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J Comp Physiol B (2004) 174: 223–236DOI 10.1007/s00360-003-0405-1 A. R. Cooper Æ S. Morris Haemoglobin function and respiratory status of the PortJackson shark, Heterodontus portusjacksoni, in responseto lowered salinity Accepted: 20 October 2003 / Published online: 8 January 2004
Springer-Verlag 2004 Abstract Haemoglobin function and respiratory status Keywords Respiration Æ Haemoglobin Æ Shark Æ of sub-adult sharks, Heterodontus portusjacksoni was Hyposaline Æ Heterodontus investigated for up to 1 week following transfer from100% to either 75% or 50% seawater. Metabolic rates Abbreviations a–v arterial–venous Æ CO2 CO2 were unusually low and arterial–venous differences in content Æ CaO2 content of O2 in arterial blood Æ CvO2 blood O2 small. Haemodilution from osmotic inflow content of O2 in venous blood Æ %E branchial O2 lowered haematocrit and reduced blood O2 content by extraction efficiency Æ fv ventilatory frequency Æ GTP up to 50%. There was no change in O2 consumption guanosine triphosphate Æ Hct haematocrit Æ [Hb] rate, blood O2 partial pressure, cardiac output, or the haemoglobin concentration Æ ITP inosine arterial-venous O2 content difference, and thus O2 triphosphate Æ met[Hb] methaemoglobin Æ _ delivery was maintained. Ventilation was acutely ele- consumption Æ NTP nucleoside triphosphate Æ OEC vated but returned to normal within 24 h. The O2 oxygen equilibrium curve Æ PaO2 partial pressure of O2 delivery to the tissues was facilitated by decreased blood in arterial blood Æ PeO2 partial pressure of expired O2-affinity that could not be simply ascribed to changes O2 Æ PiO2 partial pressure of inspired O2 Æ PinO2 inflow in the osmolyte concentration. The Hb was unaffected partial pressure of O2 Æ PO2 partial pressure of by changes in intra-erythrocyte fluid urea or trimethyl- O2 Æ PoutO2 outflow partial pressure of O2 Æ pHa arterial amine-N-oxide (TMAO) but was sensitive to changes in blood pH Æ pHpl whole blood pH Æ PV plasma NaCl. The Bohr shifts in whole blood were low and volume Æ PvCO2 partial pressure of CO2in venous there was little role for pH in modulating O2 transport.
blood Æ PvO2 partial pressure of O2in venous blood Æ _ Venous Hb saturation remained close to 65%, at the cardiac output Æ SW seawater Æ TMAO trimethylamine- steepest part of the in vivo O2 equilibrium curve, such V ventilation volume that O2 unloading could be facilitated by small reduc-tions in pressure without increasing cardiac or ventila-tory work. H. portusjacksoni tolerated 50% seawater forat least 1 month, but there was little evidence of respi- ratory responses being adaptive which instead appearedto be consequential on changes in osmotic and ionic Environmental salinity is important in determining the distribution of diverse marine elasmobranchs (Hopkinsand Cech 2003). Most marine elasmobranchs transferredto lower salinity water exhibit marked changes in plasma Communicated by G. Heldmaier and erythrocyte osmolyte composition (Holmes andDonaldson 1969; Pang et al. 1977; Shuttleworth 1988; A. R. CooperSchool of Biological Sciences, Evans 1993; Sulikowski and Maginniss 2001). Plasma University of Sydney, 2006 Sydney, dilution and loss of osmolytes can alter the O2-carrying capacity and haemoglobin oxygen-binding properties of the blood. The Port Jackson shark Heterodontus por- Morlab, School of Biological Sciences, tusjacksoni exhibits all of these responses including a net University of Bristol, Woodland Road, water influx leading to lowered haematocrit (Hct) and Bristol , BS8 1UG UK haemoglobin concentration ([Hb]) following transfer to diluted seawater (SW; Cooper and Morris 1998a, 2003).
Tel.: +44-117-9289181Fax: +44-117-9288520 This appears to be a common response of marine elasmobranchs (e.g. Goldstein and Forster 1971; Chan Haldane effect is usually very small or absent (Nikinmaa and Wong 1977a). Dilution anaemia in fish compro- and Salama 1998; review). In H. portusjacksoni no mises blood O2 transport (Gallaugher and Farrell 1998).
Haldane effect was apparent (Cooper and Morris 2003), Surprisingly, there is no truly integrative account of and through the Wyman linkage equation (Wyman respiratory response and blood function in a marine 1964), predicts virtually no Bohr shift for the Hb in these elasmobranch moved to lower salinity.
sharks (Nikinmaa 1997; review). In general, the func- The optimal haematocrit theory predicts that a de- tional properties of elasmobranch Hb appear to be cline in Hct and therefore O2 capacity would be highly conserved and do not correlate well across species accompanied by an increase in cardiac output ( _ with their aerobic demands (Wells et al. 1992; Wells views: McMahon and Wilkens 1983; Birchard 1997), but this has been questioned for teleost fish (Gallaugher Sharks appear to have acquired Hb sensitivity to et al.1995; reviewed by Gallaugher and Farrell 1998).
ATP subsequent to their evolutionary separation from Elasmobranch Hct shows some correlation between the rays (Scholnick and Mangum 1991), but otherwise species and activity levels but predicted optimal Hct was the influence of major organic and inorganic osmolytes nearly double that measured (Baldwin and Wells 1990).
on Hb function varies between species. For example, a It is unclear what the consequences would be of an decline in erythrocyte ATP increased the Hb O2-affinity osmotically induced anaemia in a shark concomitant of the school shark, Galeorhinus australis (Coates et al.
with lowered osmolyte concentrations. Increases in _ 1978), the dogfish, Squalus acanthias (Wells and Weber and in blood volume of elasmobranchs can be accom- 1983), and the carpet shark, Cephaloscyllium isabella panied by increases in ventilation (Piiper et al. 1977; Lai (Tetens and Wells 1984) but had no significant effect in et al. 1989, 1990; Farrell 1993). The oxygen consumption the electric ray, Torpedo nobiliana (Bonaventura et al.
M O2) of fishes transferred to diluted SW varies con- 1974a), the cownose ray Rhinoptera bonasus (Scholnick siderably (Rao 1968; Chan and Wong 1977b; Maxime and Mangum 1991), or the seven gilled shark, Noto- et al. 1990; Claireaux et al. 1995) and sometimes not at rynchus cepedianus (Coates et al. 1978). Urea is an all (e.g. Claireaux and Lagarde´re 1999). Hyperventila- important osmolyte in marine elasmobranchs and tion to meet increased O2 demand may influence ion potentially deleterious effects of high urea are counter- efflux if a substantial gradient exists—the ion/gas ratio acted by TMAO (Yancey and Somero 1980). In Squalus, (Perry and McDonald 1993; Gonzalez and McDonald urea apparently disrupts the binding of ATP to Hb 1994). Increased outward gradients exist only transiently regardless of the presence of TMAO (Weber 1983; for poorly regulating or osmoconforming euryhaline Weber et al. 1983a, 1983b). Thus a decline in urea, marine species when moving to more dilute water.
permitting phosphate Hb-binding, reduced the Hb O2- Transiently elevated ion loss might minimise the dura- affinity of the dogfish, S. acanthias (Weber 1983; Weber tion of osmotic perturbation of blood respiratory func- et al. 1983a, 1983b). However, urea had virtually no tion and re-establish respiratory status in marine species.
effect on the Hb of Rhinoptera bonasus (Scholnick and H. portusjacksoni transferred to 50% SW remained Mangum 1991), the dogfish, Mustelus canis (Bonaven- hyperosmotic but hypo-natric and were relatively poor tura et al. 1974a, 1974b) or in the black-tip shark Car- regulators (Cooper and Morris 1998a, 2003). As in other charhinus melanopterus (Wells et al. 1992). There is no marine elasmobranchs (Forster and Goldstein 1976; evidence to suggest any specific allosteric role for Boyd et al. 1977; Sulikowski and Maginniss 2001), the TMAO (Weber 1983; Scholnick and Mangum 1991).
intra-erythrocyte fluid [Na], [urea] and trimethylamine- Decreased NaCl induced substantial changes in Hb O2- N-oxide concentration ([TMAO]) of H. portusjacksoni affinity in the tiger shark, Galeocerdo culveri (Scholnick were reduced by up to 50% upon transfer to diluted SW and Mangum 1991) and the skate, Raja eglanteria (Cooper and Morris 1998a). Changes in [urea] and (Bonaventura et al. 1974a, 1974b) but the Hb O2-affinity [TMAO] can alter the structure and function of proteins of the rays Dasyatis sabina (Mumm et al. 1978) (Yancey and Somero 1978, 1979, 1980), including hae- and Rhinoptera bonasus (Scholnick and Mangum 1991) moglobin and Hb O2-affinity (Bonaventura et al. 1974a, 1974b; Weber 1983; Weber et al. 1983a; Scholnick and Sub-adult Port Jackson sharks, H. portusjacksoni, Mangum 1991). The in vivo changes in the Hb function occur in estuarine and inshore marine waters and can of marine elasmobranchs moving into diluted SW have tolerate 50% SW for a least 1 month (Cooper and received relatively little attention (e.g. Burke 1974).
Morris 1998a). It is not known if any aspect of O2-up- While the response of Hb and O2 binding to specific take or transport presents a limitation to this shark pe- osmolytes such as urea and TMAO may be known for netrating into lower-salinity water. The present study some elasmobranch fish (e.g. Bonaventura et al. 1974a; quantifies the changes in respiratory and metabolic sta- Martin et al. 1979; Weber 1983; Weber et al. 1983a; tus that occur during transfer from fully marine to dilute Scholnick and Mangum 1991), their role and effect over SW and in response to changes in the osmolyte and the range of physiological concentrations found in blood haematological status of H. portusjacksoni. The func- requires further elucidation.
tioning of the Hb in Port Jackson sharks may respond The buffering power of elasmobranch blood is con- adaptively to declining concentration of putative allo- siderably greater than in teleosts (Jensen 1989) and the steric regulators, or alternatively may simply be disrupted. This study characterised changes in the whole which directed the outflow back to aerated tank with a sub-sample blood and intra-erythrocyte fluid Hb O to the O2 electrode (E5047, Radiometer) in a flow-through housing.
H. portusjacksoni transferred to diluted SW as elicited by 2 analyser (PHM73, Radiometer) was connected to a com- puter running Datacan IV analysis software (SABLE SYSTEMS).
the specific effect of the decline in [NaCl], [Ca], [urea] Sharks were weighed prior to being placed into the respirometer and [TMAO] as part of the overall respiratory response.
and any air bubbles released via a sealable vent in the chamberroof. To expose sharks to either 75% or 50% SW, the appropriatevolume of SW was removed and replaced with deionised wateronce the respirometer was submerged. The outflow PO Materials and methods was maintained at >17.4 kPa by adjusting the rate of water inflow.
Flow rates ranged between 0.32 l min)1 and 2.7 l min)1 depending Experimental design on animal size (n=135). The PO2 of the inflow water (PinO2) wasrecorded before and after each experimental run to provide base The acute (24 h) and chronic (up to 168 h) respiratory status of H.
lines for the correction of any drift in the PO2 calibration. Sharks portusjacksoni transferred to diluted SW was determined using the were re-weighed at the end of each experimental run. The handling experimental protocol previously described (Cooper and Morris of fish, even for very short periods can increase _ M O2 from resting 1998a, 2003). In brief, Port Jackson sharks were acclimated for to active levels (Satchell 1991). Furthermore, the wash-out volume 1 week in full-strength SW (33–35 g l)1; PO2>18.5 kPa; 19 C).
of the respirometer can result in the distortion of the signal re- Subsequently, the sharks were either held for a further 168 h corded from rapidly changing states (Kaufmann et al. 1989).
(1 week) in either full-strength SW (100% SW) or for 1 week in M O2(lmol min)1 kg)1) was determined from 75% SW before being transferred to one of the following groups: recordings stable for at least 2 h after the sharks had been in therespirometer for at least 16 h: Control (acclimated in 100% SW and transferred to 100% SW) FR
PinO2  PoutO2 75% SW (acclimated in 100% SW and transferred to 75% SW) iii. 50% SW (acclimated in 75% SW and transferred to 50% where FR=flow rate of water through the respirometer (ml min)1) and BW=body weight (kg). The solubility coefficients of O2 (aO2)for either 100%, 75% or 50% SW at 19 C were 11.76, 12.43 and Anaesthetised sharks were cannulated via the caudal artery and 13.11 lmol l)1 kPa)1 (Cameron 1986). There was no change in O2 vein, and the cannulae flushed with heparinised (100 units ml)1, consumption during control runs without sharks. Cardiac output Sigma) shark Ringers (Cooper and Morris 1998b). The sharks were Q; ml kg)1 min)1) was calculated according to the Fick principle placed in metabolism cages in which the respiratory gas status and and using the equation; M O2=ðCaO2  Cv  O2Þ, where pH of the water in the three exposure treatments were not different CaO2)CvO2 is the arterial–venous (a–v) difference in O2 content (PO2=19.51 kPa; Cooper and Morris 2003). Blood was sampled using chilled heparinised 1-ml Hamilton gas-tight syringes at either0, 6, 12, 24, 72 or 168 h after transfer (n=6 for each time period).
Samples from sharks held in 75% SW for 1 week were used as pre- treatment values for 50% SW groups. Blood PO 2 extraction efficiency and ventilation volume with a BMS Mk2 blood micro system (Radiometer) at 19 C and connected to a PHM73 meter. The O 2 extraction efficiency (%E) was measured in sharks 2 electrode was calibrated with (n=5 for each salinity) held in either 100%, 75% or 50% SW (75% an O2-free mixture (CO2/N2) and air-saturated water. Whole blood SW acclimated) and sampled at 0, 6, 12 and 24 h. Due to the necessity O2 content (CO2) was measured using the modified Tucker of repeated sampling, the analysis of %E data used repeated mea- chamber method (Wells and Weber 1989).
sures ANOVA. The %E of H. portusjacksoni was calculated using the The ventilatory frequency (fv) of sharks (n=6 for each time period) transferred to either full-strength or diluted SW was mea- iO2)PeO2)/PiO2, and determined using a similar procedure to that of Piiper and Schumann (1967) and Grigg and sured using the impedance method to count opercular movements Read (1971). The expired O (Cooper and Morris 1998b). The O 2 (PeO2) was measured via cannulae in- serted anteriorly through the second gill flap and 5 mm into the determined from sharks (n=15 for each treatment) held in 100%, parabranchial chamber (for detail on recording from branchial flow 75% or 50% SW (75% SW acclimated), for either 24, 72 or 168 h.
in H. portusjacksoni see: Grigg 1970, Grigg and Read 1971). The The binding of O2 by the Hb was investigated in both whole blood PE45 cannula was fixed to the gill flap using histacryl surgical glue and erythrocytic fluids to separate changes due to the intrinsic (Braun) and the remaining length secured to the first dorsal fin spine.
binding properties of the Hb from any due to interactions with The sharks were allowed 24 h to recover. The inspired O allosteric regulators. Oxygen equilibrium curves (OECs) were gen- measured via cannulae from water entering the mouth of the shark erated for whole blood and intra-erythrocyte fluids from sharks transferred to either 100%, 75% or 50% SW for 72 h. The OECs iO2 and PeO2 were measured using a Radiometer O2 electrode (Cooper and Morris 1998b).
were also generated for whole blood and intra-erythrocyte fluids in There were no changes in ventilation frequency as a result of the which either the [NaCl], [urea] or [TMAO] were modified to simulate insertion of the gill flap catheters. Respiratory water flow, i.e.
in vivo changes. The intra-erythrocyte fluid nucleoside triphosphate ventilation volume ( _ concentration (NTP), haematocrit (Hct) and whole blood [Hb] w; ml min)1 kg)1), was estimated by the Fick principle using the equation: _ M O2=½aO2
ðPiO2  Pe  O2Þ.
were determined for sharks held in either 100%, 75% and 50% SW for 72 h.
w was calculated assuming that the O2 uptake is primarily branchial since cutaneous O2 uptake is less than 5% of the total M O2 in the dogfish (Toulmond et al. 1982).
O2 consumption and cardiac output M O2 of H. portusjacksoni (0.7–1.8 kg) was measured using Whole blood O2 equilibrium curves flow-through respirometry. The respirometry chamber was a 900-mm length of 300-mm diameter PVC pipe with a water-tight false Blood samples (1 ml) were taken by caudal puncture (see Cooper bottom inserted lengthwise into the chamber to reduce the water and Morris 1998b) from sharks transferred to either 100%, 75% or volume and provide a flat surface on which the sharks rested. The 50% SW for 72 h (n=6 for each salinity). Blood samples from each chamber was placed into a 450-l tank in the laboratory aquarium at treatment were pooled in a heparinised 10-ml plastic vial and a constant 19 C. Water was pumped into the inlet pipe by a inserted into a blood mixer refrigerated at 4 C. The Hct was submersible Eheim pump (Type 2213) and the flow rate adjusted by measured immediately after the blood was pooled and throughout a ball-valve. The outlet pipe was connected to a selector valve, the sampling procedure to detect cell swelling or lysis.
To construct the OECs, two aliquots of whole blood (80 ll) assay does not discriminate between ATP and other NTPs such as were tonometered in separate tubes within the BMS MkII blood guanosine triphosphate (GTP) and inosine triphosphate (ITP). As micro system (Radiometer, Denmark). The blood within the a consequence, the ATP:NTP ratio for the dogfish, Squalus tonometer tubes was equilibrated with a known PO2 and a constant acanthias (Wells and Weber 1983), was used to derive the ATP PCO2 gas mixture supplied by gas mixing pumps (Wo¨stoff, Bo- concentration used in dialysis solutions.
chum, Germany) for 25 min at 19 C. The PO2 of the gas mixturesupplied to the blood was varied using a combination of dry CO2-free air and an analytical grade O2-free (CO2/N2) gas mixture. The Intra-erythrocyte fluid O2 equilibrium curves blood PCO2 was adjusted to either 0.8, 1.5, 12.2 or 15.2 kPa.
After equilibration of the blood to each PO2, duplicate whole The diffusion chamber method (Sick and Gersonde 1969) was used to blood CO2 measurements were made using a modified Tucker produce OECs from intra-erythrocyte fluids. Blood samples (1 ml) chamber method. Duplicate pH measurements were made from were taken by caudal puncture from sharks that had been transferred blood samples tonometered to a PO2 closely coinciding with the to either 100%, 75% or 50% SW for 72 h (n=6 for each salinity).
P50 of the O2 equilibrium curve. The whole blood [Hb–O2] was Blood samples were pooled and the intra-erythrocyte fluid collected calculated as described in Wells and Weber (1989). The log P50 and as previously described (Cooper and Morris 1998a). The fluid was n50 were derived using values between 25% and 75% saturation rapidly centrifuged to remove cell debris (5 min at 13,000 g and according to the Hill equation: log (S/100-S)=logP50+(nHÆ- 4 C). The OECs were generated from 2 ll supernatant samples at logPO2), where S=the percent saturation of the pigment, P50=the 19 C and supplied with a constant PCO2 for each curve and a range PO2 at which half of the Hb is saturated (kPa) and nH Hill's of PO2 values which increased in a stepwise fashion using gas-mixing coefficient of cooperativity.
pumps (as above). Each gas setting was maintained until a steadyabsorbance was obtained indicating complete equilibrium at thatPO2. Steady absorbance differences between fully oxygenated anddeoxygenated blood were indicative of the absence of any appre- Whole blood O2 content curves following modification ciable methaemoglobin (met[Hb]) formation throughout the proce- of the plasma osmolytes dure. Complete oxygenation or deoxygenation of the film wasobtained using O2/CO2 or CO2/N2 gas mixtures, respectively.
Blood samples (1 ml) were taken from sharks (n=7 for each treat- Duplicate pH measurements were made from an 80-ll intra-eryth- ment) kept in full-strength SW and immediately pooled in a 10-ml rocyte fluid sample tonometered to a PO2 closely coinciding with the heparinised plastic vial and placed on ice. Sub-samples of a known volume were removed and centrifuged at 6,500 g for 1 min. Theplasma volume (PV) of each sub-sample was calculated from theequation PV=Whole blood volumeÆ(1)Hct/100). Half of the plasma Intra-erythrocyte O volume from each sub-sample was removed and replaced with an 2 equilibrium curves post-modification of osmolyte concentrations equal volume of the selected plasma Ringer's solutions. The bloodwas gently re-mixed and treated as described previously. The com- Further OECs were generated for Hb in intra-erythrocyte fluid in position of the ‘control' Ringer was (in mmol l)1): 12 KCl, 5 MgCl2, which either the [NaCl], [urea], [TMAO] or [Ca] was altered. Blood 10 CaCl2, 4.5 NaHCO3, 0.5 NaH2PO4, 280 NaCl, 360 urea, samples (1 ml) were taken from sharks in full-strength SW (n=5 for 90 TMAO, with lithium heparin (100 units ml)1) and adjusted to each treatment) and the intra-erythrocyte fluid dialysed in 2 l intra- pH 7.90. The NaCl-, urea-, TMAO- and urea/TMAO-free ringers erythrocyte Ringer's solution (Table 1) at 4 C for 24 h. The [Na], simply omitted the required component(s) such that the eventual [urea], [TMAO], [Ca] and [ATP] of the Ringer's solutions simulated plasma concentration was halved. The intra-erythrocyte fluid [urea] the reduction in the intra-erythrocyte fluid concentrations of sharks and [TMAO] were reduced by 48% and 52%, respectively, within transferred to diluted SW (Table 1). Any decline in [Hb], or increase 30 min of halving plasma concentrations. In contrast, intra-eryth- in met[Hb], was determined from the difference in pre- and post- rocyte fluid [Na] declined by only 22% after 30 min but after 6 h had dialysis intra-erythrocyte fluid samples (n=9). The [Hb] was mea- declined by 57%. The urea concentrations were determined using an sured as previously in Cooper and Morris (1998b) and the met[Hb] assay test kit (Boehringer Mannheim, Catalogue No. 542 946) and using procedures described by Robin and Harley (1964) and Bridges that of TMAO was determined according to Withers et al. (1994a, et al. (1985). While the post-dialysis erythrocyte [Hb] was consis- tently found to be 10% lower than pre-dialysis values, themet[Hb]:[Hb] ratios did not differ.
Plasma and intra-erythrocyte fluid total NTP Statistical analyses The plasma and intra-erythrocyte fluid NTP concentrations weremeasured using a Sigma test kit (Cat. No. 366-UV) from blood The design of the time-course for salinity exposure was fully samples taken by caudal puncture from sharks transferred to either independent (total n=96–135). Data from sharks transferred to 100%, 75% or 50% SW for 72 h (n=6 for each salinity). This ATP either 100%, 75% or 50% SW were compared, after confirming Table 1 The [NaCl], [urea], trimethylamine-N-oxide concentration (100 units ml)1) and were adjusted to pH 7.00. The experimental ([TMAO]) and [Ca] of intra-erythrocyte fluid Ringer's solutions osmolyte concentrations in Ringer's solutions are provided inbold (mmol l)1). Each solution contained KCl (60 mmol l)1), MgCl2 while the control concentrations are denoted by an asterisk l)1), ATP (3.1 mmol l)1) and lithium heparin Table 2 The whole bloodrespiratory status of transferred to either 100%,75% or 50% seawater (SW) for up to 1 week (168 h). All values are given as means±SEM.
aO2 arterial blood O2 vO2 venous blood O2 aO2 partial pressure 2 of arterial blood, PvO2 partial pressure of O *Significantly different from sharks transferred to 100% SW; #significantly different from 70% SW. (N=6 at each sample time and in each treat- ment; total n>196) homogeneity of variances, using a two-way ANOVA, and sub- and in contrast with the PO2 values, there were marked sequent to grouping by one-way ANOVA. Differences between decreases in the CO linear plots of logP 2 of sharks in either 75% or 50%SW 50 vs. pH were analysed by ANCOVA and (Table 2). In sharks moved to 75% SW, the venous blood values are expressed as means±SEM with a probability (P) value<0.05 considered significant.
O2 content (CvO2) declined significantly within 12–24 hbut in 50% SW this occurred with 6–12 h. The arterialblood O2 content (CaO2) did not decline until between 24 h and 72 h after transfer to lower salinity water. BothCaO2 and CvO2 remained lower than control values after Whole blood PO2, CO2 and [Hb–O2] 1 week (168 h; Table 2). As a consequence of the ten-dency for both CaO2 and CvO2 to decrease, there were no The arterial PO2 (PaO2), and venous PO2 (PvO2) of Port significant changes in the CaO2)CvO2 differences. After Jackson sharks transferred to either 75% or 50% SW 168 h, these a–v differences were 0.36, 0.21 and did not generally differ from those of control sharks 0.22 mmol l)1 for sharks in 100%, 75% and 50% SW, (Table 2). As a consequence, the PaO2)PvO2 differences respectively. Therefore, assuming a constant rate of blood of sharks in diluted SW did not differ from those of perfusion (below), the O2 uptake by the tissues of sharks in control sharks and thus the PO2 gradient for unloading diluted SW remained constant. This is consistent with the O2 to the tissues was not significantly reduced. However, M O2 (Table 3). The apparently constant O2 Table 3 The respiratory and cardiovascular responses of H. portusjacksoni transferred to either 100%, 75% or 50% SW for up to 1 week(168 h). All values are given as means±SEM. Hyphen denotes data unavailable. (eryth intra-erythrocyte fluid) Time (h) Treatment Hct (%) [L-lactate] Q(ml min)1 kg)1) %E plasma [L-lactate]eryth *Significantly different from sharks transferred to 100% SW; #significantly different from 70% SW. (Hct data ANOVA on arcsintransformed data). (N=6 at each sample time and in each treatment; total n>196) delivery was maintained despite a significant loss of O2 The changes in [L-lactate] were very small. The capacity of sharks transferred to either 75% or 50% SW plasma [L-lactate] of sharks transferred to either 75% or which declined within a similar 24 h period by 25% and 50% SW increased two fold and six fold, respectively, 30%, respectively, and remained lowered for the after only 6 h but never exceeded 2 mmol l)1 (Table 3).
remainder of the 1-week trial (Table 2). The decline in the While the intra-erythrocyte fluid [L-lactate] of sharks CO2 and in CO2-max tracked the reductions in Hct transferred to 75% SW did not differ from control val- (Table 3), which declined by 30% within 6–12 h and ues, that of sharks in 50% SW exhibited a four-fold 12–24 h for sharks in 75% or 50% SW, respectively, increase in intra-erythrocyte fluid [L-lactate] after 6 h, without any indication of recovery after 1 week (Table 3).
but again the concentrations remained low (Table 3).
M O2, _Q, fv and L-lactate Branchial O2 extraction efficiency and ventilationvolume The increased body weight of H. portusjacksoni aftertransfer to diluted SW resulted from water loading The %E of H. portusjacksoni was universally low and in (Cooper and Morris 1998a, 2003) and consequently the sharks moved to 75% SW showed only a very brief M O2 of H. portusjacksoni was calculated using initial elevation (Table 3). In contrast, the %E of sharks M O2 of sharks transferred to either 75% transferred to 50% SW declined by 60% to very low or 50% SW for between 1 day and 7 days did not differ values within 6 h and remained lowered for at least the from that of sharks in full-strength SW (Table 3). Con- initial 24 h (Table 3). The changes in _ sequently, the regression equations for log _ verse relationship with the changes in %E (Table 3). As l O2 min)1) vs. body mass (kg) were determined from a consequence, the _ pooled data for sharks exposed to each salinity. The log w of sharks in 75% SW did not differ from control values except immediately following M O2 of H. portusjacksoni in either full-strength or di- transfer, while the _ luted SW was directly correlated with the body mass with w of sharks in 50% SW approxi- mately doubled within only 6 h before returning to all slopes significantly different from zero (0.54–0.66; control values after 24 h (Table 3).
Q values for sharks at each salinity were calcu- M O2 values normalised for a 1.2-kg shark.
ANCOVA revealed no differences between either the Whole blood Hb functioning slope or the elevation of the regression lines (Fig. 1) andthus the overall mean M O2=10.19 lmol kg)1 min)1 The Hct of sharks held in either 75% or 50% SW for (Fig. 1). There were no changes in the calculated _ 72 h were 14% and 15%, respectively, compared to 18% sharks transferred to diluted SW, either with respect to in control sharks. During the generation of the OECs, control sharks (100%SW) or during days 1–7 (Table 3).
the Hct of control (19%±0.6) and experimental The fv of sharks transferred to either 75% or 50% SW (14.8%±0.2 and 15.4%±0.4) samples remained con- increased by 40% within 6 h but returned to control stant. Thus, the maximal CO values within 72 h (Table 3).
2 measured from sharks in 75% or 50% SW was 1.05±0.4 mmol 0.98±0.1 mmol l)1, respectively, and significantly lessthan the 1.54±0.2 mmol l)1 of sharks in 100% SW,which reflected the differences in Hct (Table 3).
Bohr factors (/) were derived from linear regression of the Dlog P50/DpH for sharks transferred to either100%, 75% or 50% SW and compared using ANCOVA(Fig. 2A). Values of / were small at )0.11, )0.12 and )0.03 for sharks held in either 100%, 75% or 50% SW,respectively, but not significantly different. There weredifferences in the elevations (affinity for O2) of theregression lines due largely to a decrease in Hb O2-affinity (at the P50) in sharks transferred to 75% SW.
There was no change in the cooperativity (n50) withchanges in the blood pH. The n50 values were thereforecalculated for sharks in each salinity and comparedusing a one-way ANOVA. The n50 values of sharks infull-strength SW (n50=1.98±0.08; Fig. 2B) were mark-edly higher than those of sharks in either 75% Fig. 1 The relationship between log M O2 and body mass in SW (n50=1.37±0.01) or 50% SW (n50=0.92±0.12), Heterodontus portusjacksoni held in either 100%, 75% or 50% respectively. Thus, the cooperativity of Hb-O2 binding seawater (SW) for up to 1 week (168 h). The horizontal broken line in H. portusjacksoni decreased, eventually to zero denotes the oxygen consumption ( _ M O2) values derived for a 1.2-kg shark. Values of individual slopes shown in parentheses (n50=1), with the decrease in water salinity.
Fig. 2A–D The in vivo whole blood Hb O2-affinity (A) and could be investigated without non-physiological buffers.
cooperativity of Hb O2-binding (B). The Bohr factor (/) and R2 In Port Jackson shark erythrocytes, the relationship from linear regression analysis for each treatment were: 100% SW, between erythrocyte pH (pHer) and plasma pH (pHpl) /=)0.11, (R2=0.91); 75% SW, /=)0.12, (R2=0.84); 50% SW,/= could be described by: pH )0.03, (R2=0.65). The in vitro whole blood Hb O and cooperativity of Hb O2-binding (D) following the manipula- and Morris 2003), which predicts that pHe=7.6 would tion of the plasma osmolyte concentrations. The broken line in be concomitant with pHi=6.95. The mean pHi of the panel C represents blood with the normal complement of osmolytes erythrocytes was essentially invariant following transfer as found in sharks kept in 100% SW. The / and R2, both to dilute SW (Cooper and Morris 2003) and a calculated from linear regression analysis for each treatment were:Ringer's-urea: /=)0.07, (R2=0.61); Ringer's-TMAO: /=)0.05, mean pH 7.06 was inserted into subsequent regression (R2=0.63); Ringer's-Na, /=)0.14, (R2=0.69); Ringer's-urea- equations to describe changes in Hb O2-affinity.
TMAO-Na, /=)0.25, (R2=0.99). Blood was pooled from six The Bohr factors of the intra-erythrocyte fluid re- sharks held in each treatment and sub-sampled for each oxygen mained low but surprisingly were positive values (reverse equilibrium curve (OEC) Bohr effect), ranging between 0.07 and 0.30 (Fig. 3A).
The response to the modification of the plasma This variation did not represent any significant change in / with respect to salinity. In view of the unexpectedvalues of />0 selected equilibrium curves were repeated The small Bohr factors in the whole blood persisted in and with identical results. Furthermore: (a) the equilib- blood in which the plasma [NaCl], [urea] or [TMAO] was rium absorbance at each PO2 was in every case verified effectively halved (/=)0.04 to )0.24). The single signif- by a steady reading, (b) the total absorbance difference icant change was the almost complete loss of a Bohr effect between oxygenated and deoxygenated solutions were when TMAO was removed (Fig. 2C). There were also the same at the end and start of each curve, (c) the significant changes in Hb O2-affinity compared to blood duplicate pH measurements made 20 min apart pro- with normal osmolyte concentrations. While a 50% vided similar values and (d) the same result was obtained reduction in plasma urea concentration produced no on diluted samples. The decreased Hb O2-affinity con- appreciable change in Hb O2-binding, a proportional comitant with lowered water salinity was somewhat reduction of either plasma [TMAO] or [NaCl] promoted more obvious in erythrocytic fluid than in the intact increased Hb O2-affinity, and reducing both TMAO and blood (Fig. 3A, C). The Hb O2-affinity of sharks trans- Na caused the largest increase in affinity (Fig. 2C). The ferred to either 75% or 50% SW was 8% and 24% reduction in plasma [TMAO] also resulted in a marked lower, respectively, than that from sharks held in increase in the cooperativity of Hb-O2 binding (mean 100%SW. While the cooperativity of Hb-O2 binding was n50=2.96±0.23) while the n50 values of other experi- higher when measured in erythrocyte fluids compared mental treatments did not differ from normal whole blood with whole blood values (n50=2.4 vs. 2.0), it did not values (Fig. 2D: mean n50; lowered urea, 1.62±0.11; alter with the change in water salinity (Fig. 3B).
lowered NaCl 2.05±0.08; lowered urea, TMAO andNa+, 1.78±0.3).
Responses to the reduction in osmolytes within Hb functioning under erythrocytic conditions erythrocyte fluid The buffering capacity of the intra-erythrocyte fluids of The reduction in intra-erythrocyte fluid [NaCl] by H. portusjacksoni limited the pH range (pH 6.6–7.0) that dialysis did not alter the Bohr factors, which remained Fig. 3A–D The in vivo intra-erythrocyte fluid Hb O2-affinity (A) control values (Table 4). Furthermore, since the intra- and cooperativity of Hb O2-binding (B). The Bohr factor (/) and erythrocyte [Hb] did not differ between control and R2, and the n50 values were: 100% SW, /=0.31, (R2=0.99), experimental sharks, there were no differences in the n50=2.38±0.03; 75% SW, /=0.07, (R2=0.96) n50=2.26±0.05;50% SW, /=0.23, (R2=0.64), n NTP:[Hb] ratio (Table 4).
50=2.40±0.09. The in vitro intra- erythrocyte fluid Hb O2-affinity (C) and cooperativity of HbO2-binding (D) following the manipulation of the [NaCl]. The Bohrfactor (/) and R2, and the n50 values were: 15:80 mmol l)1, / =0.07, (R2=0.94) n50=2.49±0.04; 25:95 mmol =0.23, (R2=0.99), n 2-binding properties of blood and Hb 50=2.66±0.034. The asterisk denotes control concentrations. Blood was pooled from six sharks held in eachtreatment and sub-sampled for each OEC The in vivo Hb O2-affinity (P50=1.92 kPa) of H. por-tusjacksoni kept in full-strength SW was higher than that positive but did result in a marked increase in Hb O2- determined by Grigg (1974) but typical of marine elas- affinity (Fig. 3C). At in vivo pH values, the decrease in mobranchs (Lenfant and Johansen 1966; Burke 1974; intra-erythrocyte [Na] from 40 mmol Martin et al. 1979; Bushnell et al. 1982; Wells and Weber 25 mmol l)1 or 15 mmol l)1 was accompanied by a 1983; Tetens and Wells 1984; Lai et al. 1990; Scholnick 17% and 33% increase in Hb O2-affinity, respectively; and Mangum 1991). The moderate cooperativity of O2- note: [Cl)]=63.5+1.17 [Na+] (Fig. 3C). The reduction binding by Hb of H. portusjacksoni (n50=1.98 in whole in intra-erythrocyte fluid [NaCl] did not alter the blood) was also similar to, or slightly greater than, that cooperativity for Hb-O2 binding and the n50 values of other elasmobranchs (Hughes and Wood 1974; remained >2.4 (Fig. 3D).
Mumm et al. 1978; Wells and Weber 1983; Lai et al.
Dialysis to reduce either intra-erythrocyte fluid [urea] 1990; Scholnick and Mangum 1991). The whole blood or [TMAO] caused no significant change in the pH Bohr factors (/=)0.11) (and thus Haldane effect) of H.
sensitivity of O2-binding (ANCOVA) and there was no portusjacksoni were typically small (Grigg 1974; Nash convincing evidence for a correlation between erythro- et al. 1976) and usual in marine elasmobranchs (reviews: cyte [urea] or [TMAO] and Hb O2-affinity (Fig. 4A, C).
Nikinmaa 1997; Wells 1999).
The cooperativity of Hb O2-binding was also unaffected The complete absence in the erythrocyte fluid of a by the reduction in [urea] or [TMAO] and generally Bohr shift, or the occurrence of reverse Bohr shifts, was n50±2.5 (Fig. 4B, D). The dialysis in urea-free Ringer's not anticipated. Previously shark Hb O2-binding has solution did not significantly change the O2-binding been quantified in buffer solutions (e.g. Weber 1983; properties of the Hb molecule (Fig. 4A, B).
Scholnick and Mangum 1991; Wells et al. 1992), whereas Neither the Hb O2-affinity nor the cooperativity of the present study retained erythrocytic fluid to preserve Hb-O2 binding were altered by the decline in intra- native osmolyte concentrations. Complete O2 equili- erythrocyte fluid [Ca]. At in vivo pH values, the log P50 bration was ensured at each step. The pH range over for low and high intra-erythrocyte fluid [Ca] were which the OECs were generated was limited by the H+ 0.45 kPa and 0.43 kPa, respectively, and Bohr factors buffering capacity of the intra-erythrocyte fluids (Tufts 0.24 and 0.23. The mean n50 values for low and high [Ca] and Perry 1998; Cooper and Morris 2003). Further were also similar at 2.39±0.05 and 2.49±0.06, respec- OECs were generated from intra-erythrocyte fluid di- luted 250 fold with control Ringer's solution and mea- The plasma and intra-erythrocyte NTP concentra- sured over the wider pH range. The results were tions of H. portusjacksoni transferred to either 75% or consistent with those from non-diluted fluid and the Hb 50% SW for 72 h did not differ significantly from showed normal alkaline and acid Bohr shifts (Table 5).
Fig. 4A–D The effect of the in vitro changes in intra-erythrocyte associated with the use of physiological salines rather fluid [urea] on the Hb O2-affinity (A) and cooperativity of Hb O2- than buffers. Some ATP degradation during dialysis binding (B). The Bohr factor (/) and R2, both calculated from may have lowered the ATP but the Hb O2-affinity was linear regression analysis for each treatment were: 0 mmol l)1[urea], typical of whole blood rather than stripped Hb.
(R2=0.99); 180 mmol l)1 [urea], /=0.34, (R2=0.92): 360 mmol The reduction in the whole blood Hb O2-affinity of l)1 [urea]*, /=0.23, (R2=0.99); 360 mmol l)1 [urea]#, /=0.06, H. portusjacksoni in dilute SW was not shown by the ion (R2=0.73). The effects of intra-erythrocyte fluid [TMAO] on the regulating bull shark Carcharhinus leucas (Burke 1974).
Hb O2-affinity (C) and cooperativity of Hb O2-binding (D). The / Analysis close to the venous O and R2, both calculated from linear regression analysis for each 2 saturation (65%) in Port treatment were: 0 mmol l)1 [TMAO], /=0.36, (R2=0.84); Jackson sharks provided the relationship: P65=6.975) l)1 [TMAO]*, /=0.23, (R2=0.99); 135 mmol 0.040%SW. Reduced salinity was also coupled in these [TMAO], /=0.12, (R2=0.96); 180 mmol l)1 [TMAO], /=0.19, sharks with a progressive reduction in the cooperativity (R2=0.99). The asterisk denotes control concentrations, the hash of Hb O2-binding. The effect of reduced water salinity mark denotes previously dialysed in urea-free Ringer's solution for24 h on resultant O2 affinity was similarly obvious in theO2-binding data for erythrocytic fluid (P50=3.57) Similar acid and alkaline Bohr shifts were apparent for the Hb of Carcharhinus milberti but only when stripped Reducing SW salinity clearly prompted changes in of phosphates (Pennelly et al. 1975). Removing phos- the affinity of H. portusjacksoni Hb for O2. Allosteric phates normally increases O effectors of shark Hb have been suggested to include 2 affinity and reduces the pH sensitivity considerably (e.g. Weber 1983). The magni- organic phosphates (e.g. Nikinmaa 1997), urea and tude of the Bohr shift in elasmobranchs ranged even TMAO (e.g. Wells 1999) and Cl) (e.g. Scholnick and within a single study from )0.35 in black-tipped reef Magnum 1991). However, the evidence for roles for shark to almost zero in shovelnosed rays (Wells et al.
these compounds is far from consistent. Urea was re- 1992). Detailed examination of data for whole cells from ported to disrupt NTP binding to Squalus Hb (Weber the cownosed ray (ray Hb seems insensitive to phos- et al. 1983a, 1983b) regardless of the presence of urea phate) (Schlonick and Mangum 1991) shows /=)0.70 (Weber 1983). Conversely, Scholnick and Mangum at high pH (pH 7.5–8.0) but at acid values (pH 6.9–7.2) (1991) found almost no-sensitivity to urea but some / was zero or even slightly positive. Similar trends were considerable response to NaCl, whereas Wells et al.
apparent in the blue shark Prionace glauca (Pennelly (1992) found no effect of NaCl or urea on Hb function in et al. 1975) and the dogfish Mustelis canis (Scholnick black-tipped sharks. Functional interpretation of O2- and Mangum 1991). Why the acid Bohr shift was so binding by shark Hb is further complicated by: O2 apparent in Port Jackson sharks is unclear but it may be sensitivity of Hb polymerisation (Fyhn and Sullivan Table 4 The total nucleosidetriphosphate (NTP) and haematological status of H.
portusjacksoni held in either100%, 75% or 50% SW for 72 h.
All values are given as means±SEM. (N=6 in each Table 5 Values for the Bohr factor (/) determined from groups of might have an effect within the physiological range and four equilibrium curves for each the mean pH values shown. The explain the observed changes in Hb solutions, it is curves are for intra-erythrocyte fluid diluted 250 fold with control apparently irrelevant in vivo for Port Jackson sharks.
Ringer's solution as provided in Table 1 and otherwise determinedusing the same methods as for undiluted fluid. The erythrocytic Removing 50% of the plasma TMAO but leaving fluid was obtained from blood pooled from six similarly treated urea at normal levels, increased cooperativity (n50=3).
TMAO depression of cooperativity is inconsistent withWeber's (1983) hypothesis that TMAO may favour Hb aggregation in sharks. In any case there were no effects of TMAO, or urea, in the erythrocytic fluid OEC andthus changes in O2 affinity seem necessarily to be med-iated by the cell membrane. The artificial plasma solu- 1975), the probability that TMAO stabilises Hb4 (Weber tion did not contain ATP since plasma levels were 1983), the interaction between urea, H+ and NTP in <0.5 mmol l)1 and erythrocytic ATP levels may have binding Hb tetramers (Nikinmaa 1997; Wells 1999) and been lowered slightly as a consequence, which may have the relative loss of cooperativity in O2-binding by Hb perturbed the O2-affinity of the manipulated whole dimers. Osmotically induced changes in erythrocyte blood. However, the salinity effect occurred in native volume and thereby mean cell haemoglobin content can whole blood and persisted in the erythrocyte fluid. It is also affect Hb ligand-binding, although in H. portus- nonetheless clear that the functional O2-binding char- jacksoni, there appears to be little chronic effect of acteristics of erythrocytes taken from sharks in low salinity on cell volume (Cooper and Morris 1998a).
salinity cannot be simply reproduced by manipulating The simulation of the in vivo reduction in plasma plasma osmolyte concentrations.
osmolyte concentrations in sharks held in 50% SW de-creased the P50 (pH 7.5) to 1.14 kPa from 2.12 kPa.
This increase in O2 affinity was, to a lesser extent, also Oxygen uptake and transport induced by lowering TMAO or NaCl alone but wasopposite to the decrease in O2-affinity seen in intact The loss of arterial O2 content in H. portusjacksoni be- whole blood and Hb in solution after exposing sharks to tween 24 h and 72 h in diluted SW, and even sooner in lower salinity.
the venous blood, was not due any reduction in PO2.
The Hb O2-affinity of most elasmobranchs is unaf- The Bohr shift was small and thus, in H. portusjacksoni, fected by changes in [urea] (Bonaventura et al. 1974a, there appeared a minimal role for red blood cell pH in 1974b; Mumm et al. 1978; Martin et al. 1979; Powers the modulation of Hb O2-affinity; this is consistent with et al. 1979; Scholnick and Mangum 1991) with the elasmobranchs generally (Nikinmaa 1997). Further- possible exception of the dogfish, S. acanthias (Weber more, changes in vivo were at most an alkalosis of et al. 1983b). The changes H. portusjacksoni Hb O2- 0.1 pH units (Cooper and Morris 2003) and thus the affinity associated with lowering urea from 360 mmol primary cause of lowered O2 capacity was a dilution l)1 to 90 mmol l)1 were indistinguishable from zero anaemia and the rapid decline in the Hct and [Hb].
(DlogP50/D[urea]=)0.003). As observed in other elas- The release of O2 to the tissues of sharks in lower mobranchs (Tetens and Wells 1984; Scholnick and salinity was facilitated by the lowered Hb O2-affinity.
Mangum 1991), the cooperativity of Hb-O2 binding was The venous O2 in H. portusjacksoni persisted close to the unaffected by changes in [urea]. Thus, changes in urea ‘shoulder' of the O2 equilibrium curves at approximately concentration do not underlie directly the affinity 65% saturation (Fig. 5). Thus, small reductions in PvO2 changes of H. portusjacksoni transferred to dilute SW.
could markedly lower the saturation of the blood and Dialysis of Hb from H. portusjacksoni reduced both increase the amount of O2 unloaded. However, as a re- Na and Cl concentration and increased the O2 affinity sult of the reduced n50, sharks in diluted SW exhibited {P50=1.303+(0.032 [Na+]) or P50=(0.028 [Cl)]))0.459}.
less sigmoidal equilibria curves and thus required a In H. portusjacksoni, the intra-erythrocyte fluid [Na+] greater reduction in PvO2 to unload an equivalent declined markedly from 34 mmol l)1 to 6 mmol l)1 amount of O2 to the tissues. A mean pH 7.90 was in- whereas [Cl)] did not change (Cooper and Morris serted into the regression equations for whole blood to 1998a). This represents a fundamental difference in the allow changes in Hb O2-affinity to be described under in response of whole cells in plasma compared to treat- vivo conditions. The log P50 of sharks held in either ments of Hb solutions. Reduction in [Cl)] reduces the 100% or 50% SW was similar at 0.28 kPa and 0.29 kPa, number of stabilising salt bonds favouring the oxy-state respectively, and approximately 15% higher than that of (Brunori et al. 1975; Jensen 1991). Consequently, the sharks held in 75% SW.
increase in Hb O2-affinity of the intra-erythrocyte fluids The blood PO2, the _ M O2 or the CaO2)CvO2 were dialysed in low [NaCl] most likely resulted from the essentially invariant despite transfer of H. portusjacksoni reduction in [Cl)]. Reduction in the intra-erythrocyte to diluted SW. Consequently, neither the rate of blood fluid [NaCl] of the dogfish, M. canis and the tiger shark, QÞ nor the utilisation or effectiveness of O2 Galeocerdo culveri (Scholnick and Mangum 1991) also uptake from the blood by the tissues was different from resulted in an increase in Hb O2-affinity. Thus while Cl) that of sharks in full-strength SW. The salinity induced 9.5 lmol min)1 kg)1 12.7 lmol min)1 kg)1 corded for H. portusjacksoni indicate a very low meta-bolic rate which was consistent the very small a–vdifferences in CO2 (0.1–0.5 mmol l)1) as compared tothe 1.1 mmol l)1 and 2.8 mmol l)1 in resting leopardand mako sharks, respectively (Lai et al. 1990; 1997).
Aside from their seasonal migration, Port Jackson sharksare slow moving, nocturnal benthic foragers and amongthe least active elasmobranchs (Last and Stevens 1994).
Sampling of mixed venous blood was not possible andthe caudal vein samples might underestimate O2 deple-tion from the blood and overestimate cardiac output.
However, there was no reduction in M_O2 of H. portus-jacksoni transferred to 50% SW and apparently no Fig. 5 Oxygen equilibrium curves derived from the in vivo change in Q_ and thus the decline in %E was likely due to relationship between the PO2 and the CO2 of H. portusjacksoni elevated PeO2 resulting from increased of _Vw. Elevated transferred to either full-strength or diluted SW. The solid curve for 100% SW is shown together with the in vivo data (open square) and eO2 would increase mean PO2 across the gills and the broken line for 50% SW with corresponding data (closed facilitate high PaO2 (for a review of diffusion vs. perfu- square). For clarity the curve for 75% SW is shown without the sion limitations across gills see: Perry and Gilmour 2002).
individual points and the standard errors are shown in one While there was a near 50% decline in the blood direction only. The a–v differences are shown as vertical bars at the CaO2 of sharks in 50% SW, they maintained the same a– right of the figure. Since the mean CaO2)CvO2 differences did notdiffer, their proportional increase with respect to the CO v difference as sharks in 100%SW. However, as a result with the decline in SW salinity of dilution anaemia reducing total O2 capacity, thissimilar CaO2)CvO2 represent a larger proportion of that anaemia consequent on increased plasma volume total O2 capacity in sharks held in 50% SW. Thus for (Cooper and Morris 1998a, 2003) caused reductions in sharks in 100% SW the O2 extracted by the tissues was Hct and in arterial and venous CO2. The relationship, 18% of the CaO2 but was 35% in sharks in 50% SW grouping all treatments, could be expressed by; [Hb– (Fig. 5). Nonetheless, sharks transferred to 50% SW O2]max=(0.083ÆHct))0.173 (R2=0.67). Despite the per- maintained a substantial venous reserve (Fig. 5).
sistent anaemia, ventilation rate returned to control Q of H. portusjacksoni in full-strength SW values between 24 h and 72 h after transfer. Conse- (55 ml min)1 kg)1) was similar to that reported for quently, the maximal [L-lactate] of H. portusjacksoni in some other sharks (e.g. Mako 47 ml min)1 kg)1, Lai 50% SW (<2 mmol l)1) was negligible compared to et al. 1997; Leopard 34 ml min)1 kg)1, Lai et al. 1990), values of between 15 mmol l)1 and 20 mmol l)1 re- but greater than the 18 ml min)1 kg)1 the congeneric ported in some elasmobranchs following hypoxia or Heterodontus francisci (Head et al. 2001). A decline in exhaustive exercise (Piiper et al. 1972; Holeton and [Hb] tends to increase the blood convection requirement Heisler 1983; Lowe et al. 1995; Routley et al. 2002).
(Q_/M_O2) in fish (Jensen 1991) but despite the marked The %E of sharks transferred to 50% SW was acutely reductions in [Hb] and CO2, the Q_ of H. portusjacksoni and severely reduced concomitant with increased PeO2 in diluted SW did not change. At the same time, the and was clearly correlated with [Hb] as described by: M O2 of H. portusjacksoni did not change upon transfer %E=(3.91Æ[Hb]))2.45, (R2=0.65). The %E values were to diluted SW. Consequently, the _ M O2 (an index of at the lower end of the literature range (e.g. Perry and mechanical work needed to supply metabolism) was McDonald 1993) including for elasmobranchs (e.g. Par- unchanged over a period of 6 days. However, the sons and Carlson 1998). Grigg (1970) pointed out that M O2 in H. portusjackonsi (4.2–4.5 l mmol)1) was due to the bottom feeding habit of H. portusjacksoni, the approximately four times larger than that in resting flow through the gill slits may periodically reverse mak- leopard sharks (Lai et al. 1990), seeming to reflect the ing the measurement of truly mixed expired PO2 quite very low values of E%.
challenging. Indeed, it is arguable the homogeneously A hyperventilatory response is not pre-requisite for mixed expired flow does not occur in these sharks.
maintaining O2 transport during longer-term experi- However, calculation of _ mental anaemia (Cameron and Davis 1970; Wood et al.
the same PiO2)PeO2 values as %E and the values of _Vw 1979; Perry and Gilmour 1996), but was acutely (27–96 l h)1) very similar to the 40–70 l h)1 reported by important for H. portusjacksoni in 50% SW. The long- Carlson and Parsons (2001). Available _ term maintenance of O2 transport by H. portusjacksoni elasmobranchs are in the range of 13–>200 lmol - did not require hyperventilation to maintain PaO2 val- min)1 kg)1 (Chan and Wong 1977b; Lai et al. 1990; ues. Thus, during the acute exposure to diluted SW, the Carlson and Parsons 1999; Meloni et al. 2002; Routley management of water influx (and possibly ion flux) may et al. 2002; Miklos et al. 2003), which is a somewhat be of greater importance than branchial O2 uptake wider range than that reported for teleosts by Gonzalez and McDonald (1994). The M O2 values between and McDonald 1992, 1994). The marine sharks in the current study probably had no severe difficulty with the Brunori M, Falcioni G, Fortuna G, Giardnia B (1975) Effect of ion gas ratio since they were transferred to 50% SW anions on the oxygen binding properties of the hemoglobincomponents from trout (Salmo irideus). Arch Biochem Biophys rather than freshwater. However, Port Jackson sharks are not good ion and osmoregulators and on transfer to Burke JD (1974) Hemoglobin stability in bull sharks. Am J Anat dilute SW appear to adopt a strategy of accelerating the excretion of osmolytes to achieve new osmotic equilib- Bushnell PG, Lutz PL, Steffensen JF, Oikari A, Gruber SH (1982) rium and thereby minimise the period of elevated water Increases in arterial blood oxygen during exercise in the lemonshark (Negaprion brevirostris). J Comp Physiol 147:41–47 influx (Cooper and Morris 2003). Bat rays increased Cameron JN (1986) Principles of physiological measurement.
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