Comp. Biochem. Physiol.. 1975. Vol. 52A. I'P. 557 to 565. Pergamon Press. Printed in Great Britain

VARIATION IN RATE OF G R O W T H AND ADRENAL CORTICOSTEROIDOGENESIS IN FIELD AND LABORATORY POPULATIONS OF THE LIZARD AMPHIBOLURUS ORNATUS P. R. BAVERSTOCK* AND S. D. BRADSHAW Department of Zoology, University of Western Australia, Nedlands, Western Australia 6009 (Received 14 August 1974)

Abstract--l. Both field and laboratory studies showed that, under conditions of hypernatraemia. slower-growing A. ornatus maintained higher levels of adrenal corticosteroids in the plasma than faster growers. 2. This apparently resulted from higher levels of ACTH secretion from the pituitary. 3. The higher levels of corticosteroids in slower growers did not appear to be responsible either for their slower growth or their enhanced ability to tolerate hypernatraemia.

INTRODUCTION THE AGAMID lizard Amphibolurus ornatus (Gray) has in recent years been the object of a series of integrated field and laboratory studies designed to elucidate important aspects of this animal's ecology and physiology (Bradshaw, 1970; Baverstock, 1975). One of the most intriguing aspects to emerge from these studies is an evident correlation between growth rate and water and electrolyte physiology of individuals of this species. Considerable variability in individual growth rates was found in a population of A. ornatus living at Bakers Hill, 80 km east of Perth in Western Australia, and differential mortality of fast and slow growers was observed during periods of seasonal stress (Bradshaw, 1971). During extended periods without rain the lizards are subject to chronic hypernatraemia, and the faster-growing individuals in the population characteristically lose weight and die (Bradshaw, 1970). Slow growers on the other hand are remarkably resistant to the long term effects of sodium loading although they succumb more readily than do fast growers to the effects of low environmental temperatures in winter. Recent laboratory studies by Baverstock (1975) have shown that hypernatraemia alone is a sufficient stimulus to induce a loss of condition in fast-growing A. ornatus and results from their inability to reduce urine production when challenged with a salt load. Such reproducible differences between the rates of urine production of different individuals of the same species, differing otherwise only in their rate of growth, suggest that hormonal factors may be involved. In particular, pituitary and adrenal hormones could be implicated in the syndrome and the present investigation was addressed to this possibility.

* Present address: Division of Animal Science, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000.

MATERIALS AND METHODS Field studies

Seasonal changes in levels of plasma corticosteroids were studied in A. ornatus in three areas, chosen because they represent extremes of climate. The climatic characteristics of the three study areas chosen and the population dynamics and physiology of the A. ornatus populations living there have been described in detail elsewhere (Bradshaw, 1971; Baverstock, 1975). The results of these studies may be summarized briefly as follows. Bakers Hill is an area that experiences severe summer droughts in some years, and winter frosts in some years. Growth rates of A. ornatus in the population are highly variable. The droughts lead to severe hypernatraemia in all animals, which in turn leads to differential weight loss and mortality of fast-growing animals. Tuttanning is an area that experiences many severe frosts each year, but droughts are relatively infrequent. In the two populations of A. ornatus studied, growth rates are as variable as at Bakers Hill, but, in contrast to the situation at Bakers Hill, fast-growing A. ornatus at Tuttanning do not suffer weight loss and high mortality during droughts because severe hypernatraemia does not occur. The third area studied, Canning Dam, is a frost-free area. The A. ornatus are uniformly slow-growing, Here, even mild droughts lead to severe hypernatraemia but individuals in the population, being all slow growers, do not suffer weight loss. Plasma corticosteroid analysis

Animals were bled by cardiac puncture with a 26 gauge needle fitted to a 1 ml "Jintan" disposable syringe containing about 0.5/~1 of heparin. The blood was centrifuged within 4 hr, the cells discarded and the plasma stored at -20°C in sealed glass capillaries. Plasma corticosteroid concentration was measured in 25,ul samples by the competitive protein-binding radioassay of Murphy (1967), as validated for reptilian plasma by Bradshaw & Fontaine-Bertrand (1970), and with the modification that the protein-binding solution was a compound of 5~ quokka (Setonix brachyurus) plasma and 4/zCi of [l,2-3H]-corticosterone per 100 ml corticosterone. Standards were assayed in duplicate. Radioactivity was measured in a Packard Scintillation Spectrometer (Model 3375) to 19/o SE using a scintillation mixture containing 70~ toluene and 30~ ethanol, with 4 g of PPO and 100 mg 557

558

P.R. BAVERSTOCKAND S. D. BRADSHAW

of POPOP/I (Hall & Cocking, 1965). Quenching was assessed by Automatic External Standardization and the average counting efficiency for tritium was 30~o. Pituitary growth hormone and prolactin Animals of known growth rate were killed by decapitation and the pituitary gland removed and immediately homogenized in a teflon-coated hand homogenizer. The samples were electrophoresed either by the disc method of Davis (1964), using a 7.5~ acrylamide running gel and Tris-glycine buffer at pH 8.3, or by the vertical slab method of Akroyd (1967), using 7.5% acrylamide and a Tris-EDTA-borate buffer at pH 8"6. Gels were stained in amido schwartz (1~o solution in 7~ acetic acid) for 24 hr in the case of "disc" or 6.5 hr in the case of "slab", and electrophoreticaUy destained. Optical densities of the putative growth hormone and prolactin bands (Nicoll & Nichols, 1971; Nicoll & Licht, 1971) were measured on an Eel "Scanner" with a slit 0'3 mm wide and attached to a gel-holder. With each gel were run several levels of standard bovine growth hormone (NIH-GH-BI5, 0'88 USP units/mg) and of standard prolactin (NIH-P-59, 30.3 i.u./mg) kindly supplied by the National Institute of Health, Bethesda. Standards were dissolved in sodium carbonate/bicarbonate buffer at pH 10.0. The optimum standard curve was obtained by recording the optical density at the point of minimum transmission. RESULTS I. Plasma corticosteroid concentration in field populations In the field, animals were usually bled within 3 min of capture. The effect of stress on steroid levels in the plasma could not be assessed in A. ornatus because its small size excluded the possibility of taking sufficient blood for more than one or two determinations of corticosteroid levels• However, we found that, in another lizard, the goanna (Varanus gouldii), corticosteroid levels rose in response to severe stress after about 4--6 min. Consequently the values reported for A. ornatus in the field probably represent the basal levels of circulating corticosteroids at the time of capture.

_

i //~

(a) All animals

15 ,o 0

o

{b) Known f a s t e r and slower grower

E 15 _o Q.

~ m,

iJl x

5

~l'

0 ONDU

1969

FMAMJJASONO

1970

JFMAMJJASOND

1971

Fig. 1. Variation in the levels of plasma corticosteroid in A. ornatus at Bakers Hill. (a) All animals (including those of unknown growth rate). (b) Faster growers (A) and slower growers (@) treated separately. Values indicate means +_ one standard error. The hatched area indicates the period of time without rain (in days).

2o 0 0B

r --0.868

,o o. .i

o

4"-

m. 0

%

I

I

I0

20

loss in body weight

Fig. 2• Relationship between corticosteroid concentration in the plasma and loss in body wt for A. ornatus of unknown growth rate at Bakers Hill in April, 1970. The loss in body wt was determined by expressing the actual body wt as a ~ of that expected for a fully hydrated animal of the same snout-vent length. The parametric correlation coefficient, r, is statistically significant (P < 1~o).

(a) Plasma corticosteroid levels in animals at Bakers Hill. A period of 49 days without rain at Bakers Hill early in 1970 resulted in significant elevation of the concentration of sodium in the plasma of A. ornatus (Baverstock, 1975). Corticosteroid levels in the plasma were also significantly elevated above winter values at this time (Fig. la). Too few animals of known growth rate were bled at the time for a comparison to be made of corticosteroid levels in the plasma of animals of known growth rate (Fig. lb). However, for seven animals of unknown growth rate, there was a strong negative correlation ( r = - 0 " 8 7 , P < 1~) between loss in body wt and concentration of corticosteroids in the plasma (Fig. 2). The simultaneous studies of animals of known growth rate revealed a positive correlation between growth rate and weight loss during the "drought" (Baverstock, 1975). Hence the negative correlation between loss in body wt and plasma corticosteroid levels suggests that, at the time, faster growers had lower levels of plasma corticosteraid. In the field, only those animals greater than 65 mm in snout-vent length could be bled by cardiac puncture without causing some mortality in animals. Any sample of animals taken from the study area at Bakers Hill consisted of only a few adults of known growth rate and, in many cases, insufficient plasma was obtained for the corticosteroid concentration to be measured. Consequently, on most occasions it was not possible to compare within samples the corticosteroid concentration in the faster and slower-growing animals. However, during the breeding season (November-December), individual animals had a greater probability of being captured and so larger samples were taken then. It was then possible to compare the corticosteroid concentration in the plasma of animals of different growth rates. In both November and December of 1970 and in November of 1971, the slow-growing animals had significantly higher levels of circulating corticosteroids (Fig. lb) (P < 5~). (b) Plasma corticosteroid levels in animals at Canning Dam. Steroid concentration in the plasma reached very high levels in Canning D a m animals, all of which

Rate of growth of the lizard Araphibolurus ornatus

O O -

I

Ii

:k

.~

o

559

I

i

I

I

I

iT t

o

,

I I ~.~~ I I I I I I I~1 N D d FMAMJJ 1969

AS

~=,~3°3~4 t ~fl" OND

J IL~-,4,~I

d FMAMJJ

A SOND

1970

d FM

1971

1972

Fig. 3. Variation in the levels of plasma corticosteroids in A. ornatus at Canning Dam. Values indicate means + one standard error. The hatched areas represent the periods of time without rain (days). 5

2. Laboratory studies (a) Corticosteroid levels in juveniles in the laboratory• • ~ ~ ........ ..--.~.-,.~ ~, Juveniles that had been reared under uniform condio ,, _-:# O tions in the laboratory, and hence whose growth rates I I I I I 1 , 1 III ill] III ........ I were known, were deprived of drinking water for a ONOJFMAMJd ASON DdF MAMJJ ASONDJF M 1969 1970 1971 number of days, after which blood samples were taken• An analysis of the corticosteroid concentrations in the plasma (Fig. 5) showed that there E {b) Populotions treated was a statistically significant negative correlation seporotely i between growth rate and corticosteroid concentration I / A'~. O u t c r o p A I Outcrop B for animals from Bakers Hill (P < 5~), Tuttanning 31(P < 0.1~) and Canning Dam (P < 1~o) when water. was withheld• If water was supplied, the correlation disappeared in the case of Tuttanning juveniles, and ONDJFM 0 NO dFM was not statistically significant in the case of the 1971 1972 1971 1972 Bakers Hill juveniles• (b) Corticosteroid levels in dehydrated adults. Adults Fig. 4. Variation in the levels of plasma corticosteroids in A. ornatus of known growth rate at Tuttanning. (a) from the three localities were dehydrated to about Results from both populations pooled. (b) Results from 709/0 of their initial body wt, thus raising plasma each population treated separately for the period of the sodium to 197_ 13.0m-equiv./i. Table 1 compares 1971/72 summer drought. A Faster growers; • slower the corticosteroid levels in the plasma of these anigrowers. Vertical lines indicate +one standard error. Points without standard errors indicate the value for single mals. The slower growers from Bakers Hill had, as individuals. The hatched area indicates the periods of time expected, a higher concentration of corticosteroids in the plasma than the faster growers• Canning Dam without rain (days). animals had high levels similar to those of the slower

1

'l(o) R e s u l t s f r o m b o t h I I p o p u l a t i o n s pooled

Ii

',

E

"

1,

are slow growers. The steroid levels were highest during the 1970/1971 summer (averaging over 10/~g/ 100 ml, Fig. 3). (c) Plasma corticosteroid levels in animals in Tuttunning. Corticosteroid concentrations in the plasma remained low throughout the study (Fig. 4a), the highest value recorded for any animal at Tuttanning being 7.2 pg/100 ml. There was no evident correlation between growth rate and corticosteroid concentration in the plasma in field animals from Tuttanning. Again, on most occasions, insufficient numbers of animals of known growth rate were taken in any one sample for a statistical comparison to be made between the corticosteroid levels in the faster and slower-growing animals within each of the Tuttanning populations. However, towards the end of the study [during a severe drought which nevertheless produced only mild hypernatraemia (Baverstock, 1975)-I sufficient numbers of animals of known growth rate were captured to allow a comparison of the corticosteroid concentrations in faster and slower-growing individuals in each population, separately. On both outcrops the pattern of corticosteroid concentration was the same (Fig. 4b). Certainly no differences were apparent that correlated with growth rate. (...t.. 52 3,.

.

Bakers Hill No water for 4 Tuttanning No water for 1 Tuttanning No water for 7 d a w [Na] pla~m8=171 days [Na] plasma = 171 day [Na plasma=t 67

[+4) rn-equiv/I 1+6) m-ecluiv/I i+_11) m-equiv/I r =-0.se~ r = +0.021 • • =-0'620 • •e 0