds with levels of gill N +,K + -TPse ctivity tht were very low (pproximtely mol DP mg protein h ). There were significnt differences mong the stocks nd etween yers (Fig. 6); however, there ws no pprent trend nd N +,K + -TPse ctivities of fish holding on the spwning grounds were not relted to migrtion distnce. Insted, the decline in enzyme ctivity ppered to e dependent on time in freshwter. This is evident when the solute chnge in enzyme ctivity is plotted ginst the estimted time in freshwter for fish smpled t the spwning grounds (Fig. 6). The exception to this trend ws the Hrrison, dt, ut these dt were limited in tht only three green fish were smpled on the Wever reek spwning grounds in. THE JOURNL OF EXPERIMENTL IOLOGY
Ionoregultory chnges in mturing slmon 475 Gill N +,K + -TPse ctivity (μmol DP mg protein h ) hnge in gill N +,K + - TPse ctivity (μmol DP mg protein h ).5..5.8.6.4. H Hrrison Shuswp hilko Quesnel, 4 5 Dys migrting Fig. 6. () Gill N +,K + -TPse ctivity ( mol DP mg protein h ) for fish rriving on the spwning grounds in the Hrrison (H), Shuswp (Sh), hilko (), Quesnel (Q) Wtersheds in nd. *Significnt difference etween yers for the sme stock. Smples with common letter do not differ significntly. Smple size rnged from 5 to, except for Hrrison, where smple size ws. () The solute chnge in gill Gill N +,K + -TPse ctivity etween fish cptured t Whonnock nd fish rriving on the spwning grounds for the different stocks exmined plotted s function of migrtion time within freshwter. Migrtion rtes re sed on estimtes from English et l. (4) nd differences in dtes etween smpling t the two loctions. Spwning slmon fter rrivl t the spwning res in ech river system, sockeye slmon typiclly hold for pproximtely 4 8 dys efore they ecome fully ripe nd egin spwning. Once spwning is completed, fish rpidly senesce nd die fter further 7 5 dys (McPhee nd Quinn, 998; Heley et l., ). Given the progressive declines in gill N +,K + -TPse ctivity s the fish migrte upriver, it is surprising tht gill N +,K + -TPse ctivities incresed while fish were holding on the spwning grounds for mny of the popultions (Figs 5). For pek run fish, the reltive increse vried, ut ws often significnt. Overll, the incresed enzyme ctivity, however, filed to prevent further significnt declines in plsm osmollity nd chloride s fish completed mturtion. N +,K + -TPse mrn expression Expression of N +,K + -TPse nd suunits showed significnt differences in fish smpled t different loctions of the migrtion (Fig. 7). Movement into freshwter, s exhiited y the smples collected t Whonnock, ws chrcterised y significnt increse in suunit mrn expression. The highest levels of gill N +,K + -TPse mrn were mesured in fish rriving t the spwning grounds. In contrst, fish tht were mturing on the spwning Sh, * Q N +,K + -TPse ctivity (μmol DP mg protein h ) Reltive N +,K + -TPse α mrn expression Reltive N +,K + -TPse α mrn expression 6 4 GS W HG H S Fig. 7. () Gill N +,K + -TPse ctivity ( mol DP mg protein h ), () N +,K + -TPse expression (reltive to EF- ) nd () N +,K + -TPse expression (reltive to EF- ) for fish cptured in sewter nd freshwter during migrtion to the spwning grounds in. Smple loctions s indicted in Fig.. Fish holding on the spwning grounds (H) nd spwners (S) were smpled on the spwning grounds of the Horsefly River (Quesnel Wtershed). Smple size rnged from 9 to. Vlues re mens ± S.E.M. grounds were chrcterised y significnt increse in suunit mrn, despite there eing no difference in expression etween sewter nd freshwter smples. Discussion The results presented in this study represent the first exmintion of ionoregultory chnges in wild migrtory dult slmon s they move from sewter to freshwter nd return to spwning grounds. Smpling dult wild fish during 6 km migrtion pth is techniclly chllenging nd requires tht resources re focused on key smpling loctions t pproprite times. The smpling loctions tht we selected for this study llowed us to ttempt to nswer four questions tht hve not een previously ddressed. Do sockeye slmon prepre for freshwter in dvnce of rrivl t the estury? decrese in gill N +,K + -TPse ctivity indictes tht sewter ionoregultory cpcity of migrting sockeye declines efore the fish enter freshwter. The chnges in, c THE JOURNL OF EXPERIMENTL IOLOGY
476 J. M. Shrimpton nd others enzyme ctivity tht occur in sewter re proly preprtory for movement into freshwter. Uchid et l. (997) lso suggest tht physiologicl nd histologicl chnges in the gill of chum slmon re preprtory for upstrem migrtion. These uthors showed tht gill N +,K + -TPse continued to decrese in mturing chum cptured efore river entry nd held in sewter; chum held in sewter showed elevted plsm osmollity nd incresed mortlity. lthough physiologicl chnges in sewter pper dptive for river entry, there is lso evidence tht hypo-osmoregultory function is directly impired in sexully mturing dult slmon (lrke nd Hirno, 995). hnges oserved in sewter slmon my e ssocited with elevted reproductive hormones. Endocrine fctors ssocited with mturtion impir ionoregultory ility in sewter (Lundqvist et l., 989; Mdsen nd Korsgrd, 989). In prllel study, n increse in reproductive hormones for Frser River sockeye tgged in Johnstone Strit ws found efore entry into freshwter (S. J. ooke, S. G. Hinch, G. T. rossin, D.. Ptterson, K. K. English, M.. Heley, S. Mcdonld, J. M. Shrimpton, J. L. Young,. Lister, G. Vn Der Krk nd. P. Frrell, mnuscript sumitted for puliction). These uthors, however, found no significnt differences in reproductive hormones mong the popultions of sockeye, suggesting tht differences etween popultions re not function of reproductive stte. Despite the decline oserved in gill N +,K + -TPse ctivity, chnges in osmollity nd chloride vlues for sewter fish do not suggest n impirment in ionoregultory performnce. Plsm osmollity nd chloride did not differ mong sewter smpling sites for the hilko nd Quesnel stocks (Figs 4 nd 5). There ws prllel decline in plsm osmolytes with the chnges in gill N +,K + -TPse ctivity for the Shuswp sockeye migrting from the Queen hrlotte Islnds (85 km from the Frser River mouth) to Johnstone Strit ( km from the Frser River mouth) (Fig. ). Lower enzyme ctivities would e expected to correspond with n increse in plsm osmollity for fish mintined in sewter, s hs een demonstrted in juvenile slmon during the prr smolt trnsformtion (Mcormick nd Sunders, 987; Shrimpton et l., 994). Wters for these two res re hyperosmotic to plsm levels, so it is difficult to interpret the declines in chloride nd osmollity. Hrrison sockeye re the only popultion to show significnt chnge in plsm chloride for sewter fish; there is significnt decline in vlues for fish cught in Johnson Strit compred to Georgi Strit (Fig. ). There is considerle influence of the Frser River within this region nd the declines in [chloride] my reflect lower environmentl slinities (Thompson, 98). Our smpling of fish in sewter for ws much more extensive thn in, ut we smpled fish in Georgi Strit in oth yers. The troll fishery in Georgi Strit is selective for lte-run fish nd few Quesnel nd no hilko fish were cught. Vlues in Georgi Strit were similr for oth yers, with the exception of gill N +,K + -TPse ctivity for Hrrison fish (Fig. ). The difference etween yers my reflect differences in holding time for fish within Georgi Strit. S. J. ooke, S. G. Hinch, G. T. rossin, D.. Ptterson, K. K. English, M.. Heley, S. Mcdonld, J. M. Shrimpton, J. L. Young,. Lister, G. Vn Der Krk nd. P. Frrell (mnuscript sumitted for puliction), however, exmined Frser River lte-run sockeye tht showed vrile dely in migrtion nd found no difference in gill N +,K + -TPse ctivities with dely time in Georgi Strit. re freshwter ionoregultory chnges complete efore fish leve the ocen? The first freshwter smpling point ws t ottonwood, which is km from the mouth of the Frser River, yet there re still sltwter intrusions t this loction. s consequence, fish smpled from ottonwood my still hve ccess to incresed slinity, ut the decrese in plsm osmollity nd [chloride] in the four popultions exmined (Figs 5) suggest tht fish smpled were in full freshwter. The switch from sewter ionoregultion to chloride uptke for fish directly trnsferred from sewter to freshwter hs een shown to tke pproximtely 4 dys (ttrm nd Eddy, 99). Perturtions in plsm osmollity nd [chloride], lthough in the opposite direction, re similr in mgnitude to those seen in smolting (lckurn nd lrke, 987); suggesting ionoregultory chnges tht re preprtory for freshwter entry hve lredy occured in sewter. Gill N +,K + -TPse ctivity showed little chnge etween the Georgi Strit nd ottonwood smples (Figs nd ); however, N +,K + -TPse mrn expression indictes tht physiologicl chnges continue fter movement into freshwter (Fig. 7). In the fish smpled, we found signficnt decline in gill N +,K + -TPse ctivity etween fish smpled t ottonwood nd Whonnock for three of the popultions. We do not know the ctul temporl difference for fish cught etween the two loctions, ut fish my trvel the 4 km in less thn 4 h (English et l., 4). Modifictions of enzyme ctivity hve dptive significnce nd occur when euryhline teleosts move etween chnging slinities (Mcormick, 995). The response normlly tkes severl dys, suggesting trnscriptionl regultion. Short-term regultion of this enzyme, however, hs een demonstrted in killifish trnsferred etween slinities (Mncer nd Mcormick, ). Elevted cyclic MP levels in rown trout hve lso een found to decrese mximl N +,K + -TPse ctivity in gills of rown trout (Tipsmrk nd Mdsen, ). The uthors suggest tht phosphoryltion my regulte N +,K + -TPse ctivity in teleosts. The significnt differences in gill N +,K + - TPse ctivity etween fish smpled t ottonwood nd Whonnock could lso e due to rpid modultion of the enzyme (Tipsmrk nd Mdsen, ). Isoform switching etween two N +,K + -TPse -suunit isoforms ( nd ) during slinity trnsfer is thought to e n importnt component in chnging the slmonid gill from n ion-soring epithelium in freshwter to n ion-secreting epithelium in sewter (Richrds et l., ). onsistent with this notion, the expression of N +,K + -TPse incresed in THE JOURNL OF EXPERIMENTL IOLOGY
Ionoregultory chnges in mturing slmon 477 wild sockeye slmon following movement from sewter in Georgi Strit to freshwter in Whonnock nd is ssocited with decrese in N +,K + -TPse ctivity (Fig. 7). These results suggest tht the gill ionoregultory chnges necessry for freshwter cclimtion, t lest in terms of mrn expression nd enzyme ctivity, re not complete efore the fish enters freshwter (Fig. 7). The lck of chnge in N +,K + - TPse mrn in the gills of wild sockeye slmon following movement from sewter to freshwter re consistent with the results of Richrds et l. (), who found tht expression of N +,K + -TPse increses trnsiently following sewter trnsfer, nd returns to the levels oserved in freshwter cclimted individuls within dys posttrnsfer. Overll, enhnced expression of N +,K + -TPse during freshwter migrtion is consistent with the isoform switching proposed y Richrds et l. (). oncurrent with the chnges in enzyme ctivity t Whonnock is reound in plsm osmollity nd [chloride]. sed on chnges in plsm prmeters nd gill N +,K + - TPse ctivity, the chloride uptke is not likely to hve chieved freshwter vlues (ttrm nd Eddy, 99), ut my e djusted s the fish move upriver. re mturing sockeye in n ionoregultory stedy stte during migrtion? Gill N +,K + -TPse ctivity, plsm osmollity nd [chloride] declined with the time nd distnce covered while migrting upriver. Migrting dult sockeye, therefore, re not in n ionoregultory stedy stte. Generlly the lowest vlues of gill N +,K + -TPse were oserved for fish pproching or first rriving on the spwning grounds. For ll stocks, enzyme ctivities nd plsm osmollities hd significntly declined from sewter vlues (Figs 5). The fctors mesured in the present study of freshwter migrting fish suggest continued physiologicl chnges throughout migrtion. Physiologicl chnges ssocited with migrtion distnce hve previously een oserved in smolting slmonids with distnce moved downstrem (Muir et l., 994). Therefore, the decline in gill N +,K + -TPse ctivity s sockeye migrte to spwning res my reflect physiologicl djustments tht continue to occur s the fish migrte. The solute chnges in gill N +,K + -TPse ctivity were relted to estimted time in freshwter. English et l. (4) used rdiotelemetry to determine migrtion rtes in mturing lte-run sockeye slmon in freshwter. They found tht sockeye fter entering freshwter migrted t firly consistent rte nd tgged sockeye mintined their chronologicl order during migrtion s ssessed y detection t monitoring sttions long the Frser River. The Hrrison sockeye, however, hold in freshwter for reltively longer period given their short river migrtion distnce (7 km). Does finl mturtion ffect ionoregultory sttus? Plsm osmollity nd chloride levels mesured in spwning fish suggest tht they re no longer le to mintin dequte homeostsis in freshwter. Sockeye re semelprous nd die shortly fter spwning. The physiologicl perturtion tht the fish experience my e of little consequence, however; gill N +,K + -TPse ctivities in spwners re generlly higher thn for fish holding on the spwning grounds (Fig. 6). The higher gill N +,K + -TPse ctivities oserved in spwners suggests tht the fish my e ttempting to compenste for the osmotic perturtion. Spwning sockeye slmon re chrcterized y mrked increse in gill N +,K + -TPse mrn compred to individuls holding on the spwning grounds. Previous work demonstrted trnsient increse in the expression of this isoform following trnsfer to sewter (Richrds et l., ) nd these uthors speculted tht the expression of N +,K + - TPse might e under the regultion of circulting glucocorticoid levels. No chnge in N +,K + -TPse expression ws oserved in response to sewter to freshwter movement; however, lrge increses in N +,K + -TPse mrn levels were oserved in spwning sockeye. Previous work hs shown tht circulting cortisol levels increse during mturtion (Donldson nd Fgerlund, 97; rruth et l., ) nd the gene is possily responsive to the higher cortisol levels. In conclusion, in the present study we found tht ionoregultory chnges preprtory for freshwter residence occur in dult sockeye slmon while they re migrting in sewter. The entry into freshwter ws ccompnied y further physiologicl djustments. Fish from the four stocks cught t Whonnock, the first smpling loction tht ws fully freshwter, however, did not differ in our mesures of ionoregultory performnce. lrge difference in freshwter migrtion distnce nd time in freshwter efore spwning existed in the four stocks of sockeye exmined. ll stocks rrived on the spwning grounds in similr physiologicl condition, s indicted y our mesurements nd consistent with mesures of energy prtitioning reported y Ptterson et l. (4) nd rossin et l. (4). Physiologicl chnges oserved in spwners suggest tht sockeye ttempt to minimize the osmotic perturtion tht is ssocited with finl mturtion. We thnk l ss, Lur Richrds, Jim ve, Jim Woodey, Mike Lpointe, rmen Mconnell, nd others from the Pcific Slmon ommission nd Fisheries nd Ocens nd for fcilitting this project. lrge numer of individuls ssisted with fish cpture, in prticulr Jyme Dvidson nd Glenn rossin. Physiologicl ssys were conducted y drin lrke nd Jyme Dvidson. ore funding ws provided y Nturl Sciences nd Engineering Reserch ouncil (NSER) Strtegic Grnt. We lso thnk the Environmentl Wtch Progrm of DFO for logistic nd finncil support. References ttrm, J.. nd Eddy, F.. (99). Recovery of chloride uptke in sewter-dpted rinow trout (Slmon girdneri) fter trnsfer to freshwter. J. Exp. iol. 48, 489-49. echm, T. D., Withler, R. E. nd Wood,.. (995). Stock identifiction THE JOURNL OF EXPERIMENTL IOLOGY
478 J. M. Shrimpton nd others of sockeye slmon y mens of ministellite DN vrition. N. m. J. Fish. Mn. 5, 49-65. lckurn, J. nd lrke, W.. (978). Revised procedure for the 4 h sewter chllenge test to mesure sewter dptility of juvenile slmon. n. Tech. Report Fish. qut. Sci. 55, 5. runer,. J., Shrimpton, J. M. nd Rndll, D. J. (99). The effect of short durtion sewter exposure on plsm ion concentrtions nd swimming performnce in coho slmon (Oncorhynchus kisutch). n. J. Fish. qut. Sci. 49, 99-45. rruth, L. L., Jones, R. E. nd Norris, D. O. (). ortisol nd pcific slmon: new look t the role of stress hormones in olfction nd homestrem migrtion. Integ. omp. iol. 4, 574-58. lrke, W.. nd Hirno, T. (995). Osmoregultion. In Physiologicl Ecology of Pcific Slmon (ed.. Groot, L. Mrgolis nd W.. lrke), pp. 7-77. Vncouver,, nd: U Press. ooke, S. J., Hinch, S. G., Frrell,. P., Lpointe, M., Jones, S. R. M., Mcdonld, S., Ptterson, D., Heley, M.. nd Vn Der Krk, G. (4). norml migrtion timing nd high en route mortlity of sockeye slmon in the Frser River, ritish olumi. Fisheries 9(), -. rossin, G. T., Hinch, S. G., Frrell,. P., Higgs, D.., Lotto,. G., Okes, J. D. nd Heley, M.. (4). Energetics nd morphology of sockeye slmon: effects of upriver migrtory distnce nd elevtion. J. Fish iol. 65, 788-8. Donldson, E. M. nd Fgerlund, U. H. M. (97). orticosteroid dynmics in Pcific slmon. Gen. omp. Endocrinol., 54-65. English, K., Sliwinski,., Lelle, M., Koski, W. R., lexnder, R., ss,. nd Woodey, J. (4). Migrtion timing nd in-river survivl of Lterun Frser River sockeye using rdio-telemetry techniques. Report prepred y LGL Limited, Sidney, for the Pcific iologicl Sttion of Fisheries nd Ocens nd. Fisher, J. P. nd Percy, W. G. (988). Growth of juvenile coho slmon (Oncorhynchus kisutch) off Oregon nd Wshington, US, in yers of differing costl upwelling. n. J. Fish qut. Sci. 45, 6-44. Heley, M.., Lke, R. nd Hinch, S. G. (). Energy expenditures during reproduction y sockeye slmon (Oncorhynchus nerk). ehviour 4, 6-8. Hinch, S. G., ooke, S. J., Heley, M.. nd Frrell,. P. (5). ehviourl physiology of fish migrtion. In ehviour nd Physiology, Fish Physiology Series, vol. 4 (ed. K. Slomn, S. lshine nd R. Wilson), pp. 9-95. msterdm: Elsevier Science. Hirno, T., Morisw, M. nd Suzuki, K. (978). hnges in plsm nd coelomic fluid composition of the mture slmon (Oncorhynchus ket) during freshwter dpttion. omp. iochem. Physiol. 6, 6-8. Hor, W. S. (988). The physiology of smolting slmonids. In Fish Physiology, vol. (ed. W. S. Hor nd D. J. Rndll), pp. 75-44. New York: cdemic Press. Lundqvist, H., org,. nd erglund, I. (989). ndrogens impir sewter dptility in smolting ltic slmon (Slmo slr). n. J. Zool. 67, 7-76. Mdsen, S. S. nd Korsgrd,. (989). Time-course effects of repetitive oestrdiol-7 nd thyroxine injections on the nturl spring smolting of tlntic slmon, Slmo slr L. J. Fish iol. 5, 9-8. Mncer, J. M. nd Mcormick, S. D. (). Rpid ctivtion of gill N +,K + -TPse ctivity in the euryhline teleost Fundulus herteroclitus. J. Exp. Zool. 87, 6-74. Mcormick, S. D. (99). Methods for the nonlethl gill iopsy nd mesurements of N +,K + -TPse ctivity. n. J. Fish qut. Sci. 5, 656-658. Mcormick, S. D. (995). Hormonl control of gill N +,K + -TPse ctivity nd chloride cell function. In Fish Physiology, vol. 4 (ed.. M. Wood nd T. J. Shuttleworth), pp. 85-5. Sn Diego, : cdemic Press. Mcormick, S. D. nd Sunders, R. L. (987). Preprtory physiologicl dpttions for mrine life of slmonids: osmoregultion, growth, nd metolism. m. Fish Soc. Symposium, -9. McKeown,.. (984). Fish Migrtion. London: room Helm Pulishers. McPhee, M. V. nd Quinn, T. P. (998). Fctors ffecting the durtion of nest defence nd reproductive life spn of femle sockeye slmon, Oncorhynchus nerk. Envir. iol. Fish. 5, 69-75. Muir, W. D., Zugg, W. S., Giorgi,. E. nd Mcutcheon, S. (994). ccelerting smolt development nd downstrem movement in yerling chinook slmon with dvnced photoperiod nd incresed temperture. quculture, 87-99. Nielsen,., restrup, K., Norum, U. nd Mdsen, S. S. (4). Future migrtory ehviour predicted from premigrtory levels of gill N + /K + - TPse ctivity in individul wild rown trout (Slmo trutt). J. Exp. iol. 7, 57-5. Ptterson, D.., Mcdonld, J. S., Hinch, S. G., Heley, M.. nd Frrell,. P. (4). The effect of exercise nd cptivity on energy prtitioning, reproductive mturtion nd fertiliztion success in dult sockeye slmon. J. Fish iol. 64, 9-59. Richrds, J. G., Semple, J. W., ystrinsky, J. S. nd Schulte, P. M. (). N + /K + -TPse -isoform switching in gills of rinow trout (Oncorhynchus mykiss) during slinity trnsfer. J. Exp. iol. 6, 4475-4486. Shrimpton, J. M., ernier, N. J., Iwm, G. K. nd Rndll, D. J. (994). Differences in mesurements of smolt development etween wild nd htchery-rered juvenile coho slmon (Oncorhynchus kisutch) efore nd fter sltwter exposure. n. J. Fish qut. Sci. 5, 7-78. Shrimpton, J. M., jörnsson,. T. nd Mcormick, S. D. (). n tlntic slmon smolt twice? Endocrine nd iochemicl chnges during smolting. n. J. Fish. qut. Sci. 57, 969-976. Thomson, R. E. (98). Ocenogrphy of the ritish olumi ost. n. Spec. Pu. Fish. qut. Sci. 56, 5. Tipsmrk,. K. nd Mdsen, S. S. (). Rpid modultion of N + /K + - TPse ctivity in osmoregultory tissues of slmonid fish. J. Exp. iol. 4, 7-79. Uchid, K., Kneko, T., Ymguchi,., Ogswr, T. nd Hirno, T. (997). Reduced hypoosmoregultory ility nd ltertion in gill chloride cell distriution in mture chum slmon (Oncorhynchus ket) migrting upstrem for spwning. Mr. iol. 9, 47-5. THE JOURNL OF EXPERIMENTL IOLOGY