Metabolic, respiratory and cardiovascular responses to acute and chronic hypoxic exposure in tadpole shrimp Triops longicaudatus

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1 1639 The Journl of Experimentl Biology 209, Published by The Compny of Biologists 2006 doi: /jeb Metbolic, respirtory nd crdiovsculr responses to cute nd chronic hypoxic exposure in tdpole shrimp Triops longicudtus S. L. Hrper 1, * nd C. L. Reiber 2 1 Deprtment of Environmentl nd Moleculr Toxicology, Oregon Stte University, Environmentl Helth Sciences Center, 1011 ALS, Corvllis, OR 97331, USA nd 2 Deprtment of Biology, University of Nevd, Ls Vegs, NV 89154, USA *Author for correspondence (e-mil: hrpers@science.oregonstte.edu) Accepted 20 Februry 2006 Hypoxic exposure experienced during sensitive developmentl periods cn shpe dult physiologicl cpbilities nd define regultory limits. Tdpole shrimp were rered under normoxic (19 21 kp O 2 ), moderte (10 13 kp O 2 ) or severe (1 3 kp O 2 ) hypoxic conditions to investigte the influence of developmentl oxygen prtil pressure (P O 2) on dult metbolic, respirtory nd crdiovsculr physiology. Developmentl P O 2 hd no effect on metbolic rte or metbolic response to hypoxic exposure in dults. All rering groups decresed O 2 consumption s wter P O 2 decresed. Hert rte, stroke volume nd crdic output were independent of P O 2 down to 5 kp O 2 in ll rering groups. Below this, crdic output ws mintined only in tdpole shrimp rered under severe hypoxic conditions. The enhnced bility to mintin crdic output ws ttributed to n increse in hemoglobin concentrtion nd O 2 -binding ffinity in those nimls. Oxygen-delivery potentil ws lso significntly Summry higher in the group rered under severe hypoxic conditions (1336 l O 2 min 1 ) when compred with the group rered under normoxic conditions (274 l O 2 min 1 ). Differences mong the rering groups tht were dependent on hemoglobin were not considered developmentl effects becuse hemoglobin concentrtion could be incresed within seven dys of hypoxic exposure independent of developmentl P O 2. Hypoxi-induced hemoglobin synthesis my be compenstory mechnism tht llows tdpole shrimp to regulte O 2 uptke nd trnsport in euryoxic (O 2 vrible) environments. The results of this study indicte tht incresed hemoglobin concentrtion, incresed O 2 -binding ffinity nd trnsient decreses in metbolic demnd my ccount for tdpole shrimp hypoxic tolernce. Key words: invertebrte, development, physiology, hypoxi, oxygen prtil pressure. Introduction Hypoxic exposure cn elicit rnge of physiologicl nd metbolic responses in qutic crustcens with oxygen conformtion nd oxygen regultion t the ends of response continuum (Hochchk, 1988; McGw et l., 1994; Reiber, 1995; Reiber nd McMhon, 1998; Wilkens, 1999). Oxygen conformers re ble to reduce metbolic demnd so tht O 2 consumption (V O 2) is coupled to environmentl O 2 prtil pressure (P O 2) (Loudon, 1988; Hochchk et l., 1999; Boutilier, 2001). Oxygen regultors, by contrst, mintin V O 2 independent of environmentl P O 2 down to point where the O 2 required for erobic processes becomes limiting (P CRIT ) (Herreid, 1980; Hochchk, 1988). Below P CRIT, either tissue oxygen demnd decreses or nerobic processes become incresingly importnt in meeting energy requirements (Hochchk, 1988). Above P CRIT, O 2 uptke nd delivery my be enhnced through vriety of compenstory mechnisms including djusting ventiltory prmeters (rtes nd volumes), incresing internl convective processes such s hert rte nd stroke volume nd chnging perfusion ptterns (McMhon, 1988; Hervnt et l., 1995; Willmer et l., 2000; Boutilier, 2001; Hochchk nd Lutz, 2001; Hopkins nd Powell, 2001; Hoppeler nd Vogt, 2001). Additionlly, some nimls cn modulte the O 2 -binding ffinity of their respirtory proteins, which enhnces O 2 uptke t the respirtory surfce nd increses totl O 2 cpcitnce (Wells nd Wells, 1984; Kobyshi et l., 1988; Hochchk et l., 1999; Willmer et l., 2000). Hypoxic exposure during development (embryonic or lrvl periods) my elicit similr suite of compenstory responses or my result in long-term or permnent chnges to the morphology nd/or physiology of the niml nd thus impct the dult hypoxic response (Bmber nd Depledge, 1997; Brdley et l., 1999; Willmer et l., 2000; Gozl nd Gozl, 2001; Peyronnet et l., 2002). The physiologicl consequences of hypoxi-induced developmentl plsticity cn be reduced

2 1640 S. L. Hrper nd C. L. Reiber metbolic rte (Hochchk et l., 1999; Boutilier, 2001), greter respirtory gs exchnge surfce re (Loudon, 1988; Loudon, 1989), incresed respirtory cpcities nd/or enhnced convection nd O 2 delivery mechnisms (Bnchero, 1987; McMhon, 1988; Grhm, 1990; Szewczk nd Jckson, 1992; Childress nd Seibel, 1998; Pelster, 1999; McMhon, 2001). Trnscriptionl regultion ccounts for mny of these long-term physiologicl nd morphologicl chnges ssocited with developmentl exposure to hypoxi. A well-documented exmple of this type of developmentl hypoxic response in crustcens is the increse of existing O 2 -binding proteins s well s the production of new higher-ffinity proteins tht result in long-term increses in O 2 -uptke nd cpcitnce (Wells nd Wells, 1984; Snyder, 1987; Kobyshi et l., 1988; Hervnt et l., 1995; Astll et l., 1997; Hou nd Hung, 1999; Wiggins nd Frppell, 2000; Brros et l., 2001). The degree to which developmentl P O 2 influences dult metbolic, respirtory nd crdiovsculr hypoxic responses ws investigted using tdpole shrimp Triops longicudtus. Severl fetures of tdpole shrimp mke them well suited to study the effects of cute nd developmentl hypoxic exposures. They re often fced with environmentl P O 2s below their P CRIT, yet the mechnisms by which they tolerte such conditions re poorly understood (Horne nd Beyenbch, 1971; Hillyrd nd Vinegr, 1972; Eriksen nd Brown, 1980; Scholnick, 1995; Scholnick nd Snyder, 1996). Tdpole shrimp hve high metbolic rtes nd respirtory structures (epipodites) tht re thought to be indequte to mintin O 2 uptke in their euryoxic hbitts (Fryer, 1988; Horne nd Beyenbch, 1971; Hillyrd nd Vinegr, 1972; Scott nd Grigrick, 1978; Eriksen nd Brown, 1980; Scholnick, 1995; Scholnick nd Snyder, 1996). A tubl, myogenic hert devoid of vsculture produces the only internl convective currents in tdpole shrimp (Ymgishi et l., 1997; Ymgishi et l., 2000). Lrge, extrcellulr hemoglobin (29 subunits) is produced in response to hypoxic exposure, but the mechnisms of this hypoxic induction re not known (Horne nd Beyenbch, 1971; Scholnick nd Snyder, 1996). Finlly, tdpole shrimp hve comprtively rpid genertion time nd menbility to lbortory culture, which mke them trctble orgnisms for developmentl investigtions (Fryer, 1988; Horne nd Beyenbch, 1971; Scott nd Grigrick, 1978). Tdpole shrimp were rered under normoxic (19 21 kp O 2 ), moderte (10 13 kp O 2 ) or severe (1 3 kp O 2 ) hypoxic conditions to determine whether differences in developmentl P O 2 were sufficient to chnge dult metbolic, respirtory or crdiovsculr system physiology nd hypoxic sensitivity. Compenstory respirtory nd crdiovsculr processes tht enhnce O 2 uptke, internl convection nd perfusion should increse in response to hypoxic exposure. We hypothesized tht tdpole shrimp rered under hypoxic conditions would hve decresed metbolic sensitivity to P O 2 chnge, incresed physiologicl responses to hypoxic exposure nd incresed hemoglobin concentrtion nd O 2 -binding ffinity reltive to those rered under normoxic conditions (Wells nd Wells, 1984; Snyder, 1987; Kobyshi et l., 1988; Hervnt et l., 1995; Astll et l., 1997; Hochchk et l., 1999; Hou nd Hung, 1999; Brros et l., 2001; Boutilier, 2001). Mterils nd methods Study orgnism nd rering conditions Sediments contining tdpole shrimp (Triops longicudtus LeConte) cysts were collected from n ephemerl pool in Brownstone Bsin (12.7 km west of Ls Vegs, NV, USA; 1425 m elevtion; N, W). Wter P O 2 nd temperture in Brownstone Bsin pool rnged from 2 to 32 kp O 2 nd 17.6 to 31.7 C, respectively (Hrper, 2003). Three 150- liter insulted quri (normoxic=19 21 kp O 2, moderte hypoxic=10 13 kp O 2 nd severe hypoxic=1 3 kp O 2 ) were set up in the lbortory using deionized wter nd sediments tken from the pool. A model GF-3/MP gs flowmeter (Cmeron Instruments Compny, Ontrio, Cnd) ws used to regulte mixture of N 2 nd room ir. Oxygen prtil pressure in ech quri ws monitored for 48-h period ech week using dt-logging dissolved O 2 meters (Model 810 Orion Dissolved Oxygen Meter; Orion Reserch, Boston, MA, USA). Ech qurium ws equipped with ultrviolet (3% UVB nd 7% UVA) enhnced lights (Super UV Reptile Dylight Lmp; 20 W; Energy Svers Unlimited Inc., Chicgo, IL, USA) tht estblished 13 h:11 h L:D cycle. A timed circulting wter bth (Model VT513; Rdiometer, Copenhgen, Denmrk) nd het exchnge coil were used to cycle temperture with the lights. The quri strted to wrm two hours fter the lights were turned on nd continued for five hours ech dy to estblish C temperture cycle. Dry sediments (100 g) contining tdpole shrimp cysts were dded to ech qurium weekly. Animls te lge, detritus nd smll invertebrtes tht htched from the sediment. Tdpole shrimp were identified s Triops longicudtus (Sssmn, 1991). Aquri were drined nd refilled monthly. Metbolism Stndrd metbolic rte ws mesured when nimls were ctive becuse they were infrequently quiescent. To ssess the confounding effect of P O 2 on ctivity, individul nimls (N=10) were plced in mrked cylindricl 10-ml flowthrough chmber nd videotped during progressive hypoxic exposure. Animls were cclimted (30 min) to the chmber under normoxic conditions. A gs flowmeter (Model GF- 3/MP) ws used to regulte the mixture of N 2 nd ir. Chmber P O 2 ws decresed from 20 to 2 kp O 2 t rte of 5 kp O 2 h 1. The number of times the niml crossed defined mrks on the chmber ws verged over one-minute intervls to produce n index of ctivity in response to P O 2. Individul nimls from ech rering group (N=7 per rering group) were plced in 125-ml closed-system drkened respirometry chmber t 28 C. The chmber hd plstic grte on the bottom under which mgnetic stirring rod ws plced to ensure thorough mixing of the chmber wter. Animls were cclimted for 30 min nd then the chmber ws seled.

3 Hypoxic responses in crustcens 1641 Oxygen content of the chmber ws monitored using model 781 Strthkelvin O 2 meter (Strthkelvin Instruments, Glsgow, UK). Oxygen consumption ws clculted bsed on the following eqution: V O 2 = (V r P WO 2 WO 2) / tm d, (1) where V O 2 is oxygen consumption, V r is the volume of wter in the respirometer, P WO 2 is the chnge in oxygen concentrtion of the wter, WO 2 is the cpcitnce of oxygen in wter, t is durtion in minutes, nd M d is the dry mss of the niml mesured in grms (Piiper et l., 1971). Individul tdpole shrimp dry mss ws determined by drying the niml t 60 C until three constnt mss mesurements were obtined. Oxygen consumption rtes were clculted for successive 5- min intervls nd expressed s mss-specific O 2 uptke ( l O 2 g 1 h 1 ). The experiment ws performed without nimls in the chmber, nd the V O 2 rtes obtined from three replictes were used to clculte men correction fctor for microbil respirtion. Animls rered under normoxic (N=10) nd severe hypoxic (N=10) conditions were used to ssess nerobic lctte metbolism in tdpole shrimp. Lctte concentrtion ws mesured for five nimls pre-tretment nd five nimls fter exposure to severe hypoxic conditions (2 kp O 2 ) for 12 h t 23 C. Lctte concentrtions in the experimentl chmber wter were lso determined. Hypoxic conditions were mintined using gs flowmeter (Model GF-3/MP) to control the mixture of N 2 nd room ir. Hemolymph ws collected in glss cpillry tubes by dorsl hert puncture. Lctte concentrtions were determined for 10 l wter smples nd 10 l hemolymph smples mixed with 1.0 ml lctte regent solution (#735-10; Trinity Biotech, St Louis, MO, USA). Hemolymph lctte concentrtions were mesured enzymticlly (Sigm Dignostics, St Louis, MO, USA; Sigm Lctte Kit #735) t 540 nm. Ventiltion Ventiltory rte nd mplitude were mesured in response to hypoxic exposure in order to ssess the effects of developmentl P O 2 on dult ventiltory hypoxic response. Adult tdpole shrimp from ech rering group (N=13 per rering group) were held in 30-ml flow-through chmber (temperture, 25 C). Tdpole shrimp were secured in the chmber with n pplictor stick nd cynocrylte glue on the lterl crpce. They were inverted in the chmber to llow the ventrl surfce to be viewed. Movements of the respirtory ppendges were videotped (60 Hz smpling speed) under dissecting microscope (Leic Stereozoom 6 Photo) using video cmer (Oscr Color Cmer Vidcm), super VHS video recording system (Pnsonic PV-54566) nd Horit time code genertor (VG 50; Horit Co., Inc., Mission Viejo, CA, USA). Tdpole shrimp were cclimted for 30 min nd then exposed to four environmentl P O 2s (20, 13.3, 10 nd 1 kp O 2 ) in rndom order. Thirty minutes ws llowed for cclimtion once new P O 2 ws chieved. Animls were not returned to normoxi between exposures. Time-encoded video ws nlyzed frme-by-frme on n editing tpe plyer (Pnsonic AG-DS550) to determine ventiltion rte nd mplitude. Ventiltion rte (frequency) ws mesured s number of ppendge bets per minute. The mplitude of ppendge bets ws determined s the men distnce tht the 4th nd 5th ppendges seprte during five subsequent ventiltory strokes (Hrper, 2003). Crdic physiology Hert rte, stroke volume nd crdic output were mesured in response to hypoxic exposure in order to ssess the effects of developmentl P O 2 on dult crdic hypoxic responses. Adult tdpole shrimp from ech rering group (N=13 per rering group) were secured in 30-ml flow-through experimentl chmber s previously described. Animls were cclimted for 30 min nd then exposed to five environmentl P O 2s (26.7, 20, 13.3, 10 nd 4 kp O 2 ) in rndom order. Thirty minutes ws llowed for cclimtion t ech P O 2. Crdic contrctions were videotped s previously described. Hert rte (fh; bets min 1 ) ws mesured when the time-encoded video ws dvnced frme-by-frme on n editing tpe plyer (Pnsonic AG-DS550, Cypress, CA). The tdpole shrimp hert ws modeled s cylinder with volume of r 2 h, where r is hlf the width of the hert nd h is length. Imges of the hert were collected during mximl [end distolic volume (EDV)] nd miniml [end systolic volume (ESV)] distention. Those imges were dimensionlly nlyzed using Scion Imging softwre (Ntionl Institutes of Helth, Bethesd, MD, USA). Stroke volume (VS; l bet 1 ) ws clculted s the difference in hert volume between EDV nd ESV. Crdic output (Q; l min 1 ) ws clculted s the product of fh nd VS. Hemoglobin Hemoglobin is the mjor protein in tdpole shrimp hemolymph (Horne nd Beyenbch, 1971). Protein concentrtions of nimls from ech rering group (N=10 per rering group) were determined to ssess the influence of developmentl P O 2 on hemoglobin production. Protein concentrtions were determined for tdpole shrimp rered under normoxic conditions nd exposed to severe hypoxic conditions for 5, 7 nd 10 dys (N=7 per dy). Likewise, protein concentrtions were determined for tdpole shrimp rered under severe hypoxic conditions nd exposed to normoxic conditions for 5, 7 nd 10 dys (N=7 per dy). Protein concentrtions were determined using Micro BCA Protein Assy Regent Kit (#23235; Pierce, Rockford, IL, USA). Protein stndrds (2.0 mg ml 1 BSA in solution of 0.9% sline nd 0.05% sodium zide) were diluted with tdpole shrimp sline (5.84 mg NCl, 7.45 mg KCl, mg CCl 2, 9.52 mg MgCl 2, 3.65 mg HCl nd 1 ml H 2 O) (Ymgishi et l., 2000) to form solutions with finl BSA concentrtions of 200, 40, 20, 10, 5, 2.5, 1 nd 0.5 g ml 1. Working regent ws prepred by mixing 12.5 ml Micro BCA Regent MA (sodium crbonte, sodium bicrbonte nd sodium trtrte in 0.2 mol l 1 NOH) nd 12 ml Micro BCA

4 1642 S. L. Hrper nd C. L. Reiber Regent MB [bicinchoninic cid (4.0%) in wter] with 0.5 ml Micro BCA Regent MC (4.0% cupric sulfte, penthydrte in wter). One milliliter of ech stndrd ws dded to ppropritely lbeled test tubes. A wter blnk nd tdpole shrimp sline were used s controls. In ech test tube, 1.0 ml working regent ws dded nd mixed. The tubes were covered with Prfilm TM nd plced in 60 C wter bth for 60 min nd then cooled to room temperture (23 C). Absorbnce ws mesured t 562 nm, with corrections mde for reference. A stndrd curve ws produced to obtin hemoglobin concentrtions of hemolymph smples. Hemoglobin O 2 -binding ffinities were mesured for nimls from ech rering group to determine differences dependent on developmentl P O 2. Hemolymph (60 l) ws collected in glss cpillry tubes from lrge tdpole shrimp (N=7 per rering group) by dorsl puncture of the hert. Hemolymph ws dded into smll flow-through tonometer tht opened into nrrow chmber (1 mm inner dimeter) inside cuvette (1 cm 1 cm 5 cm). The tonometer ws plced on its side when hemolymph ws dded nd during ech equilibrtion step. This llowed the hemolymph to flow into the bulbous region of the tonometer. A stirring fle powered by mgnetic stirrer ws plced into the tonometer to ensure thorough mixing of the hemolymph with inflowing gs mixtures. During spectrophotometric mesurements, the tonometer ws held upright so tht the hemolymph flowed into the nrrow chmber. The P O 2 of humidified inflowing gs (1000 sccm) ws djusted using gs flowmeter (Model GF-3/MP; Cmeron Instruments, Guelph, ON, Cnd). Hemolymph ws equilibrted for 20 min with normoxic ir (20 kp) nd nlyzed spectrophotometriclly using Turner spectrophotometer (Model 340; Mountin View, CA, USA) t wvelength of 570 nm. This is the wvelength of mximl bsorbnce for oxy- nd deoxy-hemoglobin for Triops (Horne nd Beyenbch, 1974). Wter ws used s reference. The bsorbnce of hemoglobin ws determined fter equilibrtion with ir of 30, 4.0, 2.7, 1.3, 1.1, 0.8, 0.5 nd 0 kp O 2. Stndrd curves were constructed from the bsorbnce of hemoglobin t 0% nd 100% sturtion. Percent sturtion for hemoglobin t ech P O 2 ws clculted using stndrd curves. Curve fitting of O 2 binding ws clculted using SigmStt 2.03 (SPSS Inc., Chicgo, IL, USA). The P 50 for ech rering group ws determined s the P O 2 t which 50% sturtion occurred (Bruno et l., 2001). Coopertivity (n H ) ws clculted s the mximl slope of log[sturtion/(1 sturtion)] ginst log[p O 2] (Bruno et l., 2001). Oxygen-dependent chnges in hemolymph ph were used to determine the significnce of Bohr shift in ltering hemoglobin O 2 -binding ffinity. Hemolymph ph ws mesured using PHR-146 Micro Combintion ph Electrode (Lzr Reserch Lbortories, Inc., Los Angeles, CA, USA) inserted into the bse of flow-through chmber (20 l). The P O 2 of inflowing humidified gs (1000 sccm) ws djusted using gs flowmeter (Model GF-3/MP) to control mixture of N 2 nd room ir. Hemolymph ph ws determined fter 10 min equilibrtion t 30, 4.0, 2.7, 1.3, 1.1, 0.8, 0.5 nd 0 kp O 2. Oxygen-crrying cpcity nd delivery potentil The O 2 -crrying cpcity of 20 l of O 2 -sturted hemolymph ws determined for nimls from ech rering group (N=7 per rering group) using methods described previously (Tucker, 1967). Hemolymph ws sturted by equilibrtion with 30 kp O 2. A potssium ferricynide solution {6 g potssium ferricynide [K 3 Fe(CN) 6 ], 3 g sponin (Sigm) nd 1 kg wter} ws dded to 10 ml glss syringe nd degssed by plugging the syringe needle with rubber stopper, pulling bck gently on the plunger to crete vcuum nd shking. Extrcted gs ws expelled nd the process ws repeted minimum of five times to ensure complete degssing. A microrespirometry chmber (400 l) with O 2 electrode (Model 781 Strthkelvin oxygen meter) ws filled with degssed potssium ferricynide solution, plugged nd stirred. After five minutes equilibrtion, the P O 2 in the chmber ws mesured. The stopper ws removed from the chmber nd 20 l of hemolymph ws injected into the chmber with the degssed potssium ferricynide. After five minutes equilibrtion, the P O 2 in the chmber ws determined. Hemolymph nd degssed potssium ferricynide solution were removed. Aerted potssium ferricynide solution ws dded to the chmber, removed nd dded gin. The chmber ws left unplugged nd the P O 2 determined fter 20 min equilibrtion. The potssium ferricynide solution ws removed from the chmber nd solution of sodium sulfite nd sodium borte [1 mg sodium sulfite (N 2 SO 3 ) nd 5 ml 0.01 mol l 1 sodium borte (N 2 B 4 O 7 ) (Sigm)] ws dded before the chmber ws plugged gin. After five minutes equilibrtion, the P O 2 of the chmber ws determined. Hemolymph O 2 content (ml O ml 1 hemolymph or Vol%) ws clculted using the eqution: O 2 content = ( P O 2/760) V(100/V s ), (2) where P O 2 is the chnge in P O 2 fter injecting the hemolymph into the degssed potssium ferricynide solution, is the solubility coefficient of O 2 in the potssium ferricynide solution, V is the chmber volume nd V s is the smple volume (Tucker, 1967). Oxygen-delivery potentil ws clculted s the product of O 2 content nd crdic output nd reported in l O 2 min 1 (Ronco et l., 1991). Clcultions for the determintion of O 2 content nd crdic output were described bove. Oxygen consumption/oxygen trnsport coupling The mount of coordintion between O 2 demnd nd delivery ws determined by compring the rtio of V O 2 to crdiovsculr trnsport. The degree of coupling ws compred mong rering groups to determine the effects of developmentl P O 2 on respirtory nd crdiovsculr system coordintion. Indices of the reltionship between V O 2 nd hemolymph O 2 trnsport (Q O 2) were clculted using the eqution:

5 Hypoxic responses in crustcens 1643 V O 2 / Q O 2 = V O 2 / C O 2, (3) where V O 2 is oxygen consumption in l O 2 g 1 h 1, C O 2 is oxygen content of fully sturted hemolymph ( l O 2 l 1 hemolymph) nd Q is crdic output ( l g 1 h 1 ) (Territo nd Altimirs, 1998; Territo nd Burggren, 1998). The rtio is unitless becuse V O 2 nd Q O 2 re both expressed in l O 2 g 1 h 1. A vlue of one suggests strong coupling between O 2 demnd nd convective trnsport. Vlues below one indicte tht circultory O 2 trnsport cpcity exceeds totl V O 2. Vlues bove one indicte tht convective trnsport my limit O 2 supply. Sttisticl nlyses All sttisticl nlyses were run with SigmStt 2.03 unless otherwise specified. Results re presented s mens ± s.e.m. with sttisticl significnce ccepted t the level of P<0.05. Multiple pirwise comprisons were mde using Bonferroni t- tests when rering effects were significnt, unless otherwise specified. Metbolism The strength of the reltionship between ctivity level nd P O 2 ws mesured using Person Product Moment Correltion. Comprisons of metbolic response to grded hypoxi were mde mong rering groups using one-wy repeted-mesures nlysis of vrince (ANOVA). Comprison of lctte concentrtions ws mde between normoxic nd severe hypoxic rering groups using Student s t-test. Ventiltion Comprisons of ventiltory mplitude nd frequency mong rering groups were mde using Kruskl Wllis one-wy ANOVA on rnks becuse dt hd unequl vrince. Multiple pirwise comprisons were mde using Tukey test. Crdic physiology Comprisons of hert rte, stroke volume nd crdic output to grded hypoxi were mde mong rering groups using onewy repeted-mesures ANOVA. Hemoglobin Comprisons of hemoglobin concentrtion were mde mong rering groups using one-wy ANOVA. The hemoglobin concentrtion of nimls tht hd been switched from normoxic to severe hypoxic, nd from severe hypoxic to normoxic conditions (fter 5, 7 nd 10 dys), ws nlyzed using one-wy repeted-mesures ANOVA. Comprison of the O 2 -binding ffinity of hemoglobin from ech rering group ws mde using Friedmn repeted-mesures ANOVA on rnks becuse of unequl vrince. Hemoglobin P 50 ws compred mong rering groups using Kruskl Wllis onewy ANOVA on rnks becuse dt were not normlly distributed. Multiple pirwise comprisons were mde using Tukey test. Hemoglobin n H ws compred mong rering groups using nlysis of covrince (SttView, 5.0.1; SttView Softwre, Cry, NC, USA). Multiple pirwise comprisons were performed using Scheffe test nd SttView. Comprisons of hemolymph ph with P O 2 were mde mong rering groups using one-wy repeted-mesures ANOVA. Oxygen-crrying cpcity nd delivery potentil Comprisons of the O 2 -crrying cpcity of hemolymph were mde mong rering groups using Kruskl Wllis onewy ANOVA on rnks becuse dt did not hve equl vrince. Multiple pirwise comprisons were mde using Tukey test. Comprisons of the O 2 -delivery potentil of hemolymph were mde mong rering groups using Kruskl Wllis one-wy ANOVA on rnks becuse dt hd unequl vrince. Multiple pirwise comprisons were mde using Tukey test. Oxygen consumption/oxygen trnsport coupling Comprisons of the O 2 consumption/oxygen trnsport coupling were mde mong rering groups using one-wy repeted-mesures ANOVA. Results Metbolism Animl ctivity ws not correlted with P O 2 nd therefore did not confound V O 2 mesurements. Developmentl P O 2 hd no effect on metbolic rte or metbolic response to hypoxic exposure (Fig. 1). All groups grdully decresed V O 2 down to P CRIT of 2 kp O 2 (691±20 l O 2 g 1 h 1 t normoxi to 274±9 l O 2 g 1 h 1 t 2 kp O 2 in normoxic nimls; 714±14 l O 2 g 1 h 1 t normoxi to 277±14 l O 2 g 1 h 1 t 2 kp O 2 in moderte hypoxic nimls; nd 728±3 6 l O 2 g 1 h 1 t normoxi to 253±12 l O 2 g 1 h 1 t 2 kp O 2 in severe hypoxic nimls). Bseline lctte levels for nimls rered under normoxic (2.971±0.29 mmol l 1 ) nd severe hypoxic (3.142±0.33 mmol l 1 ) conditions were not significntly different fter 12 h of severe hypoxic Mss-specific oxygen consumption ( l g 1 h 1 ) Wter P O2 (kp) Fig. 1. Mss-specific O 2 consumption for tdpole shrimp rered under normoxic (19 21 kp O 2 ; open circles), moderte (10 13 kp O 2 ; gry circles) or severe hypoxic (1 3 kp O 2 ; blck circles) conditions exposed to progressive hypoxi. Vlues re mens ± s.e.m. (N 7). In some cses, the error brs were smller thn the symbols.

6 1644 S. L. Hrper nd C. L. Reiber Ventiltory rte (bets min 1 ) Ventiltory mplitude (mm) A B * Prtil pressure of oxygen (kp) Fig. 2. (A) Ventiltory rte mesured s the bets per minute of respirtory ppendges nd (B) ventiltory mplitude mesured s the men mximl distnce between the 4th nd 5th ppendges during five consecutive ventiltory strokes. Tdpole shrimp rered under normoxic (19 21 kp O 2 ; open circles), modertely hypoxic (10 13 kp O 2 ; gry circles) or severe hypoxic (1 3 kp O 2 ; blck circles) conditions were exposed to four environmentl P O 2s (20, 13.3, 10 nd 1 kp O 2 ). Vlues re mens ± s.e.m. (N 13). *Significnt difference from control nimls t sme P O 2 (P<0.05). exposure (3.810±0.45 mmol l 1 nd 3.304±0.59 mmol l 1, respectively). No lctte ws observed in the ny of the wter smples. Ventiltion Ventiltion rtes were highly vrible in ll rering groups. Tdpole shrimp ventiltion rtes were not different mong rering groups nd did not differ within groups in response to hypoxic exposure (13.3, 10 nd 1 kp O 2 ) (Fig. 2A). Ventiltory mplitude did not differ within groups in response to hypoxic exposure (Fig. 2B). However, ventiltory mplitudes t 1 kp O 2 were significntly different mong the groups. Ventiltory mplitude ws less in nimls rered under severe hypoxic conditions when compred with nimls rered under normoxic conditions but only t 1 kp O 2. Crdic physiology Hert rte response to hypoxic exposure ws significntly different mong the rering groups. At low P O 2, fh decresed in tdpole shrimp rered under normoxic conditions from 281±6 bets min 1 t 20 kp O 2 to 226±5 bets min 1 t 2 kp O 2 (Fig. 3A). Animls rered under moderte hypoxi lso showed significnt decrese in fh, from 262±11 bets min 1 t 20 kp O 2 to 223±8 bets min 1 t 2 kp O 2. However, nimls rered under severe hypoxic conditions did not exhibit chnge in fh with decresed P O 2 Hert rte (bets min 1 ) Stroke volume ( l bet 1 ) Crdic output ( l min 1 ) A B b,c c C c b,c Wter P O2 (kp O 2 ) (275±9 bets min 1 t 20 kp O 2 to 238±10 bets min 1 t 2 kp O 2 ). Tdpole shrimp rered under normoxic (0.23±0.01 l bet 1 ) nd moderte hypoxic conditions (0.28±0.01 l bet 1 ) hd significnt differences in VS t 10 kp O 2 (Fig. 3B). Those rered under normoxic conditions decresed VS, wheres those rered under moderte hypoxic conditions incresed VS in response to severe hypoxic exposure. Yet neither hypoxic response ws sttisticlly significnt due to the high vribility of VS; men stndrd devition ws greter thn the hypoxic response. Crdic output ws mintined in nimls rered under severe hypoxic conditions down to 2 kp O 2 (Fig. 3C). However, nimls rered under moderte hypoxic conditions decresed Q from 64±6 l min 1 t 20 kp O 2 to 53±2 l min 1 t 2 kp O 2, nd nimls rered under normoxic conditions decresed Q from 67±4 l min 1 t 20 kp O 2 to 59±3 l min 1 t 2 kp O 2. Hemoglobin Hemoglobin concentrtions mesured s protein concentrtion were dependent on rering P O 2 nd were,b,c Fig. 3. (A) Hert rte, (B) stroke volume nd (C) crdic output of tdpole shrimp rered under normoxic (19 21 kp O 2 ; open circles), modertely hypoxic (10 13 kp O 2 ; gry circles) or severe hypoxic (1 3 kp O 2 ; blck circles) conditions exposed to normoxic (20 kp O 2 ) nd hypoxic (10, 5 nd 2 kp O 2 ) conditions. Vlues re mens ± s.e.m. (N 13). Significnce is ssumed t the level of P<0.05: significnt difference from control nimls t sme P O 2; b significnt difference from the sme niml t preceding P O 2; c significnt difference from the sme nimls t 20 kp O 2.

7 Hypoxic responses in crustcens 1645 Protein concentrtion ( g l 1 ) Normoxic Moderte hypoxic Severe hypoxic Fig. 4. Hemoglobin concentrtions of tdpole shrimp rered under normoxic (19 21 kp O 2 ), moderte (10 13 kp O 2 ) or severe hypoxic (1 3 kp O 2 ) conditions. Vlues re mens ± s.e.m. (N 10). *Significnt difference from control (normoxic) nimls (P<0.05). significntly ltered by chronic hypoxic exposure. Animls rered under normoxic (9.6±0.7 g l 1 ) nd moderte hypoxic (2.8±1.4 g l 1 ) conditions hd significntly less protein (hemoglobin) thn those rered under severe hypoxic (19.8±0.9 g l 1 ) conditions (Fig. 4). Concentrtions in tdpole shrimp rered under normoxic conditions nd trnsferred to severe hypoxic conditions incresed significntly fter 7 dys (Fig. 5). After 10 dys of severe hypoxic exposure, concentrtions were not significntly different from nimls tht hd been rered under severe hypoxic conditions. Adult tdpole shrimp rered under severe hypoxic conditions nd switched to normoxic conditions did not decrese the mount of hemoglobin in their hemolymph for the 10 dys investigted. The O 2 -binding ffinity of hemoglobin ws dependent on developmentl P O 2. Hemoglobin O 2 -binding ffinity in * * Percent sturtion Prtil pressure of oxygen (kp O 2 ) Fig. 6. Oxygen binding of hemoglobin from tdpole shrimp rered under normoxic (19 21 kp O 2 ; open circles), modertely hypoxic (10 13 kp O 2 ; gry circles) or severe hypoxic (1 3 kp O 2 ; blck circles) conditions t 25 C. The prtil pressures of O 2 required to hlf-sturte hemoglobin (P 50 ) re given in Tble 1. Vlues re mens ± s.e.m. (N 7). nimls rered under normoxic conditions ws less thn those rered under severe hypoxic conditions but not different from nimls rered under moderte hypoxic conditions (Fig. 6). Hemoglobin coopertivity ws significntly different mong rering groups (Fig. 7). Animls rered under normoxic conditions exhibited more coopertivity mong hemoglobin subunits thn those rered under moderte nd severe hypoxic conditions. Oxygen-binding ffinities nd coopertivity vlues for hemoglobin from ech rering group re given in Tble 1. Protein concentrtion ( g 100 l 1 ) Dys fter switching Fig. 5. Hemoglobin concentrtions of tdpole shrimp rered under normoxic (19 21 kp O 2 ) (open brs) conditions nd then switched s dults to severe hypoxic (1 3 kp O 2 ) conditions for 5, 7 or 10 dys. Blck brs indicte nimls rered under severe hypoxic conditions nd switched s dults to normoxic conditions for 5, 7 or 10 dys. Dy 0 is prior to switch. Vlues re mens ± s.e.m. (N 7). Significnce is ssumed t the level of P<0.05: significnt difference from normoxic nimls t the sme dy; b significnt difference within rering groups from the vlues t dy 0. b b log [sturtion/(1 sturtion)] r 2 =0.91 r 2 =0.96 r 2 = log [P O2 (kp)] Fig. 7. Hill plot of hemoglobin for tdpole shrimp rered under normoxic (19 21 kp O 2 ; open circles), modertely hypoxic (10 13 kp O 2 ; gry circles) or severe hypoxic (1 3 kp O 2 ; blck circles) conditions. Mximl slope for ech rering group represented by regression line. Coopertivities (n H ) for ech group re given in Tble 1. Vlues re mens ± s.e.m. (N 7). In some cses, the error brs were smller thn the symbols.

8 1646 S. L. Hrper nd C. L. Reiber Tble 1. Prtil pressures of O 2 required to hlf-sturte hemoglobin (Hb P 50 ), coopertivities of hemoglobin subunits (Hb n H ), hemolymph O 2 -crrying cpcities nd delivery potentils for tdpole shrimp rered under different P O 2 Hb P 50 Oxygen crrying cpcity Delivery potentil Developmentl P O 2 (kp O 2 ) (kp O 2 ) Hb n H (ml O ml 1 ) ( l O 2 min 1 ) Normoxic (19 21) ± Moderte hypoxic (10 13) * 8.3± Severe hypoxic (1 3) 0.5* 1.4* 19.4±1.6* 1336* *Significnt difference (P<0.05) from nimls rered under normoxic conditions. Oxygen-crrying cpcity nd delivery potentil Tdpole shrimp O 2 -crrying cpcity ws dependent on developmentl P O 2 (Tble 1). Animls rered under severe hypoxic conditions hd 5.2 the O 2 -crrying cpcity of those rered under normoxic conditions. Mximl O 2 -delivery potentil for tdpole shrimp ws dependent on developmentl P O 2. Oxygen-delivery potentil ws significntly lower in nimls rered under normoxic conditions (274 l O 2 min 1 ) reltive to nimls rered under severe hypoxi (1336 l O 2 min 1 ) (Tble 1). Oxygen consumption/oxygen trnsport coupling The rtio of V O 2/Q O 2 ws dependent on developmentl P O 2. Oxygen consumption/trnsport rtio ws significntly higher (consistently bove 1) in nimls rered under normoxic conditions reltive to those rered under moderte or severe hypoxic conditions (Fig. 8). Ambient P O 2 significntly ffected V O 2/Q O 2 in nimls rered under normoxic nd moderte hypoxic conditions. Animls rered under moderte hypoxic conditions hd ner coupling (1.18) of O 2 demnd to crdiovsculr supply t 20 kp O 2, which decresed significntly with severe hypoxic exposure (2 kp O 2 ). Animls rered under severe hypoxic conditions mintined V O 2/Q O 2 below 1 t ech P O 2 investigted. Discussion Metbolism The bility to regulte V O 2 my be relted to the frequency nd durtion of hypoxic exposure tht n niml typiclly encounters in its hbitt (Chen et l., 2001). Tdpole shrimp inhbit euryoxic environments tht cn fluctute dily by more thn 26.7 kp O 2 or remin lmost noxic (<1.3 kp O 2 ) for months (Horne nd Beyenbch, 1971; Scholnick, 1995). In ddition, they verticlly migrte from severely hypoxic (2 kp O 2 ) sediments to hyperoxic (32 kp O 2 ) surfce wter in reltively short period of time (Scholnick nd Snyder, 1996). Oxygen conformtion to environmentl P O 2 should be energeticlly fvorble for orgnisms frequently exposed to hypoxic conditions on mny temporl nd sptil scles (Chen et l., 2001). Tdpole shrimp would be considered oxygen conformers s they did not tightly regulte V O 2 when P O 2 ws decresed (Fig. 1). Insted, tdpole shrimp reduced O 2 demnd to mtch O 2 supply. Sensitivity to hypoxic exposure my be ltered in nimls exposed to hypoxic conditions throughout development due to ontogenetic chnges in physiologicl cpbilities nd metbolic demnd (Schulte, 2001). Adult nimls tht develop under hypoxic conditions often hve decresed metbolic rtes nd decresed hypoxic sensitivity reltive to nimls tht develop under normoxic conditions (Pichvnt et l., 2001; Sokolov nd Portner, 2001; Cech nd Crocker, 2002; Chpmn et l., 2000; Hmmond et l., 2002). Yet developmentl P O 2 did not ffect dult tdpole shrimp metbolic rte or metbolic response to cute hypoxic exposure. Tdpole shrimp tht develop under hypoxic conditions must blnce immedite O 2 demnd with long-term requirements for completing their life cycle within dys (Horne nd Beyenbch, 1971). Therefore, trnsient decreses in V O 2 in response to mbient chnges in P O 2 my be more beneficil thn permnent decreses in V O 2 tht could ultimtely limit growth nd development. Anerobic metbolism cn be used to supplement erobic metbolism nd decrese sensitivity to hypoxic exposure in mny qutic orgnisms (Truchot, 1980; Childress nd Seibel, 1998). Tdpole shrimp do not pper to utilize nerobic metbolic pthwys tht end in lctic cid production; Oxygen consumption/trnsport rtio c c b,c b,c Oxygen prtil pressure (kp) Fig. 8. Chnges in O 2 consumption/trnsport rtio with P O 2 for tdpole shrimp rered under normoxic (19 21 kp O 2 ; open circles), modertely hypoxic (10 13 kp O 2 ; gry circles) or severe hypoxic (1 3 kp O 2 ; blck circles) conditions. Vlues re mens ± s.e.m. (N 7). Significnce is ssumed t the level of P<0.05: significnt difference from control nimls t sme P O 2; b significnt difference from the sme niml t preceding P O 2; c significnt difference from the sme nimls t 20 kp O 2.

9 Hypoxic responses in crustcens 1647 hemolymph lctte levels did not increse fter 12 h of severe hypoxic exposure (1.3 kp O 2 ). However, tdpole shrimp my employ other nerobic pthwys tht were not investigted by the current study (i.e. pthwys ending in pyruvte, vleric cid or lnine). Ventiltion Orgnisms from vriety of phyl, including crustcens (Hervnt et l., 1995), birds (Frci, 1991; Min, 2000), bts (Min, 2000), humns (Gozl nd Gozl, 2001; Hoppeler nd Vogt, 2001), mussels (Chen et l., 2001), fish (Glis nd Brel, 1980; Cech nd Crocker, 2002) nd tods (Hou nd Hung, 1999) increse ventiltion rte nd/or volume in response to cute hypoxic exposure. Tdpole shrimp pprently lck this typicl ventiltory response or perhps the response ws not observed due to the length of cclimtion prior to dt collection (Fig. 2). Ventiltion rtes did not increse in response to hypoxic exposure down to 1 kp O 2. Further, developmentl P O 2 did not ffect ventiltion rtes under normoxic conditions or in response to hypoxic exposure. Tdpole shrimp ppendges re used for locomotion nd the genertion of feeding currents, in ddition to respirtory gs exchnge (Fryer, 1988). If ventiltory rtes were incresed in response to hypoxic exposure, it could cuse the tdpole shrimp to swim fster nd/or my lter the currents used for food collection. Typicl ventiltory hypoxic responses my be lcking becuse those ltertions my hve dverse effects on locomotion nd feeding. Ventiltory mplitude ws used in this study s n index of ventiltion volume. Ventiltory mplitude did not increse in response to hypoxic exposure in ny of the rering groups. Below P CRIT, mplitude ws lowest in tdpole shrimp rered under severe hypoxic conditions but ws not significntly different from mplitude under normoxic conditions. Lower ventiltory mplitude in nimls rered under severe hypoxic conditions my result from direct effects of O 2 limittion on the respirtory ppendge movement or my be strtegy to minimize metbolic demnd under severe hypoxic conditions (Min, 2000). Additionlly, ltertions in the ngle of the ppendges could ccount for ventiltory volume chnges without observed chnges in mplitude. Crdic physiology Tdpole shrimp crdiovsculr responses to hypoxic exposure follow unique pttern unlike tht documented for other crustcens (McMhon, 2001). In most crustcens, cute hypoxic exposure results in decrese in fh (brdycrdi) nd concomitnt increse in VS to mintin or increse Q (Reiber, 1995; McMhon, 2001). Smller crustcens, such s wter fles (Dphni mgn) (Pul et l., 1998) nd grss shrimp (Plemonetes pugio) (Hrper nd Reiber, 1999), increse fh (tchycrdi) to mintin Q in response to hypoxic exposure. In tdpole shrimp, however, crdic function ppers to be highly insensitive to hypoxic exposure (Fig. 3). Agin, it should be cknowledged tht this my be n rtifct of the experimentl protocol in tht n cute hypoxic response my hve been present but not observed becuse it took plce during the cclimtion period. Those nimls rered under normoxic nd moderte hypoxic conditions did not chnge fh or VS when exposed to 5 kp O 2. Below this, brdycrdi ws observed nd resulted in decresed Q. All crdic prmeters were mintined down to 1 kp O 2 in tdpole shrimp rered under severe hypoxic conditions. The difference between the crdic response of tdpole shrimp nd those of other crustcens my result from differences in mechnisms of crdic regultion. The hertbet of mny crustcens is regulted through periodic bursting ctivity of crdic gnglion, excittory neurons tht innervte the myocrdium (Ymgishi et l., 2000). Hypoxi-induced brdycrdi in those crustcens my be medited by direct effect of lck of O 2 to the crdic gnglion (Wilkens, 1999). Tdpole shrimp herts hve myogenic mechnism of regultion whereby the crdic muscle hs endogenous rhythmic properties nd does not rely on neurl impulses to contrct (Ymgishi et l., 1997; Ymgishi et l., 2000). The hert of tdpole shrimp does not respond to hypoxic exposure in the typicl compenstory mnner observed in other crustcens. Myogenic regultion pprently supports crdic function over wide rnge of P O 2s, including exposure to severe hypoxic conditions. Hemoglobin Hemoglobin concentrtions obtined in this study were comprble to previously reported concentrtions for Triops (Horne nd Beyenbch, 1974; Scholnick nd Snyder, 1996; Gudgnoli et l., 2005). Hemoglobin concentrtion ws higher in nimls tht developed under severe hypoxic conditions, yet ws not proportionl to developmentl P O 2 (Fig. 4). Animls rered under moderte hypoxic conditions produced less hemoglobin thn nimls rered under either normoxic or severe hypoxic conditions. Moderte hypoxic conditions my represent the level of P O 2 in which O 2 uptke is sufficient to meet metbolic demnd. Alterntively, it my represent trigger for genetic up- or down-regultion of hypoxi-inducible genes prior to the trnsltion of hemoglobin protein subunits. Hypoxi-induced hemoglobin synthesis ppers to be compenstory response tht llows T. longicudtus nd severl other brnchiopods to regulte O 2 uptke nd trnsport in their euryoxic hbitts. Hypoxic exposure (4 5 kp O 2 ) induced more thn 10-fold increse in hemoglobin concentrtion within 10 dys in dult D. mgn (Goldmnn et l., 1999) nd threefold increse within three weeks in dult brine shrimp (Artemi slin) (Gilchrist, 1954). Hypoxic exposure (1 3 kp O 2 ) induced significnt increse in protein concentrtions within seven dys in dult T. longicudtus (Fig. 5). Since hemoglobin is the only mjor protein in tdpole shrimp hemolymph, protein concentrtion ws ccepted to be representtive of hemoglobin concentrtion (Horne nd Beyenbch, 1971). Within 10 dys,

10 1648 S. L. Hrper nd C. L. Reiber hemoglobin concentrtions incresed to the levels observed in tdpole shrimp rered under severe hypoxic conditions. The induction of hemoglobin synthesis observed in T. longicudtus ws less thn the induction observed in A. slin or D. mgn. These results indicte tht tdpole shrimp exposed to hypoxic conditions cclimte by incresing hemoglobin concentrtion. Once the Hb is produced, though, it remins even when nimls re returned to normoxic wter (Fig. 5) (Gudgnoli et l., 2005). Hemoglobin O 2 -binding ffinity ws enhnced (decresed P 50 ) in tdpole shrimp rered under severe hypoxic conditions reltive to those rered under normoxic conditions (Fig. 6). Differences in O 2 -binding ffinity re thought to hve resulted from chnges in subunit ssembly of the functionl hemoglobin molecule (Gudgnoli et l., 2005). This hs been observed in other brnchiopods such s D. mgn nd A. slin (Bowen et l., 1969; Wring et l., 1970; Sugno nd Hoshi, 1971; Kobyshi et l., 1988; Goldmnn et l., 1999). Specific ssembly of hemoglobin subunits could be dependent on environmentl P O 2, internl chemistry or protein concentrtion (Sugno nd Hoshi, 1971; Kobyshi et l., 1988; Fgo nd Weber, 1995; Goldmnn et l., 1999). Since ech subunit differs in O 2 -binding ffinity nd coopertivity, chnges in ssembly directly ffect O 2 - binding ffinity of the functionl hemoglobin molecule (Kobyshi et l., 1988). The observed differences in hemoglobin O 2 -binding ffinity nd coopertivity support the hypothesis tht different P O 2 levels induce different hemoglobin isoforms or differentil subunit ssembly in T. longicudtus (Figs 6, 7). Oxygen-crrying cpcity nd delivery potentil Tdpole shrimp rered under severe hypoxic conditions pper to hve n enhnced bility to trnsport nd possibly store O 2 obtined from the environment due to their incresed O 2 -crrying cpcity (Tble 1). Tdpole shrimp rered under severe hypoxic conditions hd fivefold increse in hemolymph O 2 -crrying cpcity compred with those rered under normoxic conditions. Incresed hemolymph O 2 - crrying cpcity resulted from incresed hemoglobin concentrtion nd O 2 -binding ffinity observed in those nimls. Hemoglobin with high O 2 -crrying cpcity cn often serve storge function (Fgo nd Weber, 1995). Tdpole shrimp frequently surfce nd expose their respirtory ppendges to the ir wter interfce. Tdpole shrimp my obtin nd store O 2 from the higher P O 2 surfce wter for use during feeding nd hunting in severely hypoxic regions of the pool. Surfcing behvior hs been shown to increse with decresed P O 2, further supporting storge function of tdpole shrimp hemoglobin (Scholnick nd Snyder, 1996). Oxygen-delivery potentil, which tkes into ccount hemolymph O 2 content nd Q, ppered to be dependent on developmentl P O 2. Mximl O 2 -delivery potentil for tdpole shrimp ws lowest in nimls rered under normoxic conditions nd highest in those rered under severe hypoxic conditions (Tble 1). However, this ws not direct effect of developmentl environment since hemoglobin synthesis could be incresed throughout the life of the niml. Adult tdpole shrimp rered under normoxic conditions significntly incresed hemoglobin concentrtions in response to severe hypoxic exposure. Oxygen consumption/oxygen trnsport coupling The coupling of V O 2 with crdiovsculr trnsport revels the level of coordintion in tissue O 2 demnd reltive to O 2 delivery (Territo nd Burggren, 1998). In tdpole shrimp, differences in crdiovsculr contribution observed mong the rering groups were due to incresed hemoglobin concentrtion nd O 2 -binding ffinity since there ws no observed crdiovsculr hypoxic response. Oxygen consumption of tdpole shrimp rered under normoxic conditions ws not s dependent on crdiovsculr trnsport s it ws in tdpole shrimp rered under hypoxic conditions (Fig. 8). The coupling of V O 2 with trnsport ws consistently bove 1 in tdpole shrimp rered under normoxic conditions; however, V O 2/Q O 2 decresed in response to hypoxic exposure. This reduction suggests tht V O 2 ws reduced reltive to crdic output since (1) crdic output did not increse in response to hypoxic exposure nd (2) hemoglobin levels remin constnt during cute hypoxic exposure. Animls rered under normoxic conditions incresed convective processes to supply O 2 demnds or relied on diffusionl processes when exposed to severe hypoxic conditions. Crdiovsculr contribution to O 2 delivery ws gretest in nimls rered under severe hypoxic conditions. Oxygen delivery ws enhnced in those nimls vi incresed hemoglobin concentrtion nd O 2 -binding ffinity. The incresed O 2 delivery to the erobic, metboliclly ctive hert muscle my hve supported crdic contrction t normoxic rtes under hypoxic conditions. Tdpole shrimp rered under severe hypoxic conditions mintined fh nd Q when exposed to severe hypoxic conditions while the other rering groups did not. It ws concluded tht s tdpole shrimp produce more hemoglobin, they increse O 2 delivery reltive to O 2 supply nd enhnce crdic function during hypoxic exposure. Conclusions Developmentl P O 2 my not be sufficient to induce permnent chnges in dult tdpole shrimp physiologicl cpbilities. Metbolic rte, ventiltory rte nd metbolic response to cute hypoxic exposure were independent of developmentl P O 2. Differences in crdic response to hypoxic exposure mong the rering groups were probbly due to compenstory increses in hemoglobin concentrtion nd O 2 - binding ffinity rther thn to developmentl differences. Bsed on the results of this study, hypoxi-induced hemoglobin synthesis represents n effective compenstory mechnism tht llows tdpole shrimp to flexibly regulte O 2 uptke nd trnsport under euryoxic conditions. Differences tht result from incresed hemoglobin concentrtion should not

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