Cardiovascular responses of the winter flounder Pseudopleuronectes americanus to hypoxia

Camp.

Bmhrm.

Ph~\rol..

1977.

Vol

51A.

pp

123

r,, 125.

Pm,umon

in Grrar Br~rmn

Pruss. Pmied

CARDIOVASCULAR RESPONSES OF THE WINTER FLOUNDER PSEUDOPLEURONECTES AMERICANUS TO HYPOXIA* JOSEPH

J.

CECH, JR.,’ DAVID

The Research

M. ROWELL’

and

JAMES S. GLASGOW~

Institute of the Gulf of Maine, 21 Vocational South Portland, ME 04106, U.S.A.

Drive.

(Received 16 Seprrmher 1976) Abstract-l. Winter flounder exposed to hypoxic conditions (3.57 mg 02/1 x) were compared with normoxic group (7.81 mg Oz/l X) at 10°C. 2. The hypoxic fish compensated for a diminished arteriovenous O2 difference by an increased cardiac output in maintaining respiratory homeostasis. 3. This adaptive cardiovascular response pattern to hypoxia would maximize the oxygen tension gradient across the gill epithelia while buried in sea floor sediments.

INTRODUCTION

The physiological responses of teleosts to hypoxic environments have been studied in several species (see reviews by Satchell, 1971 and Hughes, 1973). Investigations regarding responses of freshwater or anadromous teleosts to low environmental oxygen tensions are more numerous than those concerning marine species, especially on demersal forms that might routinely encounter hypoxic conditions. A notable exception is the investigation of the ventilatory and cardiovascular responses of the starry flounder, Platichthys stellatus, to hypoxia and temperature change (Watters & Smith, 1973). Recent field data regarding the burrowing behavior of winter flounder, Pseudopleuronectes americanus (Walbaum), in 6-10 cm of sediment (Fletcher, 1975) raised questions concerning the physiological adjustments of this species to low environmental oxygen tensions that probably characterize subsea floor habitats (Hughes, 1973). Low oxygen concentrations also typify many polluted environments having high chemical or biochemical oxygen demands. With the obvious importance of oxygen uptake from the environment and transport to the metabolizing tissues in all aerobic organisms, it is of interest to examine the cardiovascular adjustments used by this species, Therefore, the object of this study was to compare the rate of oxygen consumption and pattern of circulatory flow in P. americanus when exposed to normoxic and hypoxic environments. MATERIALS

AND METHODS

Winter flounder (430-1090 g) were captured by otter trawl from Casco Bay, ME, and were quickly transported *Publication Number 18 of The Research Institute of the Gulf of Maine. Present Addresses: ‘Wildlife and Fisheries Biology, University of California, Davis, CA 95616; ‘Department of Zoology, University of Massachusetts, Amherst, MA 01002; 3Department of Biological Sciences, University of Maine at Portland-Gorham, 96 Falmouth Street, Portland, ME 04103, U.S.A.

to the laboratory. They were held under. essentially. natural conditions in round, 2000 1. tanks receiving a continuous flow of Casco Bay water. Fish were fed a trout meal/gelatin diet daily (Peterson et al., 1967), except for 24-32 hr before experimental use when they were starved. On the day prior to experimental use. 5 flounder were cannulated (caudal artery and caudal vein) with lengths of PE 50 polyethylene tubing (Intramedic. Clay-Adams Co., Parsippany, NJ) as described by Cech & Rowe11 (1976). The cannulated animals were then placed in Bounder-shaped,_plexiglas flow-through respirometers receiving normoxic (X = 7.81 mg 0,/l) water. Respirometers were immersed in covered, temperature-controlled experimental tanks. Cannulae extensions led out of the tanks to permit remote blood sampling without disturbing the fish. Physiological measurements were made after an overnight adjustment period. On some experimental days, the dissolved oxygen concentration of the water flowing into the respirometers was maintained at normoxic levels. On the others, dissolved oxygen was reduced evenly over a I hr period to the hypoxia level (x = 3.57 mg 0,/l) by bubbling nitrogen at a fixed flow rate through several gas diffuser stones in the interconnected experimental tanks. These fish were exposed to hypoxia at least one more hour before any experimental data were taken. Measurements on five fish typically took about 5 hr. Thus. these data represent winter flounder responses to cu. 40”~,, air-saturated sea water at 10°C and 30 ppt salinity for a period of 1-6 hr. Statistical analyses of the data showed no significant correlations between the cardiovascular variables and the length of exposure to hypoxic water. Therefore. all hypoxia fish were treated as a group when compared with the normoxic specimens. Oxygen consumption rates (tie,) were calculated from dissolved oxygen (D.O.) measurements by the Winkler method in water across the respirometer and simultaneous determinations of flow rate by timed filling of the outflow. Concurrently, measurements of arterial and venous blood pressure and heart rate (HR) were made by connecting the cannulae extensions to a pressure transducer (Statham P23BB) and recorder (Gilson ICT-SH). Systolic peaks/min in the arterial pressure trace were counted to determine

HR. Blood samples were then immediately taken from the cannulae extensions for blood gas measurements. Arterial (Pa 0,) and venous (P, 0,) oxygen tensions were measured with an oxygen electrode (IL 17026) and meter system (IL 125A). while oxygen contents (C, O2 and 123

JOS~.PHJ. C&H. JK., DAVIII M. R~W~LL AX) JAMES S. GLASC~O~

124

C, 0,) were determined using an oxygen analyzer (Lcxington Lex-OzPCon M). Blood samples were recovcrcd from O2 tension measurements and used for hematological determinatjons. Total hemoglobin concentration (Hb) was measured spectro~hotometr~cally with the cyanmethemoglobin method (Wintrobe. 1956). Onlv those fish having ?Ib’s within the ;anpe of “normal”, heaithy winter flounder from Casco Bay (Bridges (‘I ~1.. submitted) were included in this study. Immature crythrocyte percentage (Imm. RBC) was determined by counts of 200 cells in thin blood smears stained with Hemal@ stain. Mean corpuscular volume (MCV) was calculated by dividing microhematocrit percentages by hemacytometer-aided crythrocyte counts (Wintrobe, 1956). Biood pH was measured with a clinical type meter (1L 123) and microelectrode (IL.:SANZI. Cardiac o\ltput (Q) was calculated using the Fick relationship (Q = 1/02, C,+ O2 - C,O,z). and cardiac stroke volume (&, ) from Qsy = Qsi- = QINR. Cardiac work was computed as percent of total energy expenditure(s) using the method of

Hemmingsen (‘1trl. (1972). RESULTS Responses of winter flounder exposed to normoxic and hypoxic water are summarized in Table 1. One of the primary effects of hypoxia is the decreased O2 gradient across the gills, which can limit oxygen saturation of the blood. In winter flounder, this reduced saturation is demonstrated by the significant drops in arterial 0, tension and content during hypoxia (Table 1). The C,OZ drop is also reflected in the significant decrease in arterio-venous O2 content difference. Several cardiovascular adjustments to hypoxia are evident in this species’ maintenance of a relatively constant O2 uptake. The rise in cardiac output is a primary cardiovascular adjustment (Table 1). Although the mean cardiac frequency (HR) and stroke volume values both show small, nonsignificant increases, their combined increases produce a significantly elevated 0. Table

1. Comparison

Units vols vols vols mm mm

P” 0, PH.,,,., PH,,,,., Hb MCV Imm. RBC

:s: Cardiac Work Press.,,,,,, Press.,,,,,., I’,, Bo-dy wt. D-Qw.

(‘<, <‘(, ~~<, Hg Hg

grams “a, pJ 0

Q

w,

DISC‘LISSIOY Overall, the pattern of cardiovascular adjustment in winter Rounder to this level of hypoxia includes an increased cardiac output without bradycardia to maintain a constant oxygen supply. Cardiovascular responses recorded for other adult teleosts have generally been characterized by a bradycardia along with cardiac stroke volume increases, yielding no significant change in cardiac output (Satchell, 1971). However, experimental differencq regarding species used and rate of environmental O2 depletion introduce variability into available data, making direct comparisons between investigations di~cult (Marvin & Burton, 1973). Holeton & Randall (1967) measured a bradycardia, increased Qsv and approximately constant Q in the rainbow trout (&&no gairdneri) which began at c’u. SOYi el~vironmen~l O2 reduction. At a very shallow level of hypoxia, i.e. near O,-saturation, Marvin & Heath (1968) and Marvin & Burton (1973) recorded decreased l-fR and vo, in rainbow trout, bluegill sunfish (L~potnis ~??~~~r~~~~~~~~~s), and brown bullhead (~~ru~~~~~~s ~~~b~~o~~~~.~). Starry flounder (??atichth~s .sfc/latus) show approximately constant cardiac outputs with slightly decreased O2 consumption rates at levels somewhat > 5O?,, O2 reduction (Wat: tcrs & Smith, 1973). Garey (1970) has shown that Q

of cardiovascular variables in winter flounder (Pseutlople~rro~lr~l[~s arnu?%~n~s) under normoxic and hypoxic conditions*

Variable CA Oz Cv 02 A-FqDiff. p‘4 02

Another adjustment to hypoxia is indicated from the significantly decreased arterial pressure as measured at the caudal artery and no significant change in venous pressure (Table I ). This blqod pressure pattern. along with the increased Q during hypoxia, could result from either a decreased peripheral resistance in the systemic circulation or an increased vascular resistance at the gills. The bfood characteristics of total hemoglobin concentration, mean corpuscular volume and immature erythrocyte percentage showed no significant changes during hypoxia.

Normoxic 5.0 3.1 1.x 90 31 7.86 7.89 3.45 82

I

ml,mrnjkg beats!min ml/be&,/kg I,

23.1 35 0.68 5.4

mm”Hg mm Hg mi!min/kg grams m&l

27

* Values are means

3 0.39 635 7.81

k S.E. with (number

i_ * * * + i * * *

0.4(18) 0.4(17) 0.1 (17) 4(15) 3(13) 0.02 (I 8) 0.02 ( 18) 0.30 (17) 2(1Y) * l(l9) i_ 1.6(16) + I(181 k 0.117 ( 15) + 0.4 (I 3) f l(17) f
Hypoxic 3.7 2.3 1.4 28 I6 7.85 7.85 3.66 87 4 29.4 39 0.79 6.X 25 3 0.37 631 3.57

* 0.2 (16) * 0.2(17) 2 0.1 (16) * 2(17) i l(17) * 0.01 (17) & 0.03 (I 7) * 0.26 (l?i If: 3(18) * I(18) f 2.2 (16) + 2(17) f 0.06 (16) + 0.7 (16) f I(l7) + <1(17) f 0.01 (18) + 26(18) *0.05(1x)

Level of significant difference
Cardiovascular

responses of the winter flounder

declines in carp (Cyprinus carpio) at very low water O2 tensions (~40 mm Hg at 10°C). Satchel1 (1971) has pointed out that the POZ at which bradycardia appears varies with the species of fish. In the present study, it is quite possible that the level of hypoxia used was insufficient to produce the bradycardia recorded in other species (Holeton & Randall, 1967). The lack of ventral aortic (pre-gill) pressure measurements in this study prevent separation of peripheral vascular resistance changes in the gills from those in the systemic circulation. As fish typically vasoconstrict in response to hypoxia (Satcheli. 1971), the increased gill peripheral resistaqce hypothesis seems likely. However, the increased Q and insignificant rise in cardiac work during hypoxia (Table 1) support the systemic vasodilation possibility. Further work is needed to elucidate resistance changes in this species. Comparing the present study with the results from other adult teleosts. a question is raised fegarding the winter flounders’ unusual increases in Q and lack of bradycardia during hypoxia. Differences in experimental procedure may account for part of the answer. On the other hand, part may fie in the relative energetic costs of cardiac and branch% pumping while buried in sea floor sediments. To maintain an effective OZ gradient across the respiratory surfaces during hypoxia, most teleosts investigated display several-fold increases in ventilation volume (e.g. see Saunders, 1962; Holeton & Randall, 1967; Gerald & Cech, 1970; Cech & Wohlschlag, 1973; Watters & Smith, 1973). Yazdani & Alexander (1967) showed that it probably takes more energy to expire water out the blind-side (deeper) opercular opening of a partially buried flatfish than out the ocular side. They noted the presence of a connecting channel for ventilatory water between the branchial chambers. For a winter flounder buried IO-15 cm in the sediments (Fletcher, 1975), it may take considerable energy to expire and also inspire a volume of water consistent with the increases measured in other teleosts exposed to hypoxic stress. Table 1 shows that cardiac work is increased insignificantly during exposure to hypoxia. In this species, it may be energetically less expensive to increase circulatory flow than to increase the ventilatory flow several-fold through lo-15 cm of sediment. Increased circulatory flows would have high adaptive value in enhancing gas exchange by maximizing the water/blood POZ gradient across the gill membranes under these conditions. Acknotvlrd~mlents-We acknowledge the financial assistance of the U.S. Environmental Protection Agency (Grant Number R-800831); the technical assistance of J. Lavigne, D. Pedro, A. D’Amico and Dr. D. Bridges; and

125

the typing of P. Buchignani and K. Steele. We especially thank Dr. E. D. Stevens of the University of Guelph for his comments on the manuscript.

REFERENCES

D. W.. CEC~! J. J. JK. Rc PEDRO D. N. Seasonal hematological changes in winter flounder. P.~~z~d~p~~u~owcf~s u~l~~i~~~~zi~.~ (Wal baum). Submitted to Trans. Am

BRIDGES

Fish. sm.

Ctf~ J. J.. JR. & WOHI~S~HLA~ D. E. (1973) Respiratory responses of the striped mullet. MuUii cephah~s L., to hypoxic conditions. j. Fish B&I/. 5, 4211128. C’I-CH J. J.. JK. & ROWELL D. M. (1976) Vascular cannulation method for Ratfishes. Prey: Fish-Cult. (In press). GARE’I. W. F. (1962) Cardiac responses of fishes in asphyxic environments. Biol. Bull Mur. Biol. Luh.. Woods Hole 122. 362-368. GAREY W. F. (1970) Cardiac output of carp (Cyprinis carpie). Conlp. Biochem Physiol. 33. 181-l89. G~KALD J. W. & CFC.H J. J. JK. (1970) Respiratory responses of juvenile catfish (Icralilfus punctatus) to hypoxic conditions. Physiol. Zoo/. 43. 47-54. FLETCHER G. L. (1975) The effects of capture, “stress”, and storage of whole blood on the red blood ceils, plasma proteins. glucose. and electrolytes of the winter flounder (P.sL,lidopt~rtroni~~t~~s 197~206.

at~trricanus).

Can.

J.

Znof.

53.

HEMMINGSEN E. A., DOuGLAs E. I.. JOHANSEN K. & MILLAKI) R. W. (1972) Aortic blood Bow and cardiac output in the hemoglobin-free fish Churnocephalus ucerutus. Corn/~. Bioche~n. Phgsiol. 43A. 1045- 105 I. HOLETON G. F. & RANIIALL D. J. (1967) The effect of hypoxia upon the partial pressure of gases in the blood and water afferent and efferent to the gills of rainbow trout. J. rrp. Biol. 46. 317 327. HUGHES G. M. (1973) Respiratory responses of hypoxia in fish. Atn. Zuol. 13. 475489. MARVIN E. E. & HEATH A. G. (1968) Cardiac and respiratory responses to gradual hypoxia in three ecologi~lly distinct species of freshwater fish. fotnp. Bio~~~~i. Php siol. 27. 349-355.

MARVIN D. E.. JK. & B~JKTON D. T. (1973) Cardiac and respiratory responses of rainbow trout, bluegills and brown bullhead catfish during rapid hypoxia and recovery under normoxic conditions. Camp. Biochrtn. Physiol. 46A.

755 -765.

SATCHELL G. H. (1971) Circulcltiot~ in Fishrs. Cambridge Univ. Press, London. 13 1 p. SAUKDCRS R. L. (1962) The irrigation of the gills in fishes. II. Efficiency of oxygen uptake in relation to respiratory flow. activity and concentrations of oxygen and carbon dioxide. Cl111J. Zoo/. 40. 817 862. WAT~ERS K. W.. JK. & SMITH L. S. (1973) Resoiratorv dynamics of the starry Rounder. P~~~r~~~~f~~~~ .st&tus, ik response to low oxygen and high tem~rature. Mnr. Biol. 19. 1X3-148. WINTKO~E M. M. (1956) CIinicul ~~~~f~r(~~~~~.4th Edn. Lea & Febigcr. Phiiadelphia. 185 p. YAZI)A&I G. M. & ALEXANIIEKR. McN. (1967) Respiratory currents of flatfish. Nutuw. Lord. 213. 96-97.