Extracellular acid base regulation during short-term hypercapnia is effective in a shallow-water crab, but ineffective in a deep-sea crab

Transcription

1 MARINE ECOLOGY PROGRESS SERIES Vol. 33: 1 9, 2007 Pulished Mrch 2 Mr Ecol Prog Ser FEATURE ARTICLE OPEN ACCESS Extrcellulr cid se regultion during short-term hypercpni is effective in shllow-wter cr, ut ineffective in deep-se cr Eric F. Pne*, Jmes P. Brry Monterey By Aqurium Reserch Institute, 7700 Sndholdt Rod, Moss Lnding, Cliforni 95039, USA ABSTRACT: Rising levels of tmospheric cron dioxide could e cured y lrge-scle sequestrtion of CO 2 in the deep se. Such solution requires prior ssessment of the impct of hypercpnic, cidic sewter on deep-se fun. Lortory studies were conducted to ssess the short-term hypercpnic tolernce of the deep-se Tnner cr Chionoecetes tnneri, collected from 1000 m depth in Monterey Cnyon off the cost of centrl Cliforni, USA. Hemolymph cid se prmeters were monitored over 2 h of exposure to sewter equilirted with ~1% CO 2 (sewter P CO2 ~ torr or 0. kp, ph 7.1), nd compred with those of the shllow-living Dungeness cr Cncer mgister. Short-term hypercpni-induced cidosis in the hemolymph of Chionoecetes tnneri ws lmost uncompensted, with net 2 h ph reduction of 0.32 units nd net icronte ccumultion of only 3 mm. Under simultneous hypercpni nd hypoxi, short-term extrcellulr cidosis in Chionoecetes tnneri ws completely uncompensted. In contrst, Cncer mgister fully recovered its hemolymph ph over 2 h of hypercpnic exposure y net ccumultion of 12 mm icronte from the surrounding medium. The dt support the hypothesis tht deep-se nimls, which re dpted to stle environment nd exhiit reduced metolic rtes, lck the short-term cid se regultory cpcity to cope with the cute hypercpnic stress tht would ccompny lrge-scle CO 2 sequestrtion. Additionlly, the dt indicte tht sequestrtion in oxygen-poor res of the ocen would e even more detrimentl to deep-se fun. KEY WORDS: CO 2 Deep se Physiology Decpod crustce Acid se regultion Chionoecetes tnneri Cncer mgister Resle or repuliction not permitted without written consent of the pulisher The ocen s lrge-scle sorption, s well s possile future nthropogenic sequestrtion of tmospheric CO 2, will result in long-term cidifiction of the wter. The deep-se cr Chionoecetes tnneri (collected from Monterey Cnyon t 1000 m depth) is unle to regulte extrcellulr ph during short-term CO 2 exposure. The results of Pne & Brry support the hypothesis tht hypercpni will hve profound physiologicl impct on deep-se orgnisms. Photo: MBARI (200) INTRODUCTION Lrge-scle sequestrtion of cron dioxide in the deep se (Mrchetti 1977, 1979), s mens of reducing tmospheric CO 2 emissions nd mitigting greenhouse-gs-induced climte chnge, remins potentil solution to the prolem of esclting tmospheric CO 2 levels. Accordingly, the possiility of lrge-scle nthropogenic inputs of CO 2 into the deep-se hs creted intriguing nd pressing reserch questions. *Emil: epne@mri.org Inter-Reserch

2 2 Mr Ecol Prog Ser 33: 1 9, 2007 Becuse the physiology of deep-se nimls is so poorly understood, puttive deep-se CO 2 sequestrtion must e pproched with cution (Seiel & Wlsh 2001, Portner et l. 200). While in situ microcosmic CO 2 relese studies t depth hve een conducted (Brewer et l. 2000, Tmurri et l. 2000, Brry et l. 200), the direct physiologicl impcts of hypercpni, nd the cid se regultory cpcities of deep-se nimls, hve yet to e systemticlly investigted under lortory conditions. The generl hypothesis tht deep-se niml cid se regultory cpcity will e poorly dptive to hypercpnic exposure rests on 2 sic principles. The first is the trend towrd hypometolism with depth in pelgic, typiclly visul, nimls (Seiel et l. 1997). The theory holds tht limited light with depth reduces visul predtion pressure nd selects for reduced locomotory ility nd metolic cpcity (Childress et l. 1990, Childress & Seiel 199). Although this theory pplies predominntly to pelgic nimls, deep-se enthic nimls (including crustcens) lso exhiit metolic rtes typiclly n order of mgnitude lower thn their shllow-living counterprts (Childress et l. 1990, Henry et l. 1990). While this phenomenon in deep-se enthic crustcens my simply e function of very low tempertures t depth in res of steep therml grdient (Childress et l. 1990), these reduced metolic rtes oserved in deep-se enthic crustcens my still e ecologiclly relevnt (disdvntgeous) in the context of hypercpnic exposure. Secondly, cid se regultory cpcity of deep-se fun my e mldptive to hypercpni due to the nturl invrince of their chemicl environment. The deep se hs een chrcterized for severl thousnds of yers y stle physico-chemicl wter prmeters (Gge & Tyler 1991, Kennett & Ingrm 1995, Portner et l. 200). Unlike some shllow dwelling qutic nimls who hve evolved effective cid se regultory cpilities to comt sesonl, or even dily, fluctutions in wter ph, oxygen nd CO 2 concentrtion, or temperture, deep-se nimls theoreticlly should not possess such well-developed cid se regultory cpcity (Childress & Seiel 199, Seiel & Wlsh 2001). In this study, we took comprtive physiologicl pproch in testing the hypothesis tht deep-se nimls hve limited cid se regultory cpcity. We investigted the response of deep-se enthic decpod cr, Chionoecetes tnneri (Grooved Tnner cr), to cute lortory-sed hypercpnic exposure, nd compred it to tht of shllow-living decpod cr, Cncer mgister (Dungeness cr). Chionoecetes tnneri, of the rchyurn fmily Mjide, is the dominnt decpod cr of the Monterey Cnyon sefloor t 1000 m, nd the only decpod species occurring t densities comptile with remoteoperted vehicle collection t depth. It is sluggish species, lrge enough to fcilitte repetitive lood smpling, yet esily kept in the lortory with very low feeding requirements. Cncer mgister is lrge, highly undnt cr of the fmily Cncride, common to northestern Pcific costl wters. Though predominntly sutidl nd found mostly t depths to 90 m, shorewrd spring migrtions ring them into shllower wter mrgins of sndy eches nd esturies (Morris et l. 190, Airriess & McMhon 199). Hence, Cncer mgister my experience chnges in wter slinity, prtil pressures of oxygen (P O2 ), nd cron dioxide (P CO2 ), nd ph s frequently s ech tidl cycle (Airriess & McMhon 199). Our choice of Cncer mgister s n experimentl species ws lso influenced y its representtion in the existing cid se, respirtory, nd circultory literture (e.g. Johnsen et l. 1970, Airriess & McMhon 199). Additionlly, we explored the influence of oxygen vilility on the response of the deep-se Tnner cr to hypercpni. Chionoecetes tnneri ws collected from the Monterey Cnyon t depths of ~1000 m (Fig. 1), within the oxygen minimum zone (OMZ) typicl of estern Pcific wters nd the Monterey Cnyon (Fig. 2). The OMZ is the ocen zone of lowest oxygen sturtion creted y the processes of iotic oxygen consumption nd ocen circultion (Wyrtki 192). In the estern Pcific it typiclly occurs etween 500 nd 1000 m. Accordingly, we tested the response of Tnner crs to hypercpni when held in oth low oxygen (simulting OMZ levels) nd high oxygen (fully sturted sewter). Fig. 1. Loctions of ROV Ventn dive sites t ~1000 m in Monterey Cnyon (d) for collection of the deep-se decpod Grooved Tnner cr Chionoecetes tnneri

3 Pne & Brry: Acid se regultion in crs 3 Depth (m) Oxygen (mg l 1 ) Fig. 2. Oxygen profile in Monterey Cnyon, showing the oxygen minimum zone (OMZ) etween 00 nd 1200 m. Arrow: smpling depth for Chionoecetes tnneri The level of hypercpni used, (~1% CO 2 ) hs frequently een employed for oth invertertes nd vertertes from freshwter nd mrine systems (Cmeron 197, Truchot 1979, Toews et l. 193, Cliorne & Heisler 19, Lngenuch & Portner 2002, Edwrds et l. 2005). A one-unit reduction in wter ph typiclly occurs with this level of hypercpni, nd the exposure requires competent cid se regultory cpcity to offset the initil extrcellulr cidosis cused y exposure to hypercpnic sewter. MATERIALS AND METHODS Cr collection nd holding. Grooved Tnner crs Chionoecetes tnneri of oth sexes (200 to 00 g) were collected y remote-operted vehicle (ROV Ventn ) from depths of 950 to 1050 m from the sefloor of Monterey Cnyon (Fig. 1). The ROV Ventn ws operted from the RV Point Loos, under the stewrdship of the Monterey By Aqurium Reserch Institute. Once retrieved from depth, crs were held ord ship in 2 C sewter until trnsfer to the lortory. Totl time from collection to lortory trnsfer ws 3 to 5 h nd survivl ws typiclly 75% during this process. In the lortory, crs were held in 3 cuic 110 l tnks under flow-through conditions with oxygen regulted vi computer-controlled gs-regulted qurium system. In rief, oxygen ws controlled using comintion of sensors (Anderr oxygen optodes) nd gs contctors through which mixtures of nitrogen nd oxygen were used to mintin dissolved gses ner prescried setpoints for oxygen. Temperture ws lso regulted. The totl volume of the recirculting system 7 ws ~1000 l. Flow-through ws chieved y ddition of 0.25 l min 1 to ech tnk from common reservoir of sewter equilirted to set oxygen level y the processes descried ove. With the exception of hydrosttic pressure, in situ (~1000 m) sewter conditions were replicted y holding ll crs in constnt drkness in wter of 3 ± 1 C, ph of 7.5 ± 0.10, nd slinity of 3 ppt. All holding nd experimenttion ws conducted t surfce pressure. While we recognize the inconsistency of holding nimls collected from 1000 m t surfce pressure, the technicl chllenges of in vivo work with mcrofun t high hydrosttic pressure in lortory setting re formidle. We therefore weigh ny potentil depth-relted chnges in physiologicl function ginst the following oservtions regrding Chionoecetes tnneri held t surfce pressure: (1) once cclimted to the lortory, survivl is extremely high, with crs surviving >1 mo (cf. Henry et l. 1990); (2) crs molt in the lortory; nd (3) once cclimted, post-rnchil hemolymph ph of resting crs consistently flls within tight rnge (7.9 to.1) consistent with lphstt regultion in qutic nimls held t 3 C (Cmeron 19). The oxygen concentrtion of the sewter ws set to either 90 ± 5% sturtion ( High, ~350 µm), or 10 ± 5% sturtion ( Low, ~0 µm). The lower O 2 tretment mimicked in situ oxygen levels typicl of the oxygen minimum zone t depths etween roughly 00 nd 1200 m off the Cliforni cost nd within the Monterey Cnyon (Childress 1995; our Fig. 2). Pcific Dungeness crs Cncer mgister of oth sexes (500 to 1000 g) were purchsed loclly (Moss Lnding, CA) from commercil fishermn using ited trps t 30 to 0 m. Dungeness crs were held in the lortory in flowing sewter of 10 ± 1 C, ph of 7.9 ± 0.1, slinity of 3 ppt, nd n oxygen sturtion of 90 ± 5%, under 12:12 h light:drk photoperiod. All crs introduced to the lortory were llowed minimum of 3 wk to cclimte efore experimenttion. Both species of cr were fed twice weekly to stition with chopped squid; food ws withheld 72 h prior to experimenttion, nd only hrd-shelled intermolt nimls were used in experiments. In vitro experiments. The non-icronte uffering cpcity (β) of ser ws determined for Cncer mgister nd for Chionoecetes tnneri held in oth low nd high oxygen. In ll experimenttions, post-rnchil hemolymph ws ccessed y drilling smll (1 mm) hole in the dorsl crpce directly ove the hert. A ring of cynocrylte glue ws pplied round the hole nd 1 cm squre piece of dentl dm ws glued into plce (Forgue et l. 1992). Crs were llowed minimum 72 h recovery period prior to experimenttion. All hemolymph ws drwn neroiclly into ice-cold

4 Mr Ecol Prog Ser 33: 1 9, 2007 gs-tight Hmilton syringes rinsed with Cr Ringer comprised of (in mm) NCl (0), KCl (10), CCl 2 (20), MgCl 2 (9.5), nd H 3 BO 3 (3), ph 7.0 (Lng & Giner 199). For tonometric nlysis, hemolymph ws drwn nd llowed to clot on ice for 10 min. Hemolymph ws then centrifuged eroiclly t 000 g t 3 C for 3 min, producing seprted, rther thn true, serum (Dvenport 197). Seprted serum ws then dded (~150 µl) to round-ottom flsks nd equilirted with humidified gs mixtures of CO 2 nd nitrogen from prenlyzed cylinders (Airgs). Ser were equilirted for 90 min in shking wter th t the pproprite temperture (10 C for Cncer mgister serum nd 3.5 C for Chionoecetes tnneri serum). After equilirtion, smples were drwn into gstight syringes nd ph ws mesured using microelectrode nd in-line reference electrode (Microelectrodes) thermosttted to the pproprite temperture nd coupled to n Accumet (Fisher Scientific) ph meter. Totl CO 2 (C CO2 ) ws mesured y non-dispersive infrred nlysis (LI-COR model 22), following cidifiction (5% phosphoric cid) of serum nd introduction of stripped gs into n infrred nlyzer (Friederich et l. 2002). ph ws stndrdized with Rdiometer Anlyticl precision uffers djusted to the ionic strength of cr serum (~1050 mosm), while C CO2 ws stndrdized with NCO 3 (Sigm-Aldrich) dried for h t 250 C prior to mking cronte stndrds. Serum CO 2 tension (P CO2 ) nd serum [HCO 3 ], sed on mesured ph nd C CO2, were clculted y rerrngement of the Henderson-Hsselch eqution with vlues for CO 2 soluility (αco 2 ) nd pprent pk (pk ) t the pproprite temperture tken from Boutilier et l. (19) nd Truchot (197). βs were derived from liner regression of ph-icronte plots for ech species nd oxygen tretment (Truchot 1979, Cmeron 195, 19). These speciesnd oxygen-specific slopes pper on the pproprite Dvenport digrms s dshed lines (see Fig. ). In vivo experiments. Crs were plced in individul 12 (Chionoecetes tnneri) or 20 l (Cncer mgister) drkened oxes with no hed spce, modertely tightfitting lids, nd wter flow of pproximtely 50 ml min 1. All Cncer mgister experiments were run t 10 C, nd ll Chionoecetes tnneri exposures were run t 3.5 C. Cncer mgister nd Chionoecetes tnneri in the high O 2 tretment were exposed to sewter equilirted vi n exchnge column (Memrn) to gs mixture of 1% CO 2, 20% O 2 ( high O 2 ), nd lnce N 2 delivered from premixed, clirted gs cylinder (Airgs). Chionoecetes tnneri in the low O 2 tretment were exposed in similr fshion to sewter equilirted with cylinder comprising 1% CO 2, 3% O 2 ( low O 2 ), nd lnce N 2. Nominl levels of oxygen sturtion in sewter equilirted with the 2 gs mixtures were pproximtely 95 nd 1%, respectively. Wter ph ws mesured t the pproprite temperture using Rdiometer immersion electrode clirted with Rdiometer Anlyticl precision ph uffers djusted to the ionic strength of sewter. Men wter ph (from ll 3 tretments) t 2 h of hypercpnic exposure ws 7.0 ± 0.01 (SE, n = 1 mesurement). Post-rnchil hemolymph ws smpled immeditely prior to onset of hypercpnic exposure, nd then t 25 min, 50 min, 75 min, 2.5 h,.25 h, nd 2 h of hypercpni. Hemolymph ph ws mesured s descried ove, while C CO2 ws mesured s descried ove on true serum otined from neroic centrifugtion (12000 g for 1 min). Seprte liquots of seprted serum (see ove) were immeditely snp-frozen in liquid N 2 for lter nlysis of protein concentrtion, nd deproteinized in 2 volumes of ice-cold % perchloric cid prior to snp-freezing for lter nlysis of serum lctte concentrtion. Hemolymph dioxide tension (P CO2 ) nd [HCO 3 ], sed on mesured ph nd C CO2, were clculted s descried ove. Serum protein concentrtion ws nlyzed y the method of Brdford (197) using regent nd BSA stndrds from Pierce Biotechnology. Serum lctte ws mesured with commercil kit (regents nd stndrds) from Trinity Biotech. Sttistics. All mesured vlues re presented s men ± 1 SEM (n = numer of crs). Dt were tested for normlity (Shpiro-Wilk test) nd homogeneity of vrince (Levene test) prior to sttisticl tretment. Dt meeting these ssumptions were nlyzed for significnt differences using 1-wy ANOVA followed y Bonferroni s post-hoc multiple comprison test. Dt not meeting prmetric ssumptions were compred using Kruskl-Wllis test followed y multiple comprison testing ccording to the method of Dunn (19), s descried in Zr (19). Sttisticl significnce in ll cses ws ccepted t the p < 0.05 level. RESULTS Over 2 h of hypercpnic exposure, Cncer mgister exhiited lmost complete extrcellulr cid se regultion despite hemolymph ph dropping more quickly nd more shrply over the first hour of hypercpni thn in either Chionoecetes tnneri tretment (Fig. 3A). Compenstion of hemolymph ph in Cncer mgister egn t 75 min nd continued over the reminder of the exposure period, resulting in lmost complete compenstion y 2 h. In neither Chionoecetes tnneri tretment ws ny ph compenstion oserved over the first h. Only t 2 h ws ph slightly recovered in Chionoecetes tnneri cclimted to oth

5 Pne & Brry: Acid se regultion in crs 5 ΔPCO2 (torr) ΔpH A B existent in the low oxygen tretment (Fig. 3C). In neither Chionoecetes tnneri tretment ws ny pprecile gin in hemolymph icronte oserved over the first h. Viewed on ph-icronte (Dvenport) digrm, the overll pttern of hypercpnic-induced cidotic recovery in Cncer mgister ws consistent with tht of competent qutic cid se regultors. In ll 3 tretments, the first hour of hypercpnic exposure resulted in titrtion of hemolymph lood in n cidic direction, roughly down the non-icronte uffering (β) line nd consistent with CO 2 -derived respirtory cidoses (Fig. ). From 1 to h, hemolymph cid se sttus in Cncer mgister followed typicl pttern of metolic compenstion of respirtory cidosis, consistent with lrge gins in hemolymph icronte concentrtion nd movement in n lklotic direction roughly long the torr P CO2 isopleth (Fig. A). Δ[HCO 3 ] (mm) C C. mgister C. tnneri (High O 2 ) C. tnneri (Low O 2 ) Time (h) 3 2 Fig. 3. Cncer mgister nd Chionoecetes tnneri. Delt profiles of (A) ph, (B) P CO2, nd (C) [HCO 3 ] for post-rnchil hemolymph cid-se prmeters during 2 h hypercpnic (1% CO 2 ) exposure. Dt re men ± SE. Tretment mens not shring the sme letter re significntly different high nd low oxygen (Fig. 3A). The resultnt net reductions in hemolymph ph over the 2 h period were 0.0 (C. mgister), 0.32 (Chionoecetes tnneri, high O 2 ), nd 0.3 (Chionoecetes tnneri, low O 2 ) units, nd the net hemolymph ph reduction t 2 h in Cncer mgister ws significntly less thn tht of either tretment of Chionoecetes tnneri (Fig. 3A). The chnge in hemolymph P CO2 (ΔP CO2 ) profile for Cncer mgister ws consistently higher thn those of oth Chionoecetes tnneri tretments throughout the hypercpnic exposure (Fig. 3B). Internliztion of CO 2 in the 2 Chionoecetes tnneri tretments ws similr throughout the exposure period. A sustntil increse (12 mm) in the icronte concentrtion of Cncer mgister hemolymph occurred over the course of 2 h of hypercpni. In contrst, icronte gins in Chionoecetes tnneri were modest (3 mm) in the high oxygen tretment nd non- [HCO3 ] (mm) h 1.5 h C. tnneri (High O 2 ) C. tnneri (Low O 2 ) P CO2 (torr) h 2.5 h 1 h 1.5 h 2 h h 0.5 h h 1 h 2.5 h 1.5 h 0.5 h 2 h 1 h ph 2 h 0 h 0.5 h Fig.. (A) Cncer mgister nd (B) Chionoecetes tnneri. phicronte (Dvenport) digrms showing the time course of post-rnchil hemolymph cid se compenstion during 2 h hypercpnic (1% CO 2 ) exposure. Dt re men ± SE. Solid curved lines re CO 2 isopleths. Stright dshed lines re serum non-icronte uffer (β) lines determined in vitro (see text for detils) 0 h 0 h A B PCO2 (torr)

6 Mr Ecol Prog Ser 33: 1 9, 2007 Serum lctte (mm) Serum protein (g dl 1 ) A In contrst, the cid se vriles for Chionoecetes tnneri held in low oxygen remined elow the nonicronte uffering (β) line t ll time points fter 1 h. This indictes slight metolic cidosis, which compounds the uncompensted respirtory cidosis oserved in this tretment (Fig. B). The serum protein concentrtion of Cncer mgister ws significntly higher thn mesured for Chionoecetes tnneri cclimted to low O 2, oth prior to the onset of hypercpni nd throughout the exposure (Fig. 5A). Additionlly, under hypercpni, serum protein levels in Cncer mgister from 2.5 h onwrd were significntly greter thn those mesured in Chionoecetes tnneri cclimted to high oxygen (Fig. 5A). Serum lctte in Cncer mgister incresed drmticlly (~25-fold) during the first.25 h of hypercpni, only to return to seline levels y 2 h (Fig. 5B). Conversely, serum lctte in Chionoecetes tnneri ws unffected y hypercpni (Fig. 5B). DISCUSSION The deep-se Tnner cr Chionoecetes tnneri did not regulte extrcellulr cid se sttus during B C. mgister C. tnneri (High O 2 ) C. tnneri (Low O 2 ) Time (h) Fig. 5. Cncer mgister nd Chionoecetes tnneri. Time course of (A) serum protein nd (B) serum lctte during 2 h hypercpnic (1% CO 2 ) exposure. Dt re men ± SE. Tretment mens not shring the sme letter re significntly different short-term (2 h) hypercpnic exposure. Compred to the ner-complete extrcellulr compenstory response of the Dungeness cr Cncer mgister, the Tnner cr response to hypercpni ws ineffective (in high O 2 ) or non-existent (in low O 2 ) over 2 h (Figs. 3 & ). Our results indirectly support the hypothesis of Seiel & Wlsh (2001) tht deep-se nimls exchnge ions t the gills more slowly thn their shllow-living counterprts, nd re consistent with generl pttern of reduced rtes of key metolic nd enzymtic processes in deep-se decpod crs, compred to shllow-living species (Henry et l. 1990, Wlsh & Henry 1990). Bicronte cquisition, the primry mens of ph compenstion in qutic nimls, ws not oserved in Tnner crs cclimted to low oxygen, suggesting mrked reduction (or dely) in ion exchnge. In contrst, rpid, hypercpnic-induced extrcellulr icronte cquisition ( net gin of 12 mm over 2 h; Fig. 3B) oserved in Dungeness crs fits the pttern oserved in nother strong cid se regultor, the mrine shore cr Crcinus mens (Truchot 1979, 19). It should e noted tht the current work with Cncer mgister (t 10 C) nd the work y Truchot (1979, 19) with Crcinus mens (t 1 C) were conducted t sustntilly higher tempertures thn the hypercpnic experiments with Chionoecetes tnneri (3 C). In Crcinus mens exposed to ~1% CO 2 t 1 C, relevnt net trnsfers of cid se equivlents cross the rnchil epithelium were ccomplished in to 10 h. The possiility remins, therefore, tht the dptive extrcellulr response of Chionoecetes tnneri to hypercpni my ctully occur ppropritely, just over longer time period thn 2 h. The discrepncy in hemolymph ΔP CO2 profiles (Fig. 3B) during hypercpnic exposure (with Cncer mgister internlizing more CO 2 thn the 2 Chionoecetes tnneri tretments) is t odds with the common notion tht CO 2 equilirtes rpidly mong the externl medium, the extrcellulr fluid, nd the intrcellulr comprtment. A simple explntion my involve trnsitory experimentl vritions in wter CO 2 tensions cused y vrile gs exchnge with the equilirtion columns employed. While this would esily explin differences occurring in the first h of hypercpnic exposure, 2 h wter phs were very similr mong the 3 tretments, suggesting tht the wter CO 2 prtil pressures were lso very similr t 2 h. Yet the hemolymph ΔP CO2 vlue in Cncer mgister remined greter thn those of the Tnner crs. Cmeron (19) descries in detil the disequilirium conditions of inverterte lood with respect to CO 2 internliztion nd excretion, concluding tht CO 2 in inverterte lood most likely never reches equilirium conditions. Such n occurrence would certinly confound ssumptions nd discussions of CO 2 equilirium dur-

7 Pne & Brry: Acid se regultion in crs 7 ing hypercpnic exposure nd my ccount for the inconsistencies seen in Fig. 3B. It is lso possile tht the CO 2 soluility constnts nd pprent pk vlues employed, derived from literture using Crcinus mens nd temperturedjusted ccordingly, re not entirely consistent with the ctul lood chemistry of Chionoecetes tnneri. Such discrepncy could e function of ltered hydrosttic pressure dynmics involved in working with deep-se nimls t mient pressures, nd could esily explin the minor differences in the ΔP CO2 profiles oserved etween the 2 species. The key point, however, is tht Dungeness crs were le to effectively regulte their hemolymph cid se sttus despite n pprent higher internl CO 2 lod thn tht oserved in Tnner crs. In this study, cid se sttus ws monitored in the extrcellulr fluid for 2 h without mesuring intrcellulr ph (phi). There is evidence tht qutic crustcens cn regulte phi independently of extrcellulr ph (phe) (Henry & Whetly 1992, Whetly & Henry 1992), mintining constnt phi during hypercpnicinduced cidifiction of the extrcellulr fluid (Gillrd & Mln 193). The Chinese mitten cr Eriocheir sinenesis ctully incresed phi during n extrcellulr cidosis, presumly vi rpid exchnge of cid se equivlents etween the intr- nd extr-cellulr comprtments (Whiteley et l. 2001). While constncy of phi in the fce of mrked cifidiction of the extrcellulr comprtment my hve occurred in Chionoecetes tnneri exposed to hypercpni, this strtegy is one more typiclly ssocited with strong cid se regultors ntive to highly vrile environments. Therefore, we postulte tht the intrcellulr ph of Chionoecetes tnneri is likely compromised s consequence of uncompensted cidifiction of the extrcellulr comprtment, though this is n re of reserch we re ctively pursuing. Regrdless of the extent of phi regultion, mintennce of phe within firly tight limits is criticl to proper function of extrcellulr respirtory proteins, nd consequently oxygen delivery to tissues (Whetly & Henry 1992). Using the Dungeness cr, Johnsen et l. (1970) clculted Bohr shift tht would ccount for 50% increse in the hemolymph P 50 (hemolymph oxygen tension t which hemocynin is hlf-sturted) following 0. unit decrese in hemolymph ph. A similr effect with Chionoecetes tnneri hemolymph would gretly reduce oxygen crrying cpcity nd susequent delivery to respiring tissues. The inility of Chionoecetes tnneri to sustntilly ccumulte extrcellulr icronte from the environment my lso involve poor icronte retention cpcity. Even in strong cid se regultors, ph compenstion y uptke of icronte equivlents from the surrounding medium is limited y the mount of icronte tht cn e retined in the extrcellulr fluid (Truchot 1979). Mintennce of elevted concentrtions of extrcellulr icronte requires, within the kidney, commitment of sustntil frction of energeticlly costly proton pumping in order to resor vlule icronte ions tht would otherwise e lost in the urine (Truchot 197). We re currently undertking icronte infusion studies with Chionoecetes tnneri to determine the extent to which poor cid se regultion in Tnner crs is function of limited ility to cquire, or to retin, icronte. Both the net cquisition of sic equivlents from sewter, nd renl retention of icronte in the urine, require energeticlly costly epithelil ion pumping. Net cid excretion (or net se ccumultion) during hypercpni my occur t the direct expense of metolic ATP in the cse of H + -ATPses pumping protons cross epitheli, or indirectly in the cse of electroneutrl exchnge vi N + /H + or Cl /HCO 3 ntiporters (see Ahern et l for review). In the ltter cse, electroneutrl N + /H + nd Cl /HCO 3 exchnge show clerly the close coupling of ionic, osmotic, nd cid se regultion, highlighting the importnce of the primry epithelil ion pump, N + - K + -ATPse in correction of ion grdients distured y the necessity of hypercpnic cid se regultion (Ahern et l. 1999). Furthermore, in the cse of decpod crustcens, N + /H + exchnge is electrogenic (2N + /H + ), possily requiring direct synergistic interction with n ATP-dependent ion pump such s vcuolr, or V-type, H + -ATPse (Kimur et l. 199). Brnchil pumping of cid se relevnt ions to offset cid se disturnces cn e n energeticlly costly process, nd one which my e limited in deepse enthic crustcens which hve metolic rtes typiclly n order of mgnitude lower thn their shllow-living counterprts (Childress et l. 1990). Our results, showing tht the sluggish deep-se Tnner cr hs limited short-term extrcellulr cid se compenstory cpcity following hypercpni, support the theory (Seiel & Wlsh 2001, Childress & Seiel 199) tht deep-se nimls will e prticulrly sensitive to hypercpni. As indirect evidence tht the cid se regultory cpcity of deep-se nimls is mldptive to severe cid se disturnce due to lowered metolic rtes, short-term extrcellulr ph compenstion, nd extrcellulr icronte ccumultion, were modertely improved when Tnner crs were held under conditions of high oxygen, essentilly hyperoxic to their in situ conditions (Fig. 3). In shllow-living decpod crustcens, lctte uildup in the extrcellulr comprtment increses hemocynin oxygen ffinity nd cts s metolic signling compound to increse oxygen consumption rte (Lllier & Truchot 199, De wtcher et l. 1997, Bridges

8 Mr Ecol Prog Ser 33: 1 9, ). Accordingly, lctte moiliztion in Cncer mgister during hypercpni corresponds well with the need for upregultion of metoliclly costly cid se regultory trnsport phenomen (Fig. 5B). In Tnner crs held in oth oxygen levels, however, lctte ws sent from the serum prior to, nd throughout, the 2 h exposure period (Fig. 5B). This devince of the serum lctte pttern of Chionoecetes tnneri from the typicl pttern displyed y Cncer mgister (Fig. 5B) nd other shllow-living decpod crustcens (Bridges 2001), suggests tht Chionoecetes tnneri relies on nother signling molecule, such s urte or mgnesium (Bridges 2001) to effect the pproprite metolic response. Additionlly, it is possile tht Chionoecetes tnneri produces lctic cid intrcellulrly during hypercpnic stress, ut does not relese lctte from the tissue. Extrcellulr relese of the proton ccompnying lctte, however, would explin the metolic cid component evident in the Dvenport digrms for Chionoecetes tnneri (Fig. B). The dt points lying elow the pssive uffering line (1 to h in Tnner crs cclimted to high O 2 nd 1 to 2 h in Tnner crs cclimted to low O 2 ) indicte comintion of respirtory nd metolic cid input into the extrcellulr fluid. While the respirtory component is protons derived from hydrted CO 2, the metolic component could stem from lctic cid-derived protons, nd future work in our lortory will focus on tissue metolic nd cid se nlyses during hypercpnic exposure. CONCLUSIONS This report represents, to our knowledge, one of the first detiled investigtions of cid se regultory ptterns in deep-se niml. Under the cron sequestrtion scenrio, generlly involving deposition of lrge mounts of liquid CO 2 on the deep se floor, short-term tolernce of high CO 2 concentrtions will e vitl. Storge lkes of CO 2 in the deep-se enthos (Ohsumi 1993) will e ccompnied y lrge locl wter ph excursions (Adms et l. 1997). In such cses, the ~0. unit drop in serum ph in Chionoecetes tnneri exposed to 1% CO 2 nd simultneously cclimted to low O 2, over 2 h (Fig. 3A), supports the hypothesis tht the consequences of lrge-scle CO 2 sequestrtion will e physiologiclly chllenging to deep-se nimls t lest in the short term. Additionlly, the dt support the synergistic effects of hypoxi nd hypercpni (see Portner et l. 200 for review). Under lortory conditions, oxygen limittion excerted extrcellulr hypercpnic-induced cidosis in the deep-se Tnner cr. Prcticlly, then, CO 2 sequestrtion in n re of the deep-se tht is nturlly hypoxic (such s the OMZ of the estern Pcific) my hve greter detrimentl effect on deep-se fun. Even in the sence of lrge-scle sequestrtion, elevted tmospheric CO 2 is continully eing sored y the world s ocens, resulting in ph depression of pproximtely 0.1 units to dte, with projected dditionl 0. unit reduction y the yer 2100 (Hugn & Drnge 199, Cldeir & Wickett 2003, Glover & Smith 2003, Sine et l. 200). This process of pssive CO 2 influx, left unted for mny yers nd comined with long-term turnover of deep ocen wter, will eventully result in consistently hypercpnic conditions in the deep se. Accordingly, chronic hypercpnic exposures re needed to understnd how deep-se nimls will fre with such chllenge. Acknowledgements. We grtefully cknowledge the tlent nd effort of the crews of the RV Point Loos nd the ROV Ventn. Additionlly, we thnk Ptrick Whling, Kurt Buck, Chris Lover, Gernot Friederich, Eric Nelson, nd Crig Okud. These studies were supported y the Monterey By Aqurium Reserch Institute (Project ), the Ocen Cron Sequestrtion Reserch Progrm, Biologicl nd Environmentl Reserch (BER), US Deprtment of Energy (Awrd No. DE- FG03-01DF305 nd DE-FG02-0ER3721), nd the Ntionl Energy Technology Lortory (NETL), US Deprtment of Energy (Awrd No. DE-FC2-00NT0929). LITERATURE CITED Adms EE, Culfield AJ, Herzog HJ, Auerch DI (1997) Impcts of reduced ph from ocen CO 2 disposl: sensitivity of zooplnkton mortlity to model prmeters. Wste Mnge 17: Ahern GA, Duerr JM, Zhung Z, Brown RJ, Aslmkhn A, Killerew DA (1999) Ion trnsport processes of crustcen epithelil cells. Physiol Biochem Zool 72:1 1 Airriess CN, McMhon BR (199) Crdiovsculr dpttions enhnce tolernce of environmentl hypoxi in the cr Cncer mgister. J Exp Biol 190:23 1 Brry JP, Buck KR, Lover CL, Kuhnz L, Whling PJ, Peltzer ET, Wlz P, Brewer PG (200) Effects of direct ocen CO 2 injection on deep-se meiofun. J Ocenogr 0:759 7 Boutilier RG, Heming TA, Iwm GK (19) Physiochemicl prmeters for use in fish respirtory physiology. In: Hor WS, Rndll DJ (eds) Fish physiology. Acdemic Press, London, p Brdford MM (197) A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye inding. Anl Biochem 72:2 25 Brewer PG, Peltzer ET, Friederich G, Ay I, Ymne K (2000) Experiments on the ocen sequestrtion of fossil fuel CO 2 : ph mesurements nd hydrte formtion. Mr Chem 72:3 93 Bridges CR (2001) Modultion of hemocynin oxygen ffinity: properties nd physiologicl implictions in chnging world. J Exp Biol 20: Cldeir K, Wickett ME (2003) Anthropogenic cron nd ocen ph. Nture 25:35 Cmeron JN (197) Effects of hypercpni on lood cid se sttus, NCl fluxes, nd trns-gill potentil in freshwter lue crs, Cllinectes spidus. J Comp Physiol 123: Cmeron JN (195) Compenstion of hypercpnic cidosis in the

9 Pne & Brry: Acid se regultion in crs 9 Editoril responsiility: Howrd Browmn (Associte Editorin-Chief), Storeø, Norwy qutic lue cr, Cllinectes spidus: the predominnce of externl sewter over crpce cronte s the proton sink. J Exp Biol 11: Cmeron JN (19) Acid se equiliri in invertertes. In: Heisler N (ed) Acid se regultion in nimls. Elsevier, New York, p Childress JJ (1995) Are there physiologicl nd iochemicl dpttions of metolism in deep-se nimls? Trends Ecol Evol 10:30 3 Childress JJ, Seiel BA (199) Life t stle low oxygen levels: dpttions of nimls to ocenic oxygen minimum lyers. J Exp Biol 201: Childress JJ, Cowles DL, Fvuzzi JA, Mickel TJ (1990) Metolic rtes of enthic deep-se decpod crustcens decline with incresing depth primrily due to the decline in temperture. Deep-Se Res 37: Cliorne JB, Heisler N (19) Acid se regultion nd ion trnsfers in the crp (Cyprinus crpio): ph compenstion during grded long- nd short-term environmentl hypercpni, nd the effect of icronte infusion. J Exp Biol 12:1 1 Dvenport HW (197) The ABC of cid se chemistry. University of Chicgo Press, Chicgo, IL De Wtcher B, Srtoris FJ, Portner HO (1997) The neroic endproduct lctte hs ehviourl nd metolic signling function in the shore cr Crcinus mens. J Exp Biol 200: Dunn OJ (19) Multiple contrsts using rnk sums. Technometrics : Edwrds SL, Wll BP, Morrison-Shetlr A, Sligh S, Wekley JC, Cliorne JB (2005) The effect of environmentl hypercpni nd slinity on the expression of NHE-like isoforms in the gills of euryhline fish (Fundulus heteroclitus). J Exp Zool 303A: 75 Forgue J, Mssuu JC, Truchot JP (1992) When re resting wter-rethers lcking O 2? Arteril P O2 t the neroic threshold in cr. Resp Physiol :27 25 Friederich GE, Wlz PM, Burczynski MG, Chvez FP (2002) Inorgnic cron in the centrl Cliforni upwelling system during the El Niño-L Niñ event. Prog Ocenogr 5: Gge JD, Tyler PA (1991) Deep-se iology nturl history of orgnisms t the deep-se floor. Cmridge University Press, Cmridge Gillrd S, Mln A (193) Intrcellulr ph regultion in response to mient hyperoxi or hypercpni in the cryfish. Mol Physiol : Glover AG, Smith CR (2003) The deep-se floor ecosystem: current sttus nd prospects of nthropogenic chnge y the yer Environ Conserv 30: Hugn PM, Drnge H (199) Effects of CO 2 on the ocen environment. Energy Convers Mnge 37: Henry RP, Whetly MG (1992) Interction of respirtion, ion regultion, nd cid se lnce in the everydy life of qutic crustcens. Am Zool 32:07 1 Henry RP, Perry HM, Trigg CB, Hndley HL, Krrup A (1990) Physiology of two species of deep-wter crs, Chceon fenneri nd C. quinquedens: gill morphology, nd hemolymph ionic nd nitrogen concentrtions. J Crustc Biol 10: Johnsen K, Lenfnt C, Mecklenurg TA (1970) Respirtion in the cr, Cncer mgister. Z Vgl Physiol 70:1 19 Kennett JP, Ingrm BL (1995) A 20,000-yer record of ocen circultion nd climte chnge from the Snt Brr sin. Nture 377: Kimur C, Ahern GA, Busquets-Turner L, Hley SR, Ngo C, De Couet HG (199) Immunolocliztion of n ntigen ssocited with the inverterte electrogenic 2N + /1H + ntiporter. J Exp Biol 19:9 10 Lllier F, Truchot JP (199) Hemolymph oxygen trnsport during environmentl hypoxi in the shore cr, Crcinus mens. Resp Physiol 77: Lng MA, Giner H (199) Isosmotic intrcellulr regultion s mechnism of volume control in cr muscle fiers. Comp Biochem Physiol 30:5 5 Lngenuch M, Portner HO (2002) Chnges in metolic rte nd N excretion in the mrine inverterte Sipunculus nudus under conditions of environmentl hypercpni: identifying effective cid se vriles. J Exp Biol 205: Mrchetti C (1977) On geoengineering nd the CO 2 prolem. Clim Chnge 1:59 Mrchetti C (1979) Constructive solutions to the CO 2 prolem. In: Bch W, Pnkrth J, Kellogg W (eds) Mn s impct on climte. Elsevier Science, Amsterdm, p Morris RH, Aott DP, Hderlie EC (190) Intertidl invertertes of Cliforni. Stnford University Press, Stnford, CA Ohsumi T (1993) Prediction of solute cron dioxide ehviour round liquid cron dioxide pool on deep ocen sins. Energy Convers Mnge 3: Portner HO, Lngenuch M, Reipschlger A (200) Biologicl impct of elevted ocen CO 2 concentrtions: lessons from niml physiology nd erth history. J Ocenogr 0: Sine CL, Feely RA, Gruer N, Key RM nd 11 others (200) The ocen sink for nthropogenic CO 2. Science 305: Seiel BA, Wlsh PJ (2001) Potentil impcts of CO 2 injection on deep-se iot. Science 29: Seiel BA, Thuesen EV, Childress JJ, Gorodezky LA (1997) Decline in pelgic cephlopod metolism with hitt depth reflects differences in locomotory efficiency. Biol Bull 192: Tmurri MN, Peltzer ET, Friederich GE, Ay I, Ymne K, Brewer PG (2000) A field study of the effects of CO 2 ocen disposl on moile deep-se nimls. Mr Chem 72: Toews DP, Holeton GF, Heisler N (193) Regultion of the cid se sttus during environmentl hypercpni in the mrine teleost fish Conger conger. J Exp Biol 107:9 20 Truchot JP (197) Cron dioxide comining properties of the lood of the shore cr Crcinus mens (L.): cron dioxide soluility coefficient nd cronic cid dissocition constnts. J Exp Biol :5 57 Truchot JP (1979) Mechnisms of the compenstion of lood respirtory cid se disturnces in the shore cr, Crcinus mens (L.). J Exp Zool 210:07 1 Truchot JP (19) Wter cronte lklinity s determinnt of hemolymph cid se lnce in the shore cr, Crcinus mens: study t two different mient P CO2 nd P O2 levels. J Comp Physiol B 15:01 0 Truchot JP (197) Comprtive spects of extrcellulr cid se lnce. Springer-Verlg, Berlin Wlsh PJ, Henry RP (1990) Activities of metolic enzymes in the deep-wter crs Chceon fenneri nd C. quinquedens nd the shllow-wter cr Cllinectes spidus. Mr Biol 10: 33 3 Whetly MG, Henry RP (1992) Extrcellulr nd intrcellulr cid se regultion in crustcens. J Exp Zool 22: Whiteley NM, Scott JL, Breeze SJ, McCnn L (2001) Effects of wter slinity on cid se lnce in decpod crustcens. J Exp Biol 20: Wyrtki K (192) The oxygen minim in reltion to ocen circultion. Deep-Se Res 9:11 23 Zr JH (19) Biosttisticl nlysis. Prentice-Hll, Englewood Cliffs, NJ Sumitted: Octoer 10, 200; Accepted: Ferury 15, 2007 Proofs received from uthor(s): Mrch 7, 2007