Ventilation rates and activity levels of juvenile jumbo squid under metabolic suppression in the oxygen minimum zone

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1 University of Rhode Islnd iologicl Sciences Fculty Pulictions iologicl Sciences 213 Ventiltion rtes nd ctivity levels of juvenile jumo squid under metolic suppression in the oxygen minimum zone Ktj Trüench Mri R. Pegdo See next pge for dditionl uthors Follow this nd dditionl works t: Terms of Use ll rights reserved under copyright. Cittion/Pulisher ttriution Trüench, K., Pegdo, M. R., Seiel,.., & Ros, R. (213). Ventiltion rtes nd ctivity levels of juvenile jumo squid under metolic suppression in the oxygen minimum zone. Journl of Experimentl iology, 216, doi: /je.7287 ville t: This rticle is rought to you for free nd open ccess y the iologicl Sciences t DigitlCommons@URI. It hs een ccepted for inclusion in iologicl Sciences Fculty Pulictions y n uthorized dministrtor of DigitlCommons@URI. For more informtion, plese contct digitlcommons@etl.uri.edu.

2 uthors Ktj Trüench, Mri R. Pegdo, rd. Seiel, nd Rui Ros This rticle is ville t DigitlCommons@URI:

3 39 The Journl of Experimentl iology 216, Pulished y The Compny of iologists Ltd doi:1.1242/je.7287 RESERCH RTICLE Ventiltion rtes nd ctivity levels of juvenile jumo squid under metolic suppression in the oxygen minimum zone Ktj Trüench 1, *, Mri R. Pegdo 1, rd. Seiel 2 nd Rui Ros 1 1 Lortório Mrítimo d Gui, Centro de Ocenogrfi, Fculdde de Ciêncis d Universidde de Liso, v. Noss Senhor do Co, 939, Cscis, Portugl nd 2 Center for iotechnology nd Life Sciences, iologicl Sciences, University of Rhode Islnd, 12 Flgg Rod, Kingston, RI 2881, US *uthor for correspondence (kjtruench@fc.ul.pt) SUMMRY The Humoldt (jumo) squid, Dosidicus gigs, is prt-time resident of the permnent oxygen minimum zone (OMZ) in the Estern Tropicl Pcific nd, therey, it encounters oxygen levels elow its criticl oxygen prtil pressure. To etter understnd the ventiltory mechnisms tht ccompny the process of metolic suppression in these top ocenic predtors, we exposed juvenile D. gigs to the oxygen levels found in the OMZ (1% O 2, 1 kp, 1 C) nd mesured metolic rte, ctivity cycling ptterns, swimming mode, escpe jet (urst) frequency, mntle contrction frequency nd strength, stroke volume nd oxygen extrction efficiency. In normoxi, metolic rte vried etween 14 nd 29 μmol O 2 g 1 wet mss h 1, depending on the level of ctivity. The mntle contrction frequency nd strength were linerly correlted nd incresed significntly with ctivity level. dditionlly, n increse in stroke volume nd ventiltory volume per minute ws oserved, followed y mntle hyperinfltion process during high ctivity periods. Squid metolic rte dropped more thn 7% during exposure to hypoxi. Mximum metolic rte ws not chieved under such conditions nd the metolic scope ws significntly decresed. Hypoxi chnged the reltionship etween mntle contrction strength nd frequency from liner to polynomil with incresing ctivity, indicting tht, under hypoxic conditions, the jumo squid primrily increses the strength of mntle contrction nd does not regulte its frequency. Under hypoxi, jumo squid lso showed lrger infltion period (reduced contrction frequency) nd decresed relxed mntle dimeter (shortened diffusion pthwy), which optimize oxygen extrction efficiency (up to 82%/34%, without/with considertion of 6% potentil skin respirtion). dditionlly, they rethe ʻdeeplyʼ, with more powerful contrctions nd enhnced stroke volume. This deep-rething ehvior llows them to disply stle ventiltory volume per minute, nd explins the mintennce of the squidʼs cycling ctivity under such O 2 conditions. During hypoxi, the respirtory cycles were shorter in length ut incresed in frequency. This ws ccompnied y n increse in the numer of escpe jets during ctive periods nd fster switch etween swimming modes. In lte hypoxi (onset ~17±1 min), ll the ventiltory processes were significntly reduced nd followed y lethrgic stte, ehvior tht seems closely ssocited with the process of metolic suppression nd enles the squid to extend its residence time in the OMZ. Supplementry mteril ville online t Key words: hypoxi, OMZ, jet propulsion, ventiltion, jumo squid, Dosidicus gigs, metolic suppression. Received 13 Mrch 212: ccepted 7 Septemer 212 INTRODUCTION Dosidicus gigs (jumo or Humoldt squid) is the lrgest ommstrephid squid (up to 2. m length nd kg in mss) (Nesis, 1983) nd hs one the highest metolic rtes of ny niml in the ocen (Ros nd Seiel, 28). It is endemic to the Estern Pcific, nd prticulrly undnt in the highly productive wters of the Cliforni nd Peru Current systems (Nigmtullin et l., 21) nd the Cost Ric Dome (Ichii et l., 22; Wlud nd Rodhouse, 26), where it plys crucil role oth s prey (Clrke nd Pliz, 21; ití-cárdens et l., 22; Ruiz-Cooley et l., 24) nd predtor (Mrkid nd Sos-Nishizki, 23). In ddition to its ecologicl role, the Humoldt squid is n economiclly importnt species nd the trget of the world s lrgest cephlopod fishing industry (Rodhouse et l., 26). In ddition to sesonl horizontl migrtions (Mrkid et l., 2), D. gigs undergoes diel verticl migrtions into mesopelgic depths (round 2 m) during the dytime (Gilly et l., 26) (Truelood nd Seiel, 212), similr to the migrtion pttern of their primry prey myctophid fishes (Mrkid nd Sos-Nishizki, 23; Mrkid et l., 28). While t depth, D. gigs encounters the permnent oxygen minimum zone (OMZ) of the Estern Tropicl Pcific during the dy, with oxygen levels often less thn mmol l 1 (~.% O 2 ) (Kmykowski nd Zentr, 199; Morrison et l., 1999). Other top pelgic predtors (e.g. tun, swordfish, mrlin nd shrks) (rill, 1994; Prince nd Goodyer, 26; Vetter et l., 28; Nsy-Lucs et l., 29; Strmm et l., 212) seem to void these hypoxic depths, s their tolernce to hypoxic conditions is low [<1 mmol l 1 O 2 (rill, 1994; rill, 1996); ~4 mmol l 1 O 2, ig-eye tun (Lowe et l., 2)]. One might expect tht the presence of ctive, musculr squid in these hypoxic zones would e precluded, s, in other ctive squid: (i) the primry mode of locomotion, jet propulsion, is energeticlly inefficient (Weer et l., 2); (ii) the oxygen-crrying cpcity is limittive reltive to tht of fishes, requiring tht cephlopods use most of the O 2 in the lood on ech THE JOURNL OF EXPERIMENTL IOLOGY

4 36 The Journl of Experimentl iology 216 (3) cycle, leving little venous oxygen reserve ehind (Pörtner, 22); (iii) the oxygen-crrying cpcity is low ecuse of viscosity-relted constrints nd n extrcellulr low-ffinity respirtory protein (Pörtner, 22); nd (iv) ventiltory nd locomotory systems re closely tied. Regrding the lst of these, two different strtegies evolved in these mollusks to del with this restriction. Pelgic squid (like D. gigs) tht routinely depend on jet propulsion optimize their locomotory pprtus y pushing lrge wter volume through their mntle cvity, reducing oxygen extrction efficiency ( 1%) (Pörtner, 1994; Wells et l., 1988). In contrst, nekton-enthic cephlopods (i.e. cuttlefish nd octopus) developed n efficient oxygen uptke system (4 %) (Wells nd Wells, 198) y minimizing their ventiltory volume to increse diffusion (Wells et l., 1988). Squid re thought to live chroniclly on the edge of oxygen limittion (Pörtner, 22) nd re not well poised to dpt to low mient O 2 levels. Yet, the Humoldt squid hs mnged to minimize the trde-offs etween high locomotory performnce nd hypoxi tolernce vi metolic suppression (Ros nd Seiel, 28; Ros nd Seiel, 21), coupled with high-ffinity respirtory protein (Seiel, 212). Here, we exposed juvenile squid to oxygen levels found in the OMZ (1% O 2, 1 kp, 1 C, severe hypoxi) to investigte the effects on the ventiltory mechnisms tht ccompny the process of metolic suppression in D. gigs, nmely: (i) mximl, ctive, routine nd inctive metolic rtes; (ii) metolic scope; (iii) ctivity cycling ptterns nd swimming mode (with video recording); (iv) escpe jet (urst) frequency; (v) mntle contrction frequency nd strength; (vi) stroke volume, nd ventiltory volume per minute; nd (vii) oxygen extrction efficiency (with nd without potentil contriution from skin respirtion). MTERILS ND METHODS Specimen collection Juvenile Humoldt squid, D. gigs (d Origny 183), ( g wet mss) were collected vi dip net in the Gulf of Cliforni (27 N, 111 W; 28 N, 113 W), on the surfce t night, in June 211 (ord the RV New Horizon, Scripps Institute, C, US) nd were immeditely trnsferred to quri contining 1 C sewter (environmentl temperture t OMZ) on ord the vessel. It is worth noting tht ll experiments were conducted with juvenile stges, nd it is not known whether juvenile nd dult jumo squid disply similr diel verticl migrtion ehvior (i.e. whether they encounter the sme minimum oxygen levels during descent). Experimentl procedure nimls were plced in flow-through respirometry set up (27 ml volume, Loligo Systems, Tjele, Denmrk) (Ros nd Seiel, 28; Ros nd Seiel, 21), nd llowed to cclimte for 8 12 h efore mesurements of oxygen consumption were strted. Respirometers were immersed in lrge thermostticlly controlled wterth (Lud, Lud-Königshofen, Germny) t 1 C, temperture pproximting tht found t 2 m in the OMZ (Ros nd Seiel, 21). Filtered (.2 µm) nd treted ( mg l 1 streptomycin) sewter ws pumped from wter-jcketed, gs-equilirtion column through the respirometers t constnt flow rte (verge 12 ml min 1 ). The wter in the column ws uled continuously to mintin incoming wter t high (21% O 2, 21 kp,) or low P O2 (certified gs mixture with 1% O 2, 1 kp). Once the finl 1% O 2 level in the respirtion chmers hd een reched, the first 3 min were excluded, s they my represent n cclimtory phse fter djusting to new oxygen concentrtion. Oxygen concentrtions were recorded t the entrnce nd the exit of ech chmer with two Clrke-type O 2 electrodes connected to 928 Oxygen Interfce (Strthkelvin Instruments, North Lnrkshire, UK). The system ws clirted using ir- nd nitrogen-sturted sewter nd checked for electrode drift nd for microil oxygen consumption efore nd fter ech tril. ll experiments were crried out in drkness nd t tmospheric pressure. Video recordings were conducted (Sony DCR-SR78, Lison, Portugl) during respirtion runs. fterwrds, specimens were immeditely weighed on motion-compensted precision shipord lnce system (Childress nd Mickel, 198). totl of 18 specimens were investigted, with 6 replictes for the normoxic tretment (21% O 2, 21 kp) nd 12 for the hypoxic one (1% O 2, 1 kp). ecuse of differences in squid swimming ehvior (i.e. mntle contrction frequency per minute) nd respirtory profile pttern (i.e. numer of cycles) throughout hypoxi, this tretment ws sudivided into erly hypoxi (EH, hypoxi exposure time 3 16 min, N=6) nd lte hypoxi (LH, hypoxi exposure time >18 min, N=6). Exposure times re presented in Tle 1. Swimming ehvior, metolic rte nd cycling performnce Swimming ehvior Jet propulsion ws sudivided into three swimming modes, sed on previous studies (Gosline et l., 1983; rtol, 21); nmely: (1) respirtory movements, (2) stedy jetting nd (3) ursts/escpe jets vi video footge. Respirtory movements were defined y wek mntle contrctions without visile thrust. Therey, squid could e within the wter column or, under LH, even lie t the ottom of the chmers. Stedy jetting rnged from slow cruising (wek ut visile thrusts) to vigorous jetting (powerful thrusts). ursts or escpe jets were chrcterized y hyperinfltion ( % increse of relxed mntle dimeter) followed y mximum mntle contrction (Gosline nd DeMont, 198) nd fst movements. The relxed dimeter ws ssessed under normoxi t inctive metolic rte (IMR) using ImgeJ softwre (Wyne Rsnd, Ntionl Institute of Mentl Helth, ethesd, MD, US), s mntle contrctions t slow swimming speeds occur without hyperinfltion (Gosline nd DeMont, 198). The numer of escpe jets per minute ws quntified vi video nlysis. Metolic rte The numer of escpe jets per minute nd the swimming modes were linked to chnges in O 2 consumption during cycling periods. sed on this, inctive, routine, ctive nd mximum ctive metolic rte (IMR, RMR, MR nd MR mx, respectively) could e distinguished (see Tle 2, Fig. 1) with similr results to those of previous study (Ros nd Seiel, 28). RMR for normoxi nd hypoxi ws specified s the verge rte for the entirety of Tle 1. Exposure times of Dosidicus gigs to normoxic (21% O 2 ) nd hypoxic (1% O 2 ) conditions Exposure time (min) Onset of lte hypoxi N Normoxi Hypoxi (min) Men s.d Vlues re presented individully nd s mens nd stndrd devitions. Exposure times for hypoxi include cclimtion time (3 min). THE JOURNL OF EXPERIMENTL IOLOGY

5 Ventiltion under hypoxi in jumo squid 361 Tle 2. Different ctivity levels in Dosidicus gigs MR mx MR RMR IMR % RMR 3 1 to <3 1 Swimming mode ( 7%) urst frequency (ursts min 1 ) 4 4 <4 <4 ctivity levels were defined s inctive, routine, ctive nd mximum ctive metolic rte (IMR, RMR, MR nd MR mx ) nd re given in terms of urst frequency (ursts min 1 ) nd swimming ehvior (mode nd % of time for IMR), resulting in percentge clsses of oxygen consumption rte sed on RMR (see Ros nd Seiel, 28). Note tht RMRs were specified seprtely for normoxic nd hypoxic conditions nd quntified s the verge of the entire recording. Swimming modes were clssified sed on previous criteri (Gosline et l., 1983; rtol, 21): 1, respirtory movement; 2, stedy jetting; nd 3, urst/escpe jetting. ech recording. During periods of ctivity, metolic rte ws 1 3% (MR) nd >3 1% (MR mx ) higher thn the routine levels (RMR) with escpe jets 4 ursts min 1. Thus, the MR nd MR mx were quntified y verging the metolic rtes for ll peks exceeding 1% or >3%, respectively. Inctive metolic rte ws defined vi swimming mode 1 ( 7% of time) nd urst events <<4 min 1. These periods of pprent inctivity in the chmers correlted with metolic rtes 1% lower thn RMR nd verge rtes were clculted. Note tht pelgic predtors such s D. gigs rrely stop swimming in nture nd tht we did not quntify locomotion continuously for ll experiments in its entirety. However, our IMR clcultions seem to e resonle pproximtion to the criteri set for stndrd metolic rte in mmmls nd other model orgnisms. Metolic rte ws quntified s µmol O 2 g 1 wet mss h 1. The metolic scope ws clculted s the difference etween mximum nd minimum O 2 consumption. Cycling performnce ll control (normoxi) nimls nd the mjority in erly hypoxi ( out of 6) nimls showed distinct periodicity in their rte of oxygen consumption (Fig. 1). These respirtory cycles re chrcterized y steep increse in metolic rte (~first 1/3 of totl cycle length, Fig. 1) nd video nlysis confirmed tht this ws linked to n elevtion in mntle contrction frequency. The remining cycle length (Fig. 1) consisted of slow trnsition from powerful to weker mntle contrctions. Therefore, urst frequency (numer of escpe jets per minute, clculted over the entire cycle length) nd swimming modes were used to descrie those respirtory cycles. Periodicity ws further investigted y ssessing the numer of ctive nd mximum ctive cycles per hour, including their length in minutes (vi respirtory files). Mntle contrction frequency nd strength In cephlopods, locomotion nd respirtion re closely tied nd therefore counterproductive in their gols, even though in resting Sepi nd proly to some extent in squid, ventiltory nd locomotory mechnisms cn e uncoupled vi collr flp system (one et l., 1994). However, oth ventiltory nd locomotory systems re highly dependent on the frequency nd strength of mntle contrctions, which, in turn, influence the wter volume throughput. Mntle contrction frequency (ventiltion rte) ws quntified s the numer of mntle contrctions per minute O 2 (μmol l 1 ) MR mx MR RMR IMR , 1, 18, 21, 24, Fig. 1. Oscilltions in oxygen levels (ΔO 2 ) tht reflect differences etween the vlues recorded t the entrnce nd the exit of the chmer in normoxi (, 1.9 g squid), erly hypoxi (, 1% O 2,.4 g squid) nd lte hypoxi (C, 11.6 g squid) t 1 C. Solid lines in represent the thresholds for inctive (IMR), routine (RMR), ctive (MR) nd mximum metolic rte (MR mx ) (illustrted in Tle 2). lck rrows indicte the ccelertion phse of MR mx nd MR cycles, nd the verticl dshed lines mrk the respective cycle lengths. The dshed line in C represents the onset of lte hypoxi (17±1 min, N=6). Note, the vlue on the x-xis in the hypoxi plots (,C) does not represent the rel onset of hypoxi. O 2 (μmol l 1 ) C Time (s) THE JOURNL OF EXPERIMENTL IOLOGY

6 362 The Journl of Experimentl iology 216 (3) E O2 = {[( O 2.2 O 2 ) V) / V V ]} / [(O 2,IN V) / V V ], E O2 = {[( O 2. O 2 ) V) / V V ]} / [(O 2,IN V) / V V ], (2) (2C) Fig. 2. Schemtic drwing of squid with descriptions of the mesurements used to quntify ventiltion stroke volume nd hyperinfltion in Dosidicus gigs. rrows indicte cylinder length (L c,.4 of totl mntle length in D. gigs), relxed mntle rdius (r R, recorded during IMR periods in normoxi), contrcted mntle rdius (r C ) nd hyperinflted mntle rdius (r H ). (MC min 1 ) y video footge nlysis. The strength of mntle contrction ws clculted s the difference etween mximum nd minimum mntle dimeter (in mm) using ImgeJ softwre (Wyne Rsnd, Ntionl Institute of Mentl Helth, ethesd, MD, US). Hyperinfltion nd stroke volume quntifiction During escpe jets, squid powerfully contrct their rdil muscles nd, consequently, induce n increse in the outer mntle dimeter (etween % nd 1% of relxed mntle dimeter), process clled hyperinfltion (represented y the red line in Fig. 2). The relxed mntle dimeter (Fig. 2) ws recorded during the IMR periods in normoxi. Chnges in the relxed dimeter were determined s percentge of the relxed mntle dimeter specified t IMR under normoxi. Stroke volume (V w, wter throughput per mntle contrction) ws quntified sed on modified O Dor (O Dor, 1988) eqution: V w = [r H 2 (r C x) 2 ] (.4π L) (.1M/d s ), (1) where, r H represents the mximum (hyperinflted) mntle rdius (see lso Fig. 2), r C is the contrcted mntle rdius, x is the mntle thickness (mm), L is the mntle length (the upper mntle is treted s cylinder of chnging dimeter, while the lower region is rigid nd independent of the mntle rdius; in D. gigs, cylinder length L c =.4L, see Tle 3), M is the wet mss (kg), d s is the squid density (1 kg m 3 ) nd.1 is the correction fctor for viscerl mss in D. gigs (see Tle 3). The ventiltory volume per minute (V V, ml min 1 ) ws quntified y multiplying V w (ml) y ventiltory frequency (MC min 1 ). The oxygen extrction efficiency (E O2, %) ws quntified vi the following equtions, sed on the theoreticl model in I. illecerosus (Pörtner, 1994): E O2 = [( O 2 V) / V V )] / [(O 2,IN V) / V V ], (2) E O2 = {[( O 2.6 O 2 ) V) / V V )] / [(O 2,IN V) / V V ], (2D) without (Eqn 2) nd with potentil skin respirtion contriution (2%, minimum for resting squid, Eqn 2; %, mximum for resting squid, Eqn 2C; nd 6%, for squid under exercise, Eqn 2D). Here, O 2 represents the difference etween the oxygen flow in nd out of the respirtory chmer (µmol l 1 ), V is the flow rte during respirtion mesurement (l min 1 ) nd V V is the ventiltory volume per minute (ml min 1 ). Sttistics Two-wy NOV were performed to evlute significnt differences etween oxygen tretments (21% nd 1% O 2 ), metolic rtes, mntle contrction frequencies nd strengths, % of relxed dimeter nd stroke volumes. Susequently, Tukey HSD post hoc tests were conducted. In some ventiltory mesurements, no dt for the lte hypoxi tretment were recorded nd, s result, independent Student s t-tests were pplied. Liner nd polynomil regressions were performed to ssess the correltions etween mntle contrction frequency, contrction strength nd metolic rte t different ctivity levels under control nd hypoxic conditions. NCOV were conducted to detect significnt differences in the reltionship etween mntle contrction strength/frequency nd metolic rte. For ll sttisticl nlysis, STTISTIC (Tuls, OK, US) version 1. ws used. RESULTS The effect of hypoxi on D. gigs metolic rte is shown in Fig. 3. In control nimls, ctivity level only showed significnt differences (P<.) etween IMR nd MR mx, incresing from 13.7±6.1 to 28.4±12. µmol O 2 g 1 wet mss h 1 (Fig. 3; supplementry mteril Tle S1). Exposure to severe hypoxi (1% O 2, 1 kp) led to significnt decrese in O 2 consumption ( 2% of control vlues, Fig. 3). MR nd RMR were significntly reduced in EH (exposure time 3 16 min) nd IMR t LH (exposure time >18 min; two-wy NOV, P<.; supplementry mteril Tle S1). MR mx ws only chieved under normoxic conditions nd, overll, ctivity levels (MR nd MR mx ) declined with hypoxi exposure from 36% to 1% in EH to % in LH (Fig. 3, Fig. 4). RMR dominted in EH tretments, incresing from 47% (normoxi) to 77%, ut ws not present in LH (1% IMR; Fig. 4). The metolic scope lso chnged drmticlly with hypoxi (Fig. 3), decresing from 1.±.9 µmol O 2 g 1 wet mss h 1 in normoxi to 1.7±.7 µmol O 2 g 1 wet mss h 1 in EH, reching lmost (.3±.2 µmol O 2 g 1 wet mss h 1 ) in LH (one-wy NOV, Tle 3. Species-specific correction fctors for viscerl mss nd cylinder mntle length to determine stroke volume in D. gigs N Wet mss (g) L (cm) L c (cm) CF L c V T (ml) V T V vm (ml) CF vm Men s.d L, mntle length; L c, cylinder mntle length; V T, totl volume (mntle nd viscer); V vm, volume of viscerl mss. The correction fctor for viscerl mss (CF vm ) ws quntified s the proportion of V vm to V T, nd the correction fctor for cylinder mntle length (CF L c ) s the proportion of L c to L. THE JOURNL OF EXPERIMENTL IOLOGY

7 Ventiltion under hypoxi in jumo squid 363 MR (μmol O 2 g 1 wet mss h 1 ) Metolic scope (μmol O 2 g 1 wet mss h 1 ) , MR mx MR RMR IMR, Normoxi EH LH Fig. 3. Effects of hypoxi (1% O 2 ) on () metolic rte (MR) nd () metolic scope (mximum MR minimum MR) of D. gigs. Vlues re expressed s mens nd s.d. (N=6). EH, erly hypoxi; LH, lte hypoxi. Significnt differences etween the level of ctivity (cpitl letters) nd etween tretments (lowercse letters) re indicted (P<., see supplementry mteril Tle S1). MR (%) MR mx MR RMR IMR Normoxi EH LH Fig. 4. Chnges in IMR, RMR, MR nd MR mx of D. gigs, under control conditions (21% O 2 ), nd in EH nd LH (1% O 2 ). F=34.6, P<<.1). The numer of escpe jets per minute incresed significntly during MR from normoxi (11.2 jets min 1 ) to EH (14.3 jets min 1 ), ut no jets were recorded t LH (Fig. ; supplementry mteril Tle S1). Overll (disregrding the ctivity level), there ws significnt decrese in totl ursts per hour from normoxi to EH (Fig., t-test, t=., P<.). ll control nimls showed distinct periodicity in oxygen consumption rte (Fig. 1) nd video nlysis confirmed tht these cycles were correlted with urst frequency nd level of ctivity. Cycles of MR nd MR mx differed significntly in length (18±4 nd 2±8 min, respectively; Fig. 6) nd frequency (1. nd.2 cycles h 1, respectively; t-test, P<<.1; Fig. 6). In erly hypoxi, periodicity ws lso oserved (in out of 6 nimls, illustrted in Fig. 1), ut with significntly shorter cycle length (±1 min; Fig. 6) nd higher frequency (4±1 cycles h 1 ; Fig. 6; t- tests, P<.1). The effect of hypoxi (1% O 2, 1 kp) on ventiltion rte, function of mntle contrction frequency nd strength, is presented in Fig. 7. Contrction frequency significntly incresed with ctivity, from 4±7 MC min 1 in IMR to 74±3 MC min 1 in MR mx in normoxi, nd from 32±6 MC min 1 in IMR to ± MC min 1 in MR during EH (Fig. 7; supplementry mteril Tle S1). During LH, the contrction frequency decresed to 6±3 MC min 1. Contrction strength ( mntle dimeter; Fig. 7C) incresed significntly with metolic rte in normoxi nd EH (supplementry mteril Tle S1). t IMR, mntle contrction strength ws more or less constnt (round 1. mm) during EH, ut then decresed drmticlly with durtion of exposure (LH) down to.4 mm (twowy NOV, P<.; supplementry mteril Tle S1). The correltions etween contrction frequency/strength nd metolic rte re given in Fig. 7,D. In oth cses, the reltionship is liner for oth normoxic (Fig. 7: R 2 =.977, F=13.3, P=.1; Fig. 7D: R 2 =.982, F=167.6, P<.1) nd hypoxic tretments (Fig. 7: R 2 =.8, F=17, P<.; Fig. 7D: R 2 =.8, F=17.7, P<.). Interestingly, the slope for hypoxi is significntly steeper (F=12.8, P<.1, frequency; F=.2, P<., strength) thn tht for normoxi, indicting tht much higher ventiltion rtes (frequency nd strength) re required to chieve even modest metolic rtes during exposure to hypoxi. There ws lso significnt correltion etween mntle contrction frequency nd strength (Fig. 8). Under normoxic conditions, squid showed liner reltionship etween strength nd frequency (R 2 =.993, P<<.1), wheres under hypoxi (1% O 2, 1 kp) the reltionship ws polynomil (R 2 =.988, P=.1), indicting reduced reltive ventiltion frequency. There ws significnt increse in the percentge relxed mntle dimeter in MR nd MR mx (i.e. hyperinfltion) under normoxi (Fig. 9; supplementry mteril Tle S1). However, the opposite trend ws oserved in EH nd LH. The effect of hypoxi on ventiltion stroke volume V w is shown in Fig. 1. The highest vlue ws oserved during mximum ctivity (MR mx ) under normoxi (4.8 ml). During EH, there ws significnt increse in V w, with ctivity rnging from 2.7 ml (IMR) to 3.6 ml (RMR) to 4.3 ml (MR; P<.). In LH, there ws significnt drop (42%) during the inctive stte (IMR). y multiplying the V w (Fig. 1) y ventiltion (contrction) frequency (Fig. 7), we otined the ventiltory volume per minute V V (Fig. 1). s result, it ecme evident tht in EH the increse in V w (t MR nd RMR; Fig. 1) ws followed y decrese in contrction frequency (Fig. 7), leding to similr vlues of V V in normoxi nd EH (Fig. 1). In LH, this ventiltory prmeter ws significntly lower (Fig. 1, P<.; supplementry mteril Tle S1). Oxygen extrction efficiency E O2 under normoxi rnged etween 6.4% nd 13.4% (2.6% nd.4%, tking into ccount 6% potentil skin respirtion) from IMR to MR mx nd ws significntly elevted s result of exposure to hypoxi, with minimum of 43.7% (1.2% including 6% cutneous uptke) in IMR during LH, nd mximum of 81.% (33.6%) in MR during EH (Fig. 1C,D, P<.; supplementry mteril Tle S1). In Tle 4, E O2 without, nd with 2%, % nd 6% potentil skin respirtion is presented. THE JOURNL OF EXPERIMENTL IOLOGY

8 364 The Journl of Experimentl iology 216 (3) Escpe jets (no. min 1 ) Totl ursts (no. h 1 ) C C MR mx MR RMR IMR DISCUSSION Effect of hypoxi on metolic rte nd metolic scope Dosidicus gigs metolic rte vried etween 14 nd 29 µmol O 2 g 1 wet mss h 1, depending on the level of ctivity. These vlues were in greement with those from our previous studies (Ros nd Seiel, 28; Ros nd Seiel, 21), which lso indicted wide metolic scope in well-oxygented wter. Dosidicus gigs is le to mintin these high metolic rtes down to n mient O 2 level of ~2 mmol l 1 (~2 kp, ~2 m depth) (Gilly et l., 26), elow which they drmticlly drop. In the present experiments, with the temperture nd oxygen conditions found in the OMZ, squid metolic rte dropped more thn 7% under exposure to erly hypoxi with no further significnt reduction with exposure time. Moreover, MR mx ws not recorded under such conditions nd the metolic scope ws significntly decresed (Fig. 1, Fig. 3, Fig. 4). In generl, orgnisms suppress their metolism y 9% y shutting down expensive processes (i.e. protein synthesis, muscle ctivity) (Guppy nd Withers, 1999) nd supplement the remining energy demnd using comintion of ville O 2 nd neroic metolic pthwys (Seiel, 211). Cephlopods tht routinely depend on jet propulsion extrct only smll proportion ( 1%) of the ville O 2 from the ventiltory strem, ut re le to lter the O 2 extrction efficiency ( 2%) when necessry, including cutneous respirtion, for exmple Normoxi EH LH Fig.. Effect of hypoxi (1% O 2 ) on urst frequency in D. gigs. () Numer of escpe jets per minute during IMR, RMR, MR nd MR mx, nd () totl numer of urst events per hour. Vlues re expressed s mens ± s.d. (N=6). Significnt differences etween ctive nd mximum ctive cycles (cpitl letters) nd etween oxygen tretments (lowercse letters) re indicted (P<.). Cycle length (min) Cycles (no. min 1 ) MR mx Normoxi EH LH MR Fig. 6. Effect of hypoxi (1% O 2 ) on the periodicity of ctivity cycles in D. gigs. () verge cycle length for MR mx nd MR, nd () cycle frequency. Vlues re expressed s mens nd s.d. (N=; 1 of the 6 ws not cycling). Significnt differences etween MR nd MR mx cycles (cpitl letters) nd etween oxygen tretments (lowercse letters) re indicted (P<.). (Wells et l., 1988; Wells nd O Dor, 1991; Pörtner, 1994). Yet, the most common strtegy mong powerful predtors tht mnged to minimize the trde-offs etween high performnce nd hypoxi tolernce is high-ffinity respirtory protein (Lowe et l., 2; Gilly et l., 26; Seiel, 211). Hemocynin of cephlopods is usully chrcterized y low O 2 ffinity with high ph sensitivity, properties tht fcilitte O 2 relese to demnding tissues, ut presumly interfere with O 2 extrction from hypoxic or CO 2 -rich sewter (Pörtner, 1994; Pörtner, 22; Melzner et l., 27). Yet, it hs een reported (Seiel, 212) tht the respirtory protein in D. gigs is oth highly temperture nd ph sensitive, ut, in ddition, is chrcterized y high O 2 -inding ffinity. These properties fcilitte O 2 uptke in cold, deep wters where O 2 is scrce nd enhnces O 2 relese in wrmer surfce wters where O 2 nd O 2 demnd re elevted. Furthermore, Seiel (Seiel, 212) could link the hypoxi tolernce down to P crit (1.6 kp, 1 C) (Truelood nd Seiel, 212) to the optimized hemocynin function in D. gigs, nd suggests tht elow P crit (~17 m, Gulf of Cliforni) the onset of metolic suppression kicks in. However, metolic suppression is time limited, s neroic glycolysis results in the ccumultion of protons (H + ) nd orgnic compounds such s lctte nd octopine (Hochchk nd Somero, 22; Ros nd Seiel, 21) with hrmful consequences on cid se sttus (Hochchk nd Mommsen, 1983). Recent iochemicl nlysis reveled tht under EH ~6% of the depressed metolism is still chieved eroiclly ut this drops to ~4% t LH (K.T., unpulished). No significnt further drop in metolic rte etween EH nd LH could e quntified, indicting limittion in the potentil to suppress metolism nd possile switch in THE JOURNL OF EXPERIMENTL IOLOGY

9 Ventiltion under hypoxi in jumo squid 36 Contrction frequency (MC min 1 ) Contrction strength (mm) C, MR mx MR RMR IMR Normoxi EH LH C c D MR (μmol g 1 wet mss h 1 ) Fig. 7. Effect of hypoxi (1% O 2 ) on ventiltion rtes of D. gigs. () Mntle contrction (MC) frequency, nd (C) mntle contrction strength (chnge in mntle dimeter). Significnt differences etween ctivity levels (cpitl letters) nd etween tretments (lowercse letters) re indicted (P<., see supplementry mteril Tle S1). Vlues re expressed s mens nd s.d. (N=6). (,D) Ventiltion frequency nd strength, plotted ginst oxygen consumption. Dimonds, normoxi; circles, EH; tringles, LH. Yellow, IMR; red, RMR; right lue, MR; drk lue, MR mx. energy expenditure from muscle ctivity to cid se nd ion regultion processes. Verticl migrtions nd respirtory cycling ehvior Jumo squid re diel verticl migrtors tht spend their nights in shllower, well-oxygented wters (~7 m) nd dive into the OMZ during the dy (pek depth rnge 2 3 m; 2.2 kp) (Gilly et l., 26) (Truelood nd Seiel, 212). Therey, jumo squid spend more thn 7% of the dy elow 2 m ( 2% O 2, 2 kp) (Gilly et l., 26) following their min prey (myctophid fishes) (Mrkid nd Sos-Nishizki, 23; Mrkid et l., 28) nd in this wy lso escpe from: (i) unfvorle wrmer se surfce tempertures, nd (ii) elevted predtion pressure nd resource (food) competition, s most ctive fish predtors re excluded from the OMZ (Prince nd Goodyer, 26). Nonetheless, coustic dt reveled short-term cyclic verticl movements (~1 9 min), during dy nd night time, tht hve een interpreted s ctive forges (Gilly et l., 26). In previous study, we (Ros nd Seiel, 21) discovered similr periodicity in the rte of metolism in the confines of respirtion chmer (cycle length ~2 min), which ws lso oserved in the present experiments (2/6 min for ctive/mximum ctive cycles; Fig. 1, Fig. 6). s the frequency nd durtion of rief verticl movements re similr to the periodicity reported here, we rgue tht the ctivity cycles (MR mx nd MR) t normoxi my reflect the cpcity to perform migrtory forys. During erly hypoxi, the respirtory cycles were shorter in length ut incresed in frequency (Fig. 1, Fig. 6). This ws ccompnied y n increse in the numer of escpe jets in ctive periods (see MR in Fig. ) nd fster switch etween swimming modes (dt not shown). s high musculr ctivity involves the use of neroic TP production, cycling etween eroic nd neroic swimming phses over short time intervls my reduce the cost of trnsport nd permit long-term use of neroic resources, strtegy tht in the long term my imply mximized use of mient O 2 (Finke et l., 1996). Finke nd collegues ssumed tht this oscilltion etween periods of high nd low musculr ctivity my hve een relted to the hypoxi tolernce of Lolliguncul revis in costl wters. It is plusile tht D. gigs uses the sme strtegy. Furthermore, it displyed n enhnced usge of its lterl fins under progressive hypoxi, with pek t the trnsition from EH to LH (dt not shown). This lso might contriute to minimize the costs of trnsport, s lterl fins cn push lrge mss of wter especilly t low velocities (O Dor et l., 1988; Wells nd O Dor, 1991). t LH (onset ~17±1 min), Contrction frequency (MC min 1 ) Contrction strength (mm) Fig. 8. Reltionship etween mntle contrction (MC) strength (chnge in mntle dimeter) nd frequency (ventiltion rte) in D. gigs under normoxi nd hypoxi (1% O 2 ). The lck stright line nd dimonds represent normoxi, the dshed line represents hypoxi, with circles for EH nd tringles for LH. Yellow, IMR; red, RMR; right lue, MR; drk lue, MR mx. Vlues re expressed s mens (N=6). THE JOURNL OF EXPERIMENTL IOLOGY

10 366 The Journl of Experimentl iology 216 (3) % Relxed dimeter Normoxi EH LH MR mx MR RMR IMR Fig. 9. Chnges in the relxed mntle dimeter of IMR, RMR, MR nd MR mx in D. gigs, under control conditions (21% O 2 ), EH nd LH (1% O 2 ). Percentge relxed mntle dimeter ws clculted sed on the relxed dimeter under IMR normoxi. Vlues re expressed s mens nd s.d. (N=6). Red rrows indicte hyperinfltion mechnism. Significnt differences etween ctive nd mximum ctive cycles (cpitl letters) nd etween oxygen tretments (lowercse letters) re indicted (P<.). jumo squid moved into lethrgic stte nd ventiltory cycles stopped (Fig. 1C, Fig. 6). This lethrgic ehvior hs een ssocited with metolic suppression (Ros nd Seiel, 21). Gilly nd collegues (Gilly et l., 26) climed tht sustntil numer of excursions lsted for mny hours (mximum durtion of 4/8 min elow 3/2 m), during which time rhythmic diving occurred tht ppered to e s roust s tht oserved t welloxygented, shllower depths. The mjority of these extended dives c t 3 m (~1% O 2, 1 kp) peked etween 14 nd 24 min, which is in close greement with the onset of lte hypoxi (~17±1 min) in the present study. Yet, the switch to severe hypoxi within the respirtion chmers could hve een fster thn during verge, nturl descents [see fig. 11 of Gilly (Gilly et l., 26)], even though fst migrtions (i.e. hunting, escpe) in jumo squid re known, which might hve elevted stress. lso the lck of food within the chmers might hve ccelerted the depletion of neroic energy reserves, resulting in the onset of lethrgy. Ventiltory mechnisms nd oxygen extrction efficiency during hypoxi In normoxi, the D. gigs mntle contrction frequency nd strength were linerly correlted (Fig. 8) nd incresed significntly with ctivity level (Figs 7, 8). dditionlly, n incresed stroke volume nd ventiltory volume per minute (Fig. 1) were oserved, followed y hyperinfltion process during high ctivity periods (red rrows in Fig. 9). The squid Illex illecerosus nd L. oplescens, in contrst, do not vry their ventiltion vi frequency with vigorous jetting, ut vi powerful contrctions of their circulr fires (Wells et l., 1988; Weer nd O Dor, 198). liner increse in ventiltion frequency with speed (<.6 m s 1 ) ws found in I. illecerosus, ut t speeds >.6 m s 1 the correltion ws liner with cvity pressure (Weer nd O Dor, 1986). s result, I. illecerosus nd L. oplescens seem to e restricted to regulting ventiltion strength nd frequency t the sme time, while D. gigs seems to hve developed n optimized fine-tuning etween locomotion nd ventiltion, nd seems to hve the potentil to regulte mntle contrction frequency, strength nd stroke volume ccording to ctivity. Yet, EH chnged the reltionship etween ventiltion strength nd frequency from liner to polynomil with incresing ctivity V w (ml) MR mx MR RMR IMR EO2 (%) C C 1 D V V (ml min 1 ) C D C Normoxi EH LH c EO2, 6% (%) Normoxi EH LH Fig. 1. Chnges in () stroke volume V w, () ventiltory volume per minute V V, (C) O 2 extrction efficiency E O 2 nd (D) O 2 extrction efficiency with potentil skin respirtion contriution (E O 2,6%) in IMR, RMR, MR nd MR mx of D. gigs, under control conditions (21% O 2 ), EH nd LH (1% O 2 ). Vlues re expressed s mens nd s.d. (N=6). Significnt differences etween ctive nd mximum ctive cycles (cpitl letters) nd etween oxygen tretments (lowercse letters) re indicted (P<.). THE JOURNL OF EXPERIMENTL IOLOGY

11 Ventiltion under hypoxi in jumo squid 367 Tle 4. Oxygen extrction efficiencies without (%) nd with (2%, % nd 6%) considertion of potentil skin respirtion Skin respirtion Tretment ctivity % 2% % 6% Normoxi MR mx 13.4±.4 1.7± ±2.7.3±2.1 MR 1.±.4 8.4±4.4.2± ±2.2 RMR 8.6± ± ± ±1.9 IMR 6.4±3.4.1± ± ±1.4 EH MR 81.± ± ± ±8. RMR 69.1±18.4.3± ± ±7.4 IMR 8.8± ± ± ±7.1 LH IMR 43.7±16. 3.± ± ±8. Dt (mens ± s.d.) re for inctive, routine, ctive nd mximum ctive levels (IMR, RMR, MR nd MR mx ) in D. gigs, under control conditions (21% O 2 ), nd in erly nd lte hypoxi (1% O 2 ). Here, 2% skin respirtion represents the minimum resting vlue, % the mximum resting vlue nd 6% the cutneous uptke under exercise in squid (I. illecerosus, theoreticl model) (Pörtner, 1994). EH, erly hypoxi; LH, lte hypoxi (>18 min). (Fig. 8). This indictes tht D. gigs, under hypoxic conditions, primrily increses the power of mntle contrction nd does not regulte its frequency, s ws oserved for I. illecerosus nd L. oplescens under normoxi (Wells et l., 1988). In EH, jumo squid showed lrger infltion period leding to reduced contrction frequency nd decresed relxed mntle dimeter, which optimize O 2 uptke (i.e. shorter diffusion wys) vi gills nd skin (see Fig. 1C). dditionlly, they seem to rethe deeply, with more powerful contrctions (see different slopes in Fig. 7D) nd enhnced stroke volume (Fig. 1). This deeprething ehvior llows the squid to hve the sme mount of wter pssing through the gills per period of time, i.e. stle ventiltory volume per minute, nd explins the mintennce of the squid s cycling ctivity under such O 2 conditions. The O 2 extrction efficiency reched ~82% in EH t ctive metolic rtes (Fig. 1C) without considertion of cutneous uptke. Other cephlopods, such s nutilus nd squid (L. revis), lso lter the effectiveness of oxygen extrction (i.e. cutneous uptke) from the ventiltory strem s result of flling mient O 2 levels or exercise when necessry, ut t round 1 2% (once 43% in nutilus) (Wells et l., 1988; Wells, 199; Pörtner, 1994). Mximum O 2 extrction efficiency (MR t EH), considering 6% potentil skin respirtion in squid (theoreticl model, I. illecerosus under exercise) (Pörtner, 1994), ws still found to e 34% in our study (Fig. 1D). The orgniztion of squid mntle muscle structure (mitochondri-rich inner nd outer lyer) supports the importnce of skin respirtion in cephlopods (Gosline nd DeMont, 198). Under hypoxi, P O2 grdients drsticlly decline nd therefore do not fvor diffusion processes (O 2 uptke vi skin nd gills). Under LH, when jumo squid cese swimming, cutneous uptke might even decrese s result of lck of convection. Deep-rething ehvior oserved t EH, insted, might fvour cutneous uptke (inner nd outer mntle), ut here we were not le to distinguish etween O 2 uptke vi skin nd gills. In cuttlefish nd octopus, orgnisms tht re less dependent on jet propulsion, the frction of oxygen extrction from the ventiltory strem is higher [ 9% in cuttlefish (Melzner et l., 26); 4 7% in octopus (Wells nd Wells, 198; Wells, 199)], nd their O 2 uptke cn only e enhnced y n elevtion in wter throughput nd incresed diffusion grdients t the wter lood threshold (Melzner et l., 26). Other OMZ residents in the Gulf of Cliforni, like Gnthophusi ingens, cn regulte oxygen uptke minly vi n increse in ventiltion volume ( 6 times, mximum 8) t constnt (or slightly incresing) O 2 extrction efficiencies tht cn rech 48 6%, with mximum ~9% (Childress, 1971). However, these lrge chnges in ventiltion volume re not possile for squid ecuse of the constrints imposed y the mssive collgenous tissues in the mntle wlls of musculr squid (Gosline nd Shdwick, 1983; Wells et l., 1988). In contrst, t LH, ll the ventiltory processes were significntly reduced, except for O 2 extrction efficiency followed y lethrgic stte. lso, outilier nd collegues (outilier et l., 1996) reported tht Nutilus cese ventiltory movements, crdic output nd hertet to survive prolonged periods of hypoxi. In the mjority of investigted squid, ventiltion ws completely shut down, ut we cnnot rule out the possiility tht there were further very wek contrctions tht could not e ssessed y the method of nlysis used. nother explntion could e tht D. gigs hs the potentil to uncouple its ventiltory nd locomotory mechnisms vi collr flp system, s is known for resting sepi nd to some extent for squid (one et l., 1994). This originl respirtory design of cephlopods (Wells, 1988) drives expirtion vi n ctive inwrd movement of the collr flps (nd proly vi stored elstic energy during inhltion) (Gosline nd Shdwick, 1983) without the ctivity of circulr muscle fiers (one et l., 1994). Unfortuntely, the experiments were interrupted mximum of 2 min fter the jumo squid stopped moving. Nonetheless, recent dt suggest tht juvenile (R.R., unpulished) nd dult jumo squid (..S., unpulished) cn sustin severe hypoxi (1% O 2 ) for t lest 6 12 h nd therefore might use lethrgy s strtegy to extend the residence time in the OMZ. It is noteworthy tht our experiments were conducted with juveniles nd therefore our results might not pply in dults, even though metolic rtes in juvenile nd dult squid should not differ, s musculr squid disply n unusul scling reltionship (tendency to isometry). This might e consequence of: (1) tuulr geometry (mntle dimeter increses fster thn thickness with growth), exchnge surfces (surfce re 1/2 :volume 1/3 increses with size) (O Dor nd Hor, 2) nd cutneous respirtion (~6%) (Pörtner, 1994; Pörtner, 22); (2) ontogenetic differences in locomotory expenditure, ecuse the squid cost of trnsport my not decrese s much with size s tht in other nimls (e.g. mmmls, irds nd fish), where dults re more energy efficient; nd (3) energetic requirements during ll stges of the squid s short life cycle (nmely, for growth nd reproduction). High sustined production costs during ll ges my result in vlue (scling coefficient) tht does not decrese with incresing ody mss (Seiel, 27; Ros et l., 29). However, it remins uncler whether dults will djust their ventiltory mechnisms to low oxygen levels in similr wy to juveniles, especilly s little is known out the potentil of oxygen uptke vi the skin under hypoxi. THE JOURNL OF EXPERIMENTL IOLOGY

12 368 The Journl of Experimentl iology 216 (3) MR d s E O 2 IMR L L c M MC MR MR mx OMZ r C r H RMR V V V V w x LIST OF SYMOLS ND REVITIONS ctive metolic rte squid density O 2 extrction efficiency inctive metolic rte mntle length cylinder length weight mss (kg) mntle contrctions metolic rte mximum ctive metolic rte oxygen minimum zone contrcted mntle rdius mximum (hyperinflted) mntle rdius routine metolic rte flow rte ventiltory volume per minute stroke volume mntle thickness (mm) CKNOWLEDGEMENTS We would like to thnk Uni Mrkid for his ssistnce with collection of the specimens. FUNDING This work ws supported y reserch grnts to R.R. from the Portugl US Reserch Networks Progrm (FLD/US Ntionl Science Foundtion Reserch Grnt) nd The Portuguese Foundtion for Science nd Technology (FCT, Ciênci 27 Progrm) nd to..s. from the Ntionl Science Foundtion [grnt OCE-8143]. REFERENCES ití-cárdens, L., Muhli-Melo,., Cruz-Esclon, V. nd Glván-Mgñ, F. (22). 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