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The effect of body size and temperature upon oxygen consumption of the cestode Schistocephalus solidus (Müller)
Comp. Biochem. Physiol., 1966, Vol. 18, pp. 415 to 425. Pergamon Press Ltd. Printed in Great Britain
THE EFFECT OF BODY SIZE AND TEMPERATURE UPON OXYGEN CONSUMPTION OF THE CESTODE SCHISTOCEPHALUS SOLIDUS (MtJLLER) P. S P E N C E R D A V I E S and M. W A L K E Y * Department of Zoology, The University, Glasgow W.2 (Received 29 December 1965)
Abstract--1. Endogenous aerobic respiration rates of the plerocercoids of Schistocephalus solidus from the body cavity of sticklebacks were measured at a range of temperatures in winter and in summer. Respiration rates of adult worms at 40°C were also determined. 2. Respiration rate is proportional to the power - 0"478 __.0"009 of the dry body weight. The relationship is not influenced by temperature or season and is the same for adult worms. 3. Respiration rate of plerocercoids increases with temperature and is at a maximum at 40°C. This is thought to be a preadaptation for respiratory function at the elevated body temperatures of the definitive host. 4. Q10 of respiratory rate decreases at temperatures up to 30°C but then increases from 30 to 40°C, suggesting that at higher temperatures alternative enzyme pathways are utilized. 5. Respiration rate displays thermal acclimation on a seasonal basis; it is higher in winter than in summer and the Qx0 is lower in winter than in summer. 6. It is calculated that the aerobic respiration rate is significantly lower than the standard rate for free living poikilotherms. INTRODUCTION S C H I S T O C E P H A L U S SOLIDUS, in common with the great majority of cestodes,
experiences considerable change in physicochemical and biochemical conditions during the course of its life cycle. Exhibiting the characteristic pseudophyllidean pattern, development of the tapeworm proceeds from the egg via a free-living, coracidial stage to the procercoid form in the haemocoel of a copepod. T h e procercoid gives rise to a plerocercoid stage in the body cavity of a fresh-water fish, usually the three-spined stickleback. Somatic development is completed in the plerocercoid during which time the parasite increases in weight by a factor of at least 10,000. T h e tapeworm finally attains maturity in the gut of a fish-eating bird (Hopkins & Smyth, 1951) where egg production continues for a period of 7-11 days before the worm is eliminated (McCaig & Hopkins, 1963). During the changeover from plerocercoid to the adult stage in the intestine of a duck, the tapeworm experiences changes in three constituents of its physical * Present address: Department of Zoology, Queen Mary College, Mile End Road, London, E.1. 415
416
P. SPENCER DAVIES AND M . WALKEY
environment. Firstly, there is a temperature change from approximately I°C to 20°C, depending upon season, to 41°C, the body temperature of the bird host (Spector, 1956). Secondly, there is a change in pH from near neutrality (pH 7.2-7.6) in the fish body cavity (Walkey, unpublished work) through very acid conditions (pH 2.3) in the gizzard of the definitive host and back to a neutral environment (pH 6"9) in the intestine (Farner, 1942). Thirdly, although few figures are available, there is probably a difference in oxygen tension between the fish body cavity (where oxygen tension is presumably no lower than that of venous blood) and the small intestine of the duck, where Crompton et al. (1965) have shown that the partial pressure of oxygen may vary between 25 mm of mercury close to the villi and 0.5 mm in the lumen. Recent work (reviewed by Prosser & Brown, 1961) has made it increasingly clear that poikilothermic animals possess quite wide powers of metaboli~ adaptation in response to their environment. This paper is the first in a series designed to investigate the effects of some of these environmental parameters upon the metabolism of Schistocephalus. The importance of the effect of body size on metabolic rate has been well documented (Zeuthen, 1953 ; Hemmingsen, 1960) although cestodes have received little attention. Recently, however, von Brand & Ailing (1962) have shown that in both larval and adult Taenia taeniaeformis, metabolic rate is proportional to a negative power function of the weight, as in free-living animals. Very little information is available on the effects of temperature on the metabolism of cestodes, although it is now well established that the elevated temperatures experienced by the plerocercoid of S. solidus on entering the definitive host stimulates the process of maturation (Smyth, 1946, 1952; Hopkins & McCaig, 1963). In free-living poikilotherms, acclimation of metabolism to environmental temperature is a common occurrence. Therefore the present work was carried out on plerocercoids that had been acclimatized to low environmental temperatures in December and to high, summer temperatures in July. The recommendations of Zeuthen (1953) on terminology have been adopted. Thus, metabolism and respiration refer to the volumetric oxygen uptake per hour by an animal, whilst metabolic rate and respiration rate refer to the oxygen consumption per mg dry weight of animal per hour.
MATERIALS AND METHODS Plerocercoids were obtained from a population of three-spined sticklebacks, Gasterosteus aculeatus, inhabiting a small loch near Glasgow. The mean water temperature was 3°C in December and 16°C in July (Walkey, unpublished work). Adult worms were procured by feeding plerocercoids of suitable weight ( > 20 mg fresh weight) to 2-day-old ducklings. Each duckling was infected with four plerocercoids. Forty-eight hours later the birds were killed and adult Schistocephalus recovered from the intestine. Metabolic rate was determined by measurement of endogenous aerobic respiration using standard Warburg manometry.
EFFECTS OF BODY
SIZE A N D
TEMPERATURE
UPON
OXYGEN
CONSUMPTION
O F A CF~STODE
417
Following removal from its host, each worm was quickly weighed before being placed in a Warburg reaction flask. Each flask contained a single worm, 15 ml flasks being used for large worms and 5 ml flasks for the smaller ones. Respiration took place into a medium of 3 ml (2 ml in the case of small flasks) of sterile, sugar-free Tyrode solution, buffered with phosphate buffer at pH 7-2. The gas phase was atmospheric air and flasks were shaken at a rate of a hundred 5 cm strokes/min to ensure equilibration. At the completion of each experiment, the duration of which varied from 2 hr at high temperatures to 8 hr at 10°C, the worms were dried to constant weight at a temperature of 105°C. Respiratory rates were then calculated and expressed as t~l O~/mg dry wt/hr. The rate of oxygen consumption by plerocercoids was determined at temperatures of 10 °, 20 °, 30 ° and 40°C, and by adult worms at 40°C, in July and again in December. At each temperature the rates of between 10 and 20 worms were recorded. Statistical treatment o f results
As shown by Zeuthen (1953) and Hemmingsen (1960), the relationship between oxygen uptake and body weight in animals may be expressed as: Y = a X b,
(1)
where Y is oxygen uptake per unit time, X is the body weight and a and b are constants. Often it is more convenient to measure the respiratory rate on a unit weight basis and for this the relationship may be expressed as: Y / X = a X b-1.
(2)
Following the convention adopted by Davies (1966) this is rewritten as: Y ' = a X h'.
(3)
Since b is normally less than 1, b' will be a negative exponent. Hence (3) transforms to: log Y' = log a - b'log X. Thus, when respiration rate is plotted against weight on logarithmic paper, a straight-line relationship results in which b' is the regression coefficient, which describes the negative slope of the regression line, and a is the intercept on the Y axis when log X = 0. Each experiment was subjected to regression analysis and a regression equation derived (Table I). Regression coefficients ranged from -0.0873 to -0.6395 (Fig. I). In order to test whether these variations in b' value represented deviations from a population b' value, covariance analysis was carried out, analysing experiments in pairs and then in total, treating the summer and winter experiments as separate lots. It was found that, at the 5 per cent level, there was no significant difference in the 5' values derived for different temperatures during either summer or winter.
418
P. SPENCER DAVIES AND M . WALKEY TABLE 1--REGRESSION
E Q U A T I O N S A N D C O N F I D E N C E I N T E R V A L S O F R E S P I R A T I O N RATE
AND DRY W E I G H T OF
Temperature
°C
Schistocephalus
Regression equation
PLEROCERCOIDS
95% confidence i n t e r v a l o f b"
95% confidence interval of Y
Summer
Adults
10 20 30 40 40
Y Y Y Y Y
10 20 30 40 40
Y Y Y Y Y
= -0.5456X-
0.2138
= - 0"4851X+ 0'5015 = - 0 " 3 8 2 6 X + 0"8157 = -0.4282X+ 0.8872
+ + _ _ _
0.1530 0.1731 0.0922 0.0719 0.1426
+_ 0"0349 _ 0.0254 + 0'0440 _+0 ' 0 1 8 1 +_ 0 ' 0 3 2 8
= = = = =
_+ 0 ' 4 2 5 8 +_ 0 . 1 4 0 2 _+ 0 . 0 7 2 7 +_ 0 . 1 2 5 0 _+ 0"1995
_+ 0 . 0 9 0 7 _+ 0"0567 +_ 0 . 0 4 4 5 _ 0.0625 _+ 0"0277
= - 0.6310X+ 0'3219
Winter
Adults
-
0.2280X0-6395X+ 0-4508X+ 0.4213X+ 0"0873X+
0.4660 0.4609 0.5147 0.7981 0.2715
5"04.03"0
20'
f
C~
O'adults
~ f.o_ .0.9 08 ~ 07
~
~ 05 ,0.4 0 0"5-
0'2-
0"1
~
~
; ~ ~ ~ ~Ito Dry wt,
~o
3b
do 566'oT'os'o4ohoo
mg
FIG. 1. R e g r e s s i o n l i n e s o f r e s p i r a t i o n r a t e a n d w e i g h t o f p l e r o c e r c o i d s at t e m p e r a t u r e s o f 10 °, 20 °, 30 ° a n d 4 0 ° C a n d o f a d u l t s a t 4 0 ° C . D o t t e d l i n e s indicate winter experiments; solid lines indicate summer experiments.
EFFECTS OF BODY SIZE AND TEMPERATURE U P O N OXYGEN C O N S U M P T I O N OF A CESTODE
419
In addition, there was no significant difference in b' values for plerocercoids and adults at 40°C. T h e sums of squares and products of deviations from regression were therefore combined (Snedecor, 1961) and a separate common regression coefficient derived for the winter experiments and the summer experiments. This had the value of -0.5098 for summer and -0.4538 for winter worms. Covariance analysis was then carried out using the combined values, to compare the two common regression coefficients. Again, at the 5 per cent level, there
5"0-
4O3o ~
::::::::-:::::.__:
20.
:k r.~ 0-9.
adutts
.~" O.S.
~
~ - 0'7" 0"6"
° 0.5o
~
0.4-
O
0.2-
0.1
"'"" 3
4
~
6 7 e 9 II0 Dry wt,
2'0
30
"-. 10oc
40 ~'0 c~o7b ~S~O llo0
r~
FIO. 2. Regression lines of Fig. 1 replotted to the common regression coefficient of --0-4784. For explanation see text. was no significant difference between slopes in summer and winter. By a further pooling of summer and winter results an overall common regression coefficient b' = - 0.4784 + 0.009 was obtained. This regression coefficient thus describes the relationship between size and metabolic rate for both plerocercoids and adults of Schistocephalus solidus between 10 ° and 40°C in both summer and winter. Regression lines were then replotted to the common regression coefficient (Fig. 2) by substitution in the equation:
Y= Y-b'(X-X).
420
P . SPENCER DAVIES AND M. W A L K E Y
From these graphs the respiration rate of a worm of 18 mg dry weight, representing the mean weight of all worms used, was calculated for each experiment (Fig. 3). By analysing results for worms of the same weight it was then possible to compare the effects on respiration rate of variations in temperature and season. 50
//j/•
Summer
20
• Winter
• pJ
o
j,s 7-
~ ss
s JJJf
~o 4)¢ O
5"
ss S • s SS~S
i
I0
~0
I
30 Temperature,
I
4O
°C
Rate of respiration of 18 mg plerocercoid at four temperatures in winter and in summer. The respiration rate of adult worms at 40°C is indicated by [2.
F I G . 3.
RESULTS
Over the whole series of experiments, the b' values relating respiratory rate to weight range from -0.0873 to -0.6395. Covariance analyses showed that the temperature at which respiration is measured, from 10°C to 40°C, does not produce any significant change in the relationship. It is also seen that the changeover from plerocercoid to the adult condition does not affect the relationship between metabolic rate and body size. By applying the common regression coefficient, it may be stated therefore that in both summer and winter, over the temperature range 10°40°C, the respiratory rate of S. solidus is proportional to the power -0-4784 of the dry body weight. T h e value of b may be calculated from equations (1) and (2) as b = b ' + 1 which
EFFECT OF BODY SIZE AND TEMPERATURE U P O N OXYGEN C O N S U M P T I O N OF A CF-~TODE
421
equals 0.5216. Whole animal respiration of Schistocephalus therefore is proportional to the power 0.5216 of the dry body weight. TABLE 2--Qlo OF RESPIRATIONRATEOF Schistocephalus solidus Temperature range
Summer
Winter
10-20°C 20-30°C 30--40°C
2-77 2"38 2"88
2.62 1.82 2"10
3.0•
/
eSUmmet"
2.5 ¸ Q 0
--
\ k
,,ss~Winle r •~
2.0-
s s SSSS •
| 10-20
\\ j"
s
I 20-30
Temperature interval,
30=-40 "C
FIG. 4. Q10 of respiration rate of plerocercoids between 10° and 40°C in summer and winter. In both summer and winter, the rate of respiration of plerocercoids increased with increasing temperature (Fig. 3). It is interesting to note that the curve relating respiration rate and temperature for an 18 mg worm shows no sign of falling off between 30 ° and 40°C. This is illustrated more clearly by examining the Q10 of respiratory rate (Table 2, Fig. 4). There is very little difference in the Q10 of 10°-20°C and 30°-40°C particularly in the summer and in fact the highest Q10 values recorded were at 30°--40°C in summer. Both summer and winter forms exhibited a relatively low Q10 at 20°-30°C. At temperatures of 10 °, 20 ° and 30°C, the respiratory rate of an 18 mg worm is higher during the winter than during the summer. At 40°C, however, this difference is reversed and the summer worms have a higher rate of respiration. Covariance analysis showed that the differences between the summer and winter worms were significant
422
P. SPENCER DAVIES AND M . WALKEY
( P < 1 per cent) at 10 ° and 20°C but were not significant at 30 ° and 40°C. The rate/temperature curve for winter worms is therefore displaced upwards, a process termed "translation" (Prosser & Brown, 1961), and in addition it is rotated about an axis so that it intersects with the summer curve at some point between 30 ° and 40°C. Rotation is brought about by a change in Qx0 between summer and winter; the Q~0 of winter worms is lower over each 10°C temperature interval between 10 ° and 40°C (Fig. 4) than during the summer. DISCUSSION The relationship between size and respiration follows the same pattern as other poikilotherms where respiration is generally proportional to a power function of the body weight. In a detailed review, Hemmingsen (1960) showed that over the whole phylogenetic size range of poikilotherms the value of the exponent was 0-75, although the experimentally determined value for any species may deviate slightly from this. Attempts have also been made to relate respiration to body surface area, that is to a power 0.67 of the body weight (Benedict, 1938). In tapeworms the body form may be compared to a flattened cylinder (Wardle & McLeod, 1952), so that increase in surface area should be very nearly proportional to increase in body weight. Therefore b values for tapeworms should be approximately unity. The experimentally determined value of b = 0-5216 clearly does not support this theory. Working with the cestode Taenia taeniaeformis, yon Brand & Ailing (1962) obtained b values ranging from 0.67 to 0.9 with no significant difference between the values recorded for aerobic or anaerobic respiration. In the present work it has also been shown that the relationship is not affected by either temperature or season. It is probably best to conclude therefore that the b values obtained may be regarded merely as deviations from the phylogenetic b value of 0.75, the fundamental explanation of which is still not understood. Respiration rate of Schistocephalus plerocercoids increases with each successive temperature interval between 10 ° and 40°C (Fig. 3). Thus the thermal death point and inactivation temperature of respiratory enzyme systems are apparently in excess of 40°C, that is more than 20°C above the normal environmental temperature of the plerocercoid in the body cavity of its fish host. Since most free-living poikilotherms live at environmental temperatures only a few degrees below their thermal death-point (Heilbrunn, 1952), plerocercoids evidently possess enzyme systems preadapted to function at the higher temperatures experienced within the definitive host. Similar preadaptation was reported by Vernberg (1961), who found that larval stages of two species of trematodes in the mud-flat snail, Nassarius obsoleta, possessed upper thermal death-points closely correlated with the body temperatures of their respective definitive hosts. Examination of the Q10 of respiratory rate reveals further evidence of adaptation and acclimation. The low winter Q1. values may be compared with the low values found in free-living poikilotherms when acclimated to low temperatures (Rao & Bullock, 1954). In addition, the low value of Qlo recorded between
EFFECT O F B O D Y SIZE A N D T E M P E R A T U R E U P O N O X Y G E N C O N S U M P T I O N O F A CESTODE
423
20 ° and 30°C may be compared with a similar phenomenon recorded by Davies (1966) and Vernberg & Hunter (1961), who found that, in the general decrease of Qlo with increasing temperature, a particularly low value was recorded over the normal habitat range of temperature of animals investigated. This may be regarded as a compensatory process resulting in the metabolism being held at a fairly constant level in the normal habitat. Vernberg (1961) observed a similar levelling of Q10 of respiration of the larval stages of two trematodes in Nassarius obsoleta, but these corresponded to the environmental temperatures of the adult worms, rather than that within their mollusc host. The elevated Qlo between 30 ° and 40°C (2-88 and 2.10) represents something of an anomaly since, as was pointed out by B~lehr~dek (1930), Q10 values of biological systems normally decrease with increase in temperature. It is possible therefore that above approximately 30°C an alternative metabolic pathway or enzyme system is utilized, which has different temperature characteristics from the normal low temperature system. This hypothesis fits well with the suggestion by Hopkins & McCaig (1963) that the crossing of a temperature barrier at 34°C provides the sole stimulus for the metabolic switch inhibiting somatic growth and initiating the final phases of oogenesis, vitellogenesis and spermatogenesis. The similarity in respiratory rates of adults and plerocercoids at 40°C indicates that adult worms are able to metabolize aerobically. However, the biochemical significance of this is uncertain, especially in view of the findings of Hopkins (1952) and Smyth (1956) that abnormal maturation patterns result in Schistocephalus if the parasite is cultured in a medium through which air is bubbled. When the correlations of respiration and temperature in summer and winter are compared it is seen that the curve for summer worms is displaced or translated downwards (Fig. 3). Furthermore, since the mean Qt0 values are greater in summer, the two lines intersect at a temperature between 30 ° and 40°C. This is a clear case of acclimation to seasonal environmental temperature and falls within the Type IV A classification of acclimatory processes as summarized by Prosser (1958) and Prosser & Brown (1961). Acclimation of this type, involving translation with rotation, has also been recorded in goldfish (Kanungo & Prosser, 1959) and in the trematode Zo6gonus rubellus (Vernberg & Vemberg, 1965). The latter result is of particular interest since in the same work it was reported that larval stages of Himasthla quissetensis, a trematode infecting a homoeothermic definitive host (cf. Z. rubellus in fish), exhibited a metabolic temperature relationship with translation but no rotation (Type II of Prosser). The net result of acclimation in Schistocephalus is that a plerocercoid metabolizes at the same rate at 15°C in the summer as it does at 10°C in the winter. In the metabolic economy of the worm the low summer respiratory rate could be regarded as a compensatory process limiting the expenditure of energy at the higher environmental temperatures. Although the importance of this to the parasite is less clear, its significance in the host/parasite relationship may lie in a relatively lower nutritional demand by the parasite on the host at the higher summer temperatures, i.e. at a time when the host itself is experiencing a higher metabolic demand.
424
P. SPENCER DAVIES AND M. WALKEY
The absolute level of respiration of Schistocephalus plerocercoids remains for discussion. Hemmingsen (1960) derived a value for the regression line of standard energy metabolism and fresh weight at 20°C for free-living poikilotherms. This has the value: logcal/hr= -3.161 + 0.068 + 0"75X, where X is the fresh body weight. By substitution in the above equation it is possible to determine the caloric expenditure per hour of an 18 mg dry weight ( = approx. 70mg fresh weight) plerocercoid to be equal to 10-4'°26cal/hr. Assuming that carbohydrate is the metabolic substrate, and that 1 ml of oxygen will yield 5.05 × 10 -3 cal (Krogh, 1916), the predicted oxygen consumption may be calculated as 19/zl/hr. In the present work (Fig. 3) it is seen that the endogenous aerobic respiratory uptake of an 18 mg dry weight worm is equal to 8"6/zl O2/hr in winter and 5.9/xl O2/hr in summer. It would appear therefore that Schistocephalus plerocercoids, under the experimental conditions employed, have a significantly lower level of oxidative metabolism than that of free-living poikilotherms. It should be noted, however, that in a number of other parasitic helminths, anaerobic metabolism continues even at atmospheric oxygen tensions (Rogers, 1962). The total energy expenditure of S. solidus may therefore be considerably higher than that accounted for by aerobic oxidative metabolism alone. Acknowledgements--We wish to thank Miss B. Tagg for technical assistance and Mr. Odd Vagle for assistance in the production of a programme for the University of Glasgow K D F 9 computer. We should also like to thank Dr. C. A. Hopkins for his helpful criticism of the manuscript. One of us (M. W.) undertook part of the work while in receipt of a grant from the University of London Central Research Fund. REFERENCES BELHR~DEK J. (1930) Temperature coefficients in biology. Biol. Rev. 5, 30-58. BENEDICT F. G. (1938) Vital Energetics. A Study in Comparative Basal Metabolism. Publication 503. Carnegie Institute, Washington, D.C. YON BRAND T. 8c ALLING D. W. (1962) Relations between size and metabolism in larval and adult Taenia taeniaeformis. Comp. Biochem. Physiol. 5, 141-148. CROMPTON D. W. T., SHRIMPTON D. H. & SILVER I. A. (1965) Measurements of the oxygen tension in the lumen of the small intestine of the domestic duck. J. exp. Biol. 43, 473-478. DAVIES P. SPENCER (1966) Physiological ecology of Patella--I. The effect of body size and temperature on respiration rate. J. mar. Biol. Ass. U.K. (In press.) F~mNER D. S. (1942) T h e hydrogen ion concentration in avian digestive tracts. Poult. Sci. 21, 445-450. HEILBRUNN L. V. (1952) An Outline of General Physiology. 3rd edn. Saunders, Philadelphia and London. HEMMINGSEN A. M. (1960) Energy metabolism as related to body size and respiratory surfaces, and its evolution. Rep. Steno metal Hosp. 9 (II), 7-110. HOPKINS C. A. (1952) Studies on cestode metabolism--II. T h e utilization of glycogen by Schistocephalus solidus in vitro. Expl Parasitol. 1, 196-213. HOPKINS C. A. & MCCAIG M. L. O. (1963) Studies on Schistocephalus solidus--I. T h e correlation of development in the plerocercoid with infectivity to the definitive host. Expl Parasitol. 13, 235-243.
15
EFFECT O F BODY SIZE AND TEMPERATURE U P O N OXYGEN C O N S U M P T I O N OF A CESTODE
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HOPKINS C. A. & SMYTH J. D. (1951) Notes on the morphology and life history of Schistocephalus solidus (Cestoda: Diphyllobothriidae). Parasitol. 41, 283-291. KANtmCO M. S. & PROSSER C. L. (1959) Physiological and biochemical adaptation of goldfish to cold and warm temperatures--I. Standard and active oxygen consumption of cold and warm-acclimated goldfish at various temperatures, aT. cell. comp. Physiol. 54, 259-263. KROGH A. (1916) The Respiratory Exchanges of Animals and Man. Longmans, Green, London. McCAIO M. L. O. & HOPKINS C. A. (1963) Studies on Schistocephalus solidus--II. Establishment and longevity in the definitive host. Expl Parasitol. 13, 273-283. PROSSER C. L. (1958) General summary: the nature of physiological adaptation. In Physiological Adaptation (Edited by PEOSSER C. L.), pp. 50-78. Am. Physiol. Sue., Washington. PROSSER C. L. & BROWN F. A. (1961) Comparative Animal Physiology. 2nd edn. Sounders, Philadelphia and London. RAO K. P. & BULLOCK T. H. (1954) Qx0 as a function of size and habitat temperature in poikilotherms. Am. Nat. 88, 33--44. ROGERS W. P. (1962) The Nature of Parasitism, p. 161. Academic Press, New York and London. SMYTH J. D. (1946) Studies on tapeworm physiology--I. T h e cultivation of Schistocephalus solidus in vitro. 07. exp. Biol. 23, 47-70. SMYTH J. D. (1952) Studies on tapeworm physiology--VI. Effect of temperature on the maturation in vitro of Schistocephalus solidus, ft. exp. Biol. 29, 304-309. SMYTH J. D. (1956) Studies on tapeworm physiology--IX. A histochemical study of egg shell formation in Schistocephalus solidus (Pseudophyllidea). Expl Parasitol. 5, 519-540. SNEDECOR G. W. (1961) Statistical Methods applied to Experiments in Agriculture and Biology, p. 398. 5th edn. Iowa State Univ. Press, U.S.A. SPECTOR W. S. (Ed.) (1956) Handbook of Biological Data. Saunders, Philadelphia and London. VERNBERG W. B. (1961) Studies on oxygen consumption in digenetic trematodes--VI. T h e influence of temperature on larval trematodes. Expl Parasitol. 11, 270-275. VERNBERG W. B. & HUNTER W. S. (1961) Studies on oxygen consumption in digenetic trematodes--V. T h e influence of temperature on three species of adult trematodes. Expl Parasitol. 11, 34-38. VERNBERG W. B. & VERNBERG J. F. (1965) Interrelationships between parasites and their h o s t s - - I . Comparative metabolic patterns of thermal acclimation of larval trematodes with that of their host. Cutup. Biochem. Physiol. 14, 557-566. WAROLE R. A. & McLBoD J. A. (1952) The Zoology of Tapeworms. Univ. Minnesota Press. ZEUTHEN E. (1953) Oxygen uptake as related to body size in organisms. Q. Rev. Biol. 28, 1-12.
THE EFFECT OF BODY SIZE AND TEMPERATURE UPON OXYGEN CONSUMPTION OF THE CESTODE SCHISTOCEPHALUS SOLIDUS (MtJLLER) P. S P E N C E R D A V I E S and M. W A L K E Y * Department of Zoology, The University, Glasgow W.2 (Received 29 December 1965)
Abstract--1. Endogenous aerobic respiration rates of the plerocercoids of Schistocephalus solidus from the body cavity of sticklebacks were measured at a range of temperatures in winter and in summer. Respiration rates of adult worms at 40°C were also determined. 2. Respiration rate is proportional to the power - 0"478 __.0"009 of the dry body weight. The relationship is not influenced by temperature or season and is the same for adult worms. 3. Respiration rate of plerocercoids increases with temperature and is at a maximum at 40°C. This is thought to be a preadaptation for respiratory function at the elevated body temperatures of the definitive host. 4. Q10 of respiratory rate decreases at temperatures up to 30°C but then increases from 30 to 40°C, suggesting that at higher temperatures alternative enzyme pathways are utilized. 5. Respiration rate displays thermal acclimation on a seasonal basis; it is higher in winter than in summer and the Qx0 is lower in winter than in summer. 6. It is calculated that the aerobic respiration rate is significantly lower than the standard rate for free living poikilotherms. INTRODUCTION S C H I S T O C E P H A L U S SOLIDUS, in common with the great majority of cestodes,
experiences considerable change in physicochemical and biochemical conditions during the course of its life cycle. Exhibiting the characteristic pseudophyllidean pattern, development of the tapeworm proceeds from the egg via a free-living, coracidial stage to the procercoid form in the haemocoel of a copepod. T h e procercoid gives rise to a plerocercoid stage in the body cavity of a fresh-water fish, usually the three-spined stickleback. Somatic development is completed in the plerocercoid during which time the parasite increases in weight by a factor of at least 10,000. T h e tapeworm finally attains maturity in the gut of a fish-eating bird (Hopkins & Smyth, 1951) where egg production continues for a period of 7-11 days before the worm is eliminated (McCaig & Hopkins, 1963). During the changeover from plerocercoid to the adult stage in the intestine of a duck, the tapeworm experiences changes in three constituents of its physical * Present address: Department of Zoology, Queen Mary College, Mile End Road, London, E.1. 415
416
P. SPENCER DAVIES AND M . WALKEY
environment. Firstly, there is a temperature change from approximately I°C to 20°C, depending upon season, to 41°C, the body temperature of the bird host (Spector, 1956). Secondly, there is a change in pH from near neutrality (pH 7.2-7.6) in the fish body cavity (Walkey, unpublished work) through very acid conditions (pH 2.3) in the gizzard of the definitive host and back to a neutral environment (pH 6"9) in the intestine (Farner, 1942). Thirdly, although few figures are available, there is probably a difference in oxygen tension between the fish body cavity (where oxygen tension is presumably no lower than that of venous blood) and the small intestine of the duck, where Crompton et al. (1965) have shown that the partial pressure of oxygen may vary between 25 mm of mercury close to the villi and 0.5 mm in the lumen. Recent work (reviewed by Prosser & Brown, 1961) has made it increasingly clear that poikilothermic animals possess quite wide powers of metaboli~ adaptation in response to their environment. This paper is the first in a series designed to investigate the effects of some of these environmental parameters upon the metabolism of Schistocephalus. The importance of the effect of body size on metabolic rate has been well documented (Zeuthen, 1953 ; Hemmingsen, 1960) although cestodes have received little attention. Recently, however, von Brand & Ailing (1962) have shown that in both larval and adult Taenia taeniaeformis, metabolic rate is proportional to a negative power function of the weight, as in free-living animals. Very little information is available on the effects of temperature on the metabolism of cestodes, although it is now well established that the elevated temperatures experienced by the plerocercoid of S. solidus on entering the definitive host stimulates the process of maturation (Smyth, 1946, 1952; Hopkins & McCaig, 1963). In free-living poikilotherms, acclimation of metabolism to environmental temperature is a common occurrence. Therefore the present work was carried out on plerocercoids that had been acclimatized to low environmental temperatures in December and to high, summer temperatures in July. The recommendations of Zeuthen (1953) on terminology have been adopted. Thus, metabolism and respiration refer to the volumetric oxygen uptake per hour by an animal, whilst metabolic rate and respiration rate refer to the oxygen consumption per mg dry weight of animal per hour.
MATERIALS AND METHODS Plerocercoids were obtained from a population of three-spined sticklebacks, Gasterosteus aculeatus, inhabiting a small loch near Glasgow. The mean water temperature was 3°C in December and 16°C in July (Walkey, unpublished work). Adult worms were procured by feeding plerocercoids of suitable weight ( > 20 mg fresh weight) to 2-day-old ducklings. Each duckling was infected with four plerocercoids. Forty-eight hours later the birds were killed and adult Schistocephalus recovered from the intestine. Metabolic rate was determined by measurement of endogenous aerobic respiration using standard Warburg manometry.
EFFECTS OF BODY
SIZE A N D
TEMPERATURE
UPON
OXYGEN
CONSUMPTION
O F A CF~STODE
417
Following removal from its host, each worm was quickly weighed before being placed in a Warburg reaction flask. Each flask contained a single worm, 15 ml flasks being used for large worms and 5 ml flasks for the smaller ones. Respiration took place into a medium of 3 ml (2 ml in the case of small flasks) of sterile, sugar-free Tyrode solution, buffered with phosphate buffer at pH 7-2. The gas phase was atmospheric air and flasks were shaken at a rate of a hundred 5 cm strokes/min to ensure equilibration. At the completion of each experiment, the duration of which varied from 2 hr at high temperatures to 8 hr at 10°C, the worms were dried to constant weight at a temperature of 105°C. Respiratory rates were then calculated and expressed as t~l O~/mg dry wt/hr. The rate of oxygen consumption by plerocercoids was determined at temperatures of 10 °, 20 °, 30 ° and 40°C, and by adult worms at 40°C, in July and again in December. At each temperature the rates of between 10 and 20 worms were recorded. Statistical treatment o f results
As shown by Zeuthen (1953) and Hemmingsen (1960), the relationship between oxygen uptake and body weight in animals may be expressed as: Y = a X b,
(1)
where Y is oxygen uptake per unit time, X is the body weight and a and b are constants. Often it is more convenient to measure the respiratory rate on a unit weight basis and for this the relationship may be expressed as: Y / X = a X b-1.
(2)
Following the convention adopted by Davies (1966) this is rewritten as: Y ' = a X h'.
(3)
Since b is normally less than 1, b' will be a negative exponent. Hence (3) transforms to: log Y' = log a - b'log X. Thus, when respiration rate is plotted against weight on logarithmic paper, a straight-line relationship results in which b' is the regression coefficient, which describes the negative slope of the regression line, and a is the intercept on the Y axis when log X = 0. Each experiment was subjected to regression analysis and a regression equation derived (Table I). Regression coefficients ranged from -0.0873 to -0.6395 (Fig. I). In order to test whether these variations in b' value represented deviations from a population b' value, covariance analysis was carried out, analysing experiments in pairs and then in total, treating the summer and winter experiments as separate lots. It was found that, at the 5 per cent level, there was no significant difference in the 5' values derived for different temperatures during either summer or winter.
418
P. SPENCER DAVIES AND M . WALKEY TABLE 1--REGRESSION
E Q U A T I O N S A N D C O N F I D E N C E I N T E R V A L S O F R E S P I R A T I O N RATE
AND DRY W E I G H T OF
Temperature
°C
Schistocephalus
Regression equation
PLEROCERCOIDS
95% confidence i n t e r v a l o f b"
95% confidence interval of Y
Summer
Adults
10 20 30 40 40
Y Y Y Y Y
10 20 30 40 40
Y Y Y Y Y
= -0.5456X-
0.2138
= - 0"4851X+ 0'5015 = - 0 " 3 8 2 6 X + 0"8157 = -0.4282X+ 0.8872
+ + _ _ _
0.1530 0.1731 0.0922 0.0719 0.1426
+_ 0"0349 _ 0.0254 + 0'0440 _+0 ' 0 1 8 1 +_ 0 ' 0 3 2 8
= = = = =
_+ 0 ' 4 2 5 8 +_ 0 . 1 4 0 2 _+ 0 . 0 7 2 7 +_ 0 . 1 2 5 0 _+ 0"1995
_+ 0 . 0 9 0 7 _+ 0"0567 +_ 0 . 0 4 4 5 _ 0.0625 _+ 0"0277
= - 0.6310X+ 0'3219
Winter
Adults
-
0.2280X0-6395X+ 0-4508X+ 0.4213X+ 0"0873X+
0.4660 0.4609 0.5147 0.7981 0.2715
5"04.03"0
20'
f
C~
O'adults
~ f.o_ .0.9 08 ~ 07
~
~ 05 ,0.4 0 0"5-
0'2-
0"1
~
~
; ~ ~ ~ ~Ito Dry wt,
~o
3b
do 566'oT'os'o4ohoo
mg
FIG. 1. R e g r e s s i o n l i n e s o f r e s p i r a t i o n r a t e a n d w e i g h t o f p l e r o c e r c o i d s at t e m p e r a t u r e s o f 10 °, 20 °, 30 ° a n d 4 0 ° C a n d o f a d u l t s a t 4 0 ° C . D o t t e d l i n e s indicate winter experiments; solid lines indicate summer experiments.
EFFECTS OF BODY SIZE AND TEMPERATURE U P O N OXYGEN C O N S U M P T I O N OF A CESTODE
419
In addition, there was no significant difference in b' values for plerocercoids and adults at 40°C. T h e sums of squares and products of deviations from regression were therefore combined (Snedecor, 1961) and a separate common regression coefficient derived for the winter experiments and the summer experiments. This had the value of -0.5098 for summer and -0.4538 for winter worms. Covariance analysis was then carried out using the combined values, to compare the two common regression coefficients. Again, at the 5 per cent level, there
5"0-
4O3o ~
::::::::-:::::.__:
20.
:k r.~ 0-9.
adutts
.~" O.S.
~
~ - 0'7" 0"6"
° 0.5o
~
0.4-
O
0.2-
0.1
"'"" 3
4
~
6 7 e 9 II0 Dry wt,
2'0
30
"-. 10oc
40 ~'0 c~o7b ~S~O llo0
r~
FIO. 2. Regression lines of Fig. 1 replotted to the common regression coefficient of --0-4784. For explanation see text. was no significant difference between slopes in summer and winter. By a further pooling of summer and winter results an overall common regression coefficient b' = - 0.4784 + 0.009 was obtained. This regression coefficient thus describes the relationship between size and metabolic rate for both plerocercoids and adults of Schistocephalus solidus between 10 ° and 40°C in both summer and winter. Regression lines were then replotted to the common regression coefficient (Fig. 2) by substitution in the equation:
Y= Y-b'(X-X).
420
P . SPENCER DAVIES AND M. W A L K E Y
From these graphs the respiration rate of a worm of 18 mg dry weight, representing the mean weight of all worms used, was calculated for each experiment (Fig. 3). By analysing results for worms of the same weight it was then possible to compare the effects on respiration rate of variations in temperature and season. 50
//j/•
Summer
20
• Winter
• pJ
o
j,s 7-
~ ss
s JJJf
~o 4)¢ O
5"
ss S • s SS~S
i
I0
~0
I
30 Temperature,
I
4O
°C
Rate of respiration of 18 mg plerocercoid at four temperatures in winter and in summer. The respiration rate of adult worms at 40°C is indicated by [2.
F I G . 3.
RESULTS
Over the whole series of experiments, the b' values relating respiratory rate to weight range from -0.0873 to -0.6395. Covariance analyses showed that the temperature at which respiration is measured, from 10°C to 40°C, does not produce any significant change in the relationship. It is also seen that the changeover from plerocercoid to the adult condition does not affect the relationship between metabolic rate and body size. By applying the common regression coefficient, it may be stated therefore that in both summer and winter, over the temperature range 10°40°C, the respiratory rate of S. solidus is proportional to the power -0-4784 of the dry body weight. T h e value of b may be calculated from equations (1) and (2) as b = b ' + 1 which
EFFECT OF BODY SIZE AND TEMPERATURE U P O N OXYGEN C O N S U M P T I O N OF A CF-~TODE
421
equals 0.5216. Whole animal respiration of Schistocephalus therefore is proportional to the power 0.5216 of the dry body weight. TABLE 2--Qlo OF RESPIRATIONRATEOF Schistocephalus solidus Temperature range
Summer
Winter
10-20°C 20-30°C 30--40°C
2-77 2"38 2"88
2.62 1.82 2"10
3.0•
/
eSUmmet"
2.5 ¸ Q 0
--
\ k
,,ss~Winle r •~
2.0-
s s SSSS •
| 10-20
\\ j"
s
I 20-30
Temperature interval,
30=-40 "C
FIG. 4. Q10 of respiration rate of plerocercoids between 10° and 40°C in summer and winter. In both summer and winter, the rate of respiration of plerocercoids increased with increasing temperature (Fig. 3). It is interesting to note that the curve relating respiration rate and temperature for an 18 mg worm shows no sign of falling off between 30 ° and 40°C. This is illustrated more clearly by examining the Q10 of respiratory rate (Table 2, Fig. 4). There is very little difference in the Q10 of 10°-20°C and 30°-40°C particularly in the summer and in fact the highest Q10 values recorded were at 30°--40°C in summer. Both summer and winter forms exhibited a relatively low Q10 at 20°-30°C. At temperatures of 10 °, 20 ° and 30°C, the respiratory rate of an 18 mg worm is higher during the winter than during the summer. At 40°C, however, this difference is reversed and the summer worms have a higher rate of respiration. Covariance analysis showed that the differences between the summer and winter worms were significant
422
P. SPENCER DAVIES AND M . WALKEY
( P < 1 per cent) at 10 ° and 20°C but were not significant at 30 ° and 40°C. The rate/temperature curve for winter worms is therefore displaced upwards, a process termed "translation" (Prosser & Brown, 1961), and in addition it is rotated about an axis so that it intersects with the summer curve at some point between 30 ° and 40°C. Rotation is brought about by a change in Qx0 between summer and winter; the Q~0 of winter worms is lower over each 10°C temperature interval between 10 ° and 40°C (Fig. 4) than during the summer. DISCUSSION The relationship between size and respiration follows the same pattern as other poikilotherms where respiration is generally proportional to a power function of the body weight. In a detailed review, Hemmingsen (1960) showed that over the whole phylogenetic size range of poikilotherms the value of the exponent was 0-75, although the experimentally determined value for any species may deviate slightly from this. Attempts have also been made to relate respiration to body surface area, that is to a power 0.67 of the body weight (Benedict, 1938). In tapeworms the body form may be compared to a flattened cylinder (Wardle & McLeod, 1952), so that increase in surface area should be very nearly proportional to increase in body weight. Therefore b values for tapeworms should be approximately unity. The experimentally determined value of b = 0-5216 clearly does not support this theory. Working with the cestode Taenia taeniaeformis, yon Brand & Ailing (1962) obtained b values ranging from 0.67 to 0.9 with no significant difference between the values recorded for aerobic or anaerobic respiration. In the present work it has also been shown that the relationship is not affected by either temperature or season. It is probably best to conclude therefore that the b values obtained may be regarded merely as deviations from the phylogenetic b value of 0.75, the fundamental explanation of which is still not understood. Respiration rate of Schistocephalus plerocercoids increases with each successive temperature interval between 10 ° and 40°C (Fig. 3). Thus the thermal death point and inactivation temperature of respiratory enzyme systems are apparently in excess of 40°C, that is more than 20°C above the normal environmental temperature of the plerocercoid in the body cavity of its fish host. Since most free-living poikilotherms live at environmental temperatures only a few degrees below their thermal death-point (Heilbrunn, 1952), plerocercoids evidently possess enzyme systems preadapted to function at the higher temperatures experienced within the definitive host. Similar preadaptation was reported by Vernberg (1961), who found that larval stages of two species of trematodes in the mud-flat snail, Nassarius obsoleta, possessed upper thermal death-points closely correlated with the body temperatures of their respective definitive hosts. Examination of the Q10 of respiratory rate reveals further evidence of adaptation and acclimation. The low winter Q1. values may be compared with the low values found in free-living poikilotherms when acclimated to low temperatures (Rao & Bullock, 1954). In addition, the low value of Qlo recorded between
EFFECT O F B O D Y SIZE A N D T E M P E R A T U R E U P O N O X Y G E N C O N S U M P T I O N O F A CESTODE
423
20 ° and 30°C may be compared with a similar phenomenon recorded by Davies (1966) and Vernberg & Hunter (1961), who found that, in the general decrease of Qlo with increasing temperature, a particularly low value was recorded over the normal habitat range of temperature of animals investigated. This may be regarded as a compensatory process resulting in the metabolism being held at a fairly constant level in the normal habitat. Vernberg (1961) observed a similar levelling of Q10 of respiration of the larval stages of two trematodes in Nassarius obsoleta, but these corresponded to the environmental temperatures of the adult worms, rather than that within their mollusc host. The elevated Qlo between 30 ° and 40°C (2-88 and 2.10) represents something of an anomaly since, as was pointed out by B~lehr~dek (1930), Q10 values of biological systems normally decrease with increase in temperature. It is possible therefore that above approximately 30°C an alternative metabolic pathway or enzyme system is utilized, which has different temperature characteristics from the normal low temperature system. This hypothesis fits well with the suggestion by Hopkins & McCaig (1963) that the crossing of a temperature barrier at 34°C provides the sole stimulus for the metabolic switch inhibiting somatic growth and initiating the final phases of oogenesis, vitellogenesis and spermatogenesis. The similarity in respiratory rates of adults and plerocercoids at 40°C indicates that adult worms are able to metabolize aerobically. However, the biochemical significance of this is uncertain, especially in view of the findings of Hopkins (1952) and Smyth (1956) that abnormal maturation patterns result in Schistocephalus if the parasite is cultured in a medium through which air is bubbled. When the correlations of respiration and temperature in summer and winter are compared it is seen that the curve for summer worms is displaced or translated downwards (Fig. 3). Furthermore, since the mean Qt0 values are greater in summer, the two lines intersect at a temperature between 30 ° and 40°C. This is a clear case of acclimation to seasonal environmental temperature and falls within the Type IV A classification of acclimatory processes as summarized by Prosser (1958) and Prosser & Brown (1961). Acclimation of this type, involving translation with rotation, has also been recorded in goldfish (Kanungo & Prosser, 1959) and in the trematode Zo6gonus rubellus (Vernberg & Vemberg, 1965). The latter result is of particular interest since in the same work it was reported that larval stages of Himasthla quissetensis, a trematode infecting a homoeothermic definitive host (cf. Z. rubellus in fish), exhibited a metabolic temperature relationship with translation but no rotation (Type II of Prosser). The net result of acclimation in Schistocephalus is that a plerocercoid metabolizes at the same rate at 15°C in the summer as it does at 10°C in the winter. In the metabolic economy of the worm the low summer respiratory rate could be regarded as a compensatory process limiting the expenditure of energy at the higher environmental temperatures. Although the importance of this to the parasite is less clear, its significance in the host/parasite relationship may lie in a relatively lower nutritional demand by the parasite on the host at the higher summer temperatures, i.e. at a time when the host itself is experiencing a higher metabolic demand.
424
P. SPENCER DAVIES AND M. WALKEY
The absolute level of respiration of Schistocephalus plerocercoids remains for discussion. Hemmingsen (1960) derived a value for the regression line of standard energy metabolism and fresh weight at 20°C for free-living poikilotherms. This has the value: logcal/hr= -3.161 + 0.068 + 0"75X, where X is the fresh body weight. By substitution in the above equation it is possible to determine the caloric expenditure per hour of an 18 mg dry weight ( = approx. 70mg fresh weight) plerocercoid to be equal to 10-4'°26cal/hr. Assuming that carbohydrate is the metabolic substrate, and that 1 ml of oxygen will yield 5.05 × 10 -3 cal (Krogh, 1916), the predicted oxygen consumption may be calculated as 19/zl/hr. In the present work (Fig. 3) it is seen that the endogenous aerobic respiratory uptake of an 18 mg dry weight worm is equal to 8"6/zl O2/hr in winter and 5.9/xl O2/hr in summer. It would appear therefore that Schistocephalus plerocercoids, under the experimental conditions employed, have a significantly lower level of oxidative metabolism than that of free-living poikilotherms. It should be noted, however, that in a number of other parasitic helminths, anaerobic metabolism continues even at atmospheric oxygen tensions (Rogers, 1962). The total energy expenditure of S. solidus may therefore be considerably higher than that accounted for by aerobic oxidative metabolism alone. Acknowledgements--We wish to thank Miss B. Tagg for technical assistance and Mr. Odd Vagle for assistance in the production of a programme for the University of Glasgow K D F 9 computer. We should also like to thank Dr. C. A. Hopkins for his helpful criticism of the manuscript. One of us (M. W.) undertook part of the work while in receipt of a grant from the University of London Central Research Fund. REFERENCES BELHR~DEK J. (1930) Temperature coefficients in biology. Biol. Rev. 5, 30-58. BENEDICT F. G. (1938) Vital Energetics. A Study in Comparative Basal Metabolism. Publication 503. Carnegie Institute, Washington, D.C. YON BRAND T. 8c ALLING D. W. (1962) Relations between size and metabolism in larval and adult Taenia taeniaeformis. Comp. Biochem. Physiol. 5, 141-148. CROMPTON D. W. T., SHRIMPTON D. H. & SILVER I. A. (1965) Measurements of the oxygen tension in the lumen of the small intestine of the domestic duck. J. exp. Biol. 43, 473-478. DAVIES P. SPENCER (1966) Physiological ecology of Patella--I. The effect of body size and temperature on respiration rate. J. mar. Biol. Ass. U.K. (In press.) F~mNER D. S. (1942) T h e hydrogen ion concentration in avian digestive tracts. Poult. Sci. 21, 445-450. HEILBRUNN L. V. (1952) An Outline of General Physiology. 3rd edn. Saunders, Philadelphia and London. HEMMINGSEN A. M. (1960) Energy metabolism as related to body size and respiratory surfaces, and its evolution. Rep. Steno metal Hosp. 9 (II), 7-110. HOPKINS C. A. (1952) Studies on cestode metabolism--II. T h e utilization of glycogen by Schistocephalus solidus in vitro. Expl Parasitol. 1, 196-213. HOPKINS C. A. & MCCAIG M. L. O. (1963) Studies on Schistocephalus solidus--I. T h e correlation of development in the plerocercoid with infectivity to the definitive host. Expl Parasitol. 13, 235-243.
15
EFFECT O F BODY SIZE AND TEMPERATURE U P O N OXYGEN C O N S U M P T I O N OF A CESTODE
425
HOPKINS C. A. & SMYTH J. D. (1951) Notes on the morphology and life history of Schistocephalus solidus (Cestoda: Diphyllobothriidae). Parasitol. 41, 283-291. KANtmCO M. S. & PROSSER C. L. (1959) Physiological and biochemical adaptation of goldfish to cold and warm temperatures--I. Standard and active oxygen consumption of cold and warm-acclimated goldfish at various temperatures, aT. cell. comp. Physiol. 54, 259-263. KROGH A. (1916) The Respiratory Exchanges of Animals and Man. Longmans, Green, London. McCAIO M. L. O. & HOPKINS C. A. (1963) Studies on Schistocephalus solidus--II. Establishment and longevity in the definitive host. Expl Parasitol. 13, 273-283. PROSSER C. L. (1958) General summary: the nature of physiological adaptation. In Physiological Adaptation (Edited by PEOSSER C. L.), pp. 50-78. Am. Physiol. Sue., Washington. PROSSER C. L. & BROWN F. A. (1961) Comparative Animal Physiology. 2nd edn. Sounders, Philadelphia and London. RAO K. P. & BULLOCK T. H. (1954) Qx0 as a function of size and habitat temperature in poikilotherms. Am. Nat. 88, 33--44. ROGERS W. P. (1962) The Nature of Parasitism, p. 161. Academic Press, New York and London. SMYTH J. D. (1946) Studies on tapeworm physiology--I. T h e cultivation of Schistocephalus solidus in vitro. 07. exp. Biol. 23, 47-70. SMYTH J. D. (1952) Studies on tapeworm physiology--VI. Effect of temperature on the maturation in vitro of Schistocephalus solidus, ft. exp. Biol. 29, 304-309. SMYTH J. D. (1956) Studies on tapeworm physiology--IX. A histochemical study of egg shell formation in Schistocephalus solidus (Pseudophyllidea). Expl Parasitol. 5, 519-540. SNEDECOR G. W. (1961) Statistical Methods applied to Experiments in Agriculture and Biology, p. 398. 5th edn. Iowa State Univ. Press, U.S.A. SPECTOR W. S. (Ed.) (1956) Handbook of Biological Data. Saunders, Philadelphia and London. VERNBERG W. B. (1961) Studies on oxygen consumption in digenetic trematodes--VI. T h e influence of temperature on larval trematodes. Expl Parasitol. 11, 270-275. VERNBERG W. B. & HUNTER W. S. (1961) Studies on oxygen consumption in digenetic trematodes--V. T h e influence of temperature on three species of adult trematodes. Expl Parasitol. 11, 34-38. VERNBERG W. B. & VERNBERG J. F. (1965) Interrelationships between parasites and their h o s t s - - I . Comparative metabolic patterns of thermal acclimation of larval trematodes with that of their host. Cutup. Biochem. Physiol. 14, 557-566. WAROLE R. A. & McLBoD J. A. (1952) The Zoology of Tapeworms. Univ. Minnesota Press. ZEUTHEN E. (1953) Oxygen uptake as related to body size in organisms. Q. Rev. Biol. 28, 1-12.