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A study of the sources of nutrients for embryonic development in a viviparous lizard, Sphenomorphus quoyii
A STUDY OF THE SOURCES OF NUTRIENTS FOR EMBRYONIC DEVELOPMENT IN A VIVIPAROUS LIZARD, SPHENUMORPHUS
QUOYII
JANE THOMPSON* School of Biological
Sciences,
University of Sydney, N.S.W. 2006, Australia
(Recriced 7 May 1981) Abstract ‘-1. The Eastern water skink is a viviparous lizard with a simple placenta and a large yolk sac. 2. Changes in wet, dry and ash weights of developing follicles and of the egg and egg components during gestation were analysed. 3. The total wet weight of the whole egg (embryo, yolk plus embryonic fluids and extraembryonic membranes) increases significantly during development. 4. This increase is due to uptake of water and inorganic ions from the mother. 5. Both the water and inorganic ions are incorporated into the developing embryo. 6. Lipid is the principal organic material metabolized and it is estimated that there is sufficient lipid in the yolk for embryonic growth and metabolism. Relatively little protein is metabolized. 7. The results show that embryos take up water and some inorganic ions from the mother but there is no evidence to suggest a dependence on the mother for organic nutrients.
INTRODUCTION
egg increases during gestation for any viviparous reptile. Hall (1969) claimed that there was such an increase in the snake, Regina grahami, but his results are not statistically significant. In two species of viviparous snake, Thamnophis sirtulis and Psrudechis porphyriacus, dry weight does not change significantly during development (Clark & Sisken, 1956; Shine, 1977 respectiveIy). In these species there may be addition of nutrients from the mother since some reduction in (ash-free) dry weight would be expected during gestation as a result of metabolism. In P. porphyriacus there is also an increase in the ash content of eggs during development suggesting an uptake of minerals from the mother (Shine, 1977). Clark & Sisken (1956) made a similar claim for 7: sirtnlis but their conclusion is based on too few samples for statistical significance. In this study of S. quqvii changes in the chemical composition of pre-ovulatory follicles and in the eggs during the entire gestation period were examined. A preliminary account of some of these results has been reported elsewhere (Thompson, 1977a).
Pfacentation has been described in a number of species of reptile (Weekes. 1935; Bauchot, 1965). However, very little is known of the function of the placenta, in particular, whether it is a route for transfer of metabolites from the mother to the embryo. In general, the eggs of viviparous reptiles contain a large quantity of yolk, although there are some species where it appears to be reduced (Weekes, 1935; Bellairs, 1970) or where yolk sparing occurs (Bellairs et al., 1955). In any case, the yolk is probably the principal source of nutrients for the embryo but it is possible that there may, in addition, be some exchange of metabolites between the maternal and embryonic circulations. The Eastern water skink is a typical viviparous lizard with a simple placenta and large yolk sac (Harrison & Weekcs, 1925; Weekes, 1927). The aim of this study was to determine whether there was uptake of metabolites from the mother or whether the yolk contained suflicient nutrients for complete development of the embryo. Dessaur & Fox (1959) pointed out that in assessing the possible role of the reptilian placenta in exchange of metabolites it would be invaluable to compare the chemical composition of eggs at different stages of embryonic development. Few such studies have been reported in the literature. In the garter snake, T&PInopf1i.ssirtulis, there is sufficient lipid present in the yolk but there may be some transfer of amino acids across the placenta (Clark et al., 1955; Clark & Sisken, 1956). In some species, water is gained from the maternal circulation (Clark et al., 1955; Coin & Goin, 1971; Branson & Baker, 1974; Shine, 1977). However, there is no evidence that the total dry weight of the
MATERIALS
* Present address: Zoology Department, Australian National University, P.O. Box 4, Canberra, A.C.T. 2600, Australia.
AND
METHODS
Fifty-four pregnant females with a total of 309 embryos were collected from a study area about 30 km south of Sydney at regular intervals during one breeding season. Lizards were killed with an intra-peritoneal injection of Nembutal within four days of capture. The liver was removed and weighed. In non-pregnant females ovaries were removed and the developing follicles separated and weighed. Dry weight of follicles was determined by drying to constant weight in a vacuum drier at 25°C. Dried samples were then heated in crucibles in a mufBe furnace at 42O’C for 24 hr to determine the ash weight. In pregnant females. the oviducts were removed and the eggs released by teasing away the oviducts with a pair of fine forceps.
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Follicle
wet ?Gight,
mg
30.3
400
quoyii
I
I
I
100
200
300
Follicle
Follicle
Fig. 1. Changes
88z:
A
wet
weight,
wet
weight,
mg
mg
in dry weight (a), water content (b). organic weight (c) and ash weight (d) of follicles of S with growth of the follicles. The data are combined in Fig, I (e) for comparison.
Stages studied ranged from immediately after ovulation to full term embryos. The wet weights of the whole eggs, which included embryo, yolk sac and extra-embryonic membranes, were recorded. One or two whole eggs from each female were used for immediate determination of total
dry and ash weight as above. In females carrying early stage eggs, the remainder of the eggs were frozen at - 20 6 for later analysis. In later stage samples. after removing the extra-embryonic fluids and membranes. the embryo and yolk sac were separated, weighed and then stored as above.
4c
Nutrients Anu/ytical
for lizard embryos
procrdurrs
Total nitrogen in dried yolk sacs and embryos was determined using the micro Kjeldahl technique. Total protein was then estimated as total nitrogen x 6.25. This calculation assumes that the conversion factor 6.25 is appropriate for lizard protein and that all nitrogen is in the form of protein. In support of this assumption, Tomita (1929) estimated that 98.5”;, of the total nitrogen in the yolk of Thu~~~s.soc~/wlys sp. was in the form of protein and, further, excretory nitrogen probably represents only a small fraction of total nitrogen (Clark ef ul., 1957). Total lipid in dried samples of early stage eggs or in later stages in dried yolk sacs and embryos was determined uiing the diphasic extraction procedure of Bligh & Dyer (1959) modified for small samples. Dried samples were ground in a mortar and pestle and a known weight transferred to a 15 ml conical centrifuge tube to which 0.8 ml IN I\;aCI was added. mixed and left to stand overnight at 4 C. One ml chloroform and 2ml methanol (AR grade) were then added and mixed. Another 1 ml chloroform was added, mixed, followed by 1 ml distilled water and the mixing repeated. The resulting emulsion was then centrifuged at 4 C for IOmin after which it separated into an upper aqueous layer and a lower one containing lipids with a thick white layer. presumed to be mainly protein, at the interface. The top layer was removed and discarded and the interface was displaced gently so that the bottom layer could be removed and transferred to a pre-weighed small g&s drying vessel. The chloroform was then evaporated in a desiccator under vacuum and the weight of lipid determmed.
E.dimution of the uyr 0f embrps In captured adult females follicles developed approximately synchronously and, during the breeding season, at any one time. pregnant females carried embryos at approximately the same stage of gestation. Therefore the age of embryos was estimated by taking Day 1 as the day on which I first found females which had ovulated, assuming that fertilization took place at ovulation. RESULTS
Changes in composition of developing follicles Follicles ranged from very early stages where yolk deposition was just beginning (wet wt approx. 13 mg)
511
to fully developed follicles (wet wt approx. 380mg) near ovulation representing a period of approximately three weeks. Changes in all measured variables are shown in Fig. 1. Dry weight of the follicles increased significantly during this period and the rate of deposition of solids was constant. The water content of the follicles (wet wt-dry wt) also increased constantly and there was a significant and linear increase in organic weight (dry wt-ash wt) parallel with the increase in dry weight. The ash content of the follicle also increased linearly with growth of the whole follicle. Relatively, there was a decrease in the proportion of water in the follicle while the proportion of dry matter, organic matter and ash increased. Changes follicles
in lirer weight qf ftimales
with developing
The ratio of liver weight to body weight (corrected for weight of follicles) decreased with growth of the follicles (Fig. 2). Gross changes in the whole egg und egg components 1. The whole egg. Changes in the composition of the whole egg, which includes the embryo, yolk, extraembryonic fluids and membranes are shown in Fig. 3. Total wet weight increased approximately 4-fold during gestation. The pattern of growth was triphasic with the most rapid increase in weight in the middle of the gestation period. The increase in total wet weight is due mainly to uptake of water. The total water content increased in the same manner as the total wet weight, however, there was a significant decrease in total dry weight. Organic weight also decreased significantly and the amount by which it declined was greater than the decline in dry weight. This can be accounted for by the fact that the total ash weight increased by approximately 4 mg. Relatively, the proportion of water in the eggs changed more than any other measured variable. 2. The embryo and yolk sac’. Growth of the embryo proceeds at the expense of the yolk sac (Fig. 4). The wet weight of the embryo increased sigmoidally,
45-
30 0
100
I 200
1 300
400
Follicle wet weight, mg Fig. 2. Change in liver weight of S. quoyii females as a function of stage of development of the follicles.
JANE THOMPSC>N
512
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,
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1
40 , Estimated
60 age,
,
80
days
0
Solids
0
1 20
I 40
Estimated
60
4 OnpofllC So
age, days
Fig. 3. Changes in wet weight (a). dry weight (b), water content (c), organic weight (d) and ash weight (e) of whole eggs (embryo, yolk sac plus extra-embryonic fluids and membranes) of S. yuoyii with age of the embryo. The data are combined in Fig. 3 (f) for comparison. whereas
the weight of the yolk sac increased to about the time of n~id-gestation and then declined steadily. The wet weight of the embryo at term was greater than the initial wet weight of the yolk. However, the dry weight of the embryo did not exceed the initial dry weight of the yolk. Thus the gain in weight of the
embryo can be attributed, in part, to addition of water as can the increase in total wet weight of the yolk at mid-gestation. The change in organic matter in the yolk and embryo followed a similar pattern to the dry weight changes. However, there was an increase in the ash
.
Nutrients for lizard embryos
3X3-
.(d)
i
S
250-
200-
150-
o-
I
(e) . 30-
. 25-
.
0
. i
20! 15-
IO-
5-
OO
Estimated age, days
Estimated age, days Fig. 4. Changes in the wet weight (a), dry weight (b), water content (c), organic weight (d) and ash weight (e) of the yolk (a) and embryo (0) of S. quoyiiduring gestation.
content of the embryo above that initially in the yolk. The gain is approximately equal to the gain observed in the whole egg (Fig. 3). Protein and lipid composition of the embryo and yolk SUC
The amount of protein in the embryo at term was
similar to that initially in the yolk while the lipid content of the full term embryo was significantly less (Fig. 5). There was a decrease in the total amount of lipid in the yolk and embryo combined, the most ranid rate of decline being in the last third of Restatidn while the total proteyn content of the cornLined embryo and yolk sac decreased only slightly.
JANE THOMPSON
514
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60 days
I
80
1
Fig. 5. Changes in the protein (a) and lipid content (b) of the yoIk (e) and embryo (0) of S. quoyii with age of the embryo. Fig. 5 (c) shows the total protein (II) and lipid (0) in the whole egg (yolk and embryo) as a function of age of the embryo.
Nutrients for lizard embryos
515
3
Days
since ovubtion
Fig. 6. Changes in the liver weight of pregnant females of S. quo~ii during gestation.
Chortges in iiwr
wright
qf j~mu1e.s during gestation
There was no significant change in the ratio of liver wsight to total body weight during gestation (Fig. 6). DlSCL’SSIOiV
Yolk substances are laid down in the ovarian follicle prior to ovulation. The pattern of growth of the ovarian follicles in S. quovii differs from that in the Ribbon Snake, T~*arnn~~h~.~ sauritus (Dessaur 8~ Fox, 1959). The measurements on folficies from s. 4~0~~~ correspond to the second phase described by Dessaur & Fox; no measurements were made on the very early white translucent follicles in my study. In S. quoyii, there was a steady decrease in percentage water in the follicles as solids accumulate whereas in Thrrmnopkis sauritus the percentage water increased rapidly at first and then at a lower rate during this phase of follicular growth. There was a significant decrease in the weight of the li\er in females with enlarging follicles. similar to that
observed in Thamnophis suuritus (Dessaur & Fox, 1959). Since S. yuqii has no obvious fat bodies which are a source of yolk material in some lizards and snakes (Dessaur & Fox, 1959: Hahn & Tinkle, 1965). the liver is probably the principal source of yolk substances in S. quoyii. Changes gestution
in chemicul
composition
“j, Lipid
the
rgy
dwiny
On a dry weight basis the egg of S. quoyii consists mainly of fat (2%;) and protein (SSq<>)at ovulation. These values are similar to those reported for other reptiles (Table I). Minerals account for 67% of the dry matter in the egg. Material unaccounted for may include carbohydrate although this is expected to be very low and probably less than 3”,, (Tomita, 1929; Fukuda, 1939). In addition, there may be some inaccuracies in measurements leading to less than lOO”/;, recovery. The most obvious change in the whole egg during gestation was an increase in the total amount and in the proportion of water. There are two possible
Table 1. Lipid and protein content of reptilian eggs Species
of
01,Protein
32 14.3 25.1
55.6
30
40
66
51
28 30
66 SO
39.2 36-43 36 30 28
48.3 50 55
Author Tomita (1929) Lynn & von Brand (1945) von Brand & Lynn (1947) Fukuda (1939) Clark & Sisken (1956) Clark et ul. (1955) Dessaur & Fox (1956) Hahn & Tinkle (1965) Ricklefs & Cullen (1973) Hadley & Christie (1974) Avery (1975)
JANE THOMPSON
516 Table 2. Water
content
of eggs of viviparous
squamates
‘
content at ovulation
51 54
84 81
40 40
80
sources of water: the maternal circulation and metabolic water. The amount by which water content increased is more than would be expected from the nletabolism of lipid. One mmoi of a typical fatty acid, paimitic acid {moi.wt 261) yields 288 mg Hz0 (Conn & Stumpf, 1973). The expected yield from 40mg of lipid would therefore be approximately 45 mg HZ0 which is much less than the measured increase of about 800 mg H,O. Thus, there is definite evidence in S. q~~~)~i~ for uptake of water from the mother. Similar increases in the water content of developing eggs have been reported in both oviparous and viviparous reptiles (Packard t’t al., 1977). Increase in water content of eggs of viviparous species are compared in Table 2. There is some disagreement in the literature regarding the adaptive significance of water absorption and storage in reptilian eggs. In egg laying species, accumulated water may act merely as a buffer against the dangers of potential desiccation (Fitch & Fitch, 1967; Badham, 1971: Bustard, 1971a,b) and water uptake and storage may not be essential for the normal development of the embryo (Bustard, 1966, 1971a; Dmi’el, 1967). However, in Anolis mralinmsis, absorption and storage of water is important for the deveioping embryo, since hatchiings are smaller when hatched from eggs deprived of water (Gordon, 1960) so water absorption may be a critical factor in the normal development of these and other reptilian eggs (Packard et ui.. 1977). The hatchiings of some oviparous lizards, snakes and turtles weigh more than freshly laid eggs (Cunningham & Hucne. 1938; Brown, 1956; Mendelssohn, 1963: Bustard, 1965) which is possible only if part of the water absorbed is incorporated into the embryo. On the other hand. hatchlings of some species weigh less than freshly laid eggs (Lynn & von Brand, 1945; Bustard. 1967; Dmi’ei, 1967 ; Mendelssohn, 1963, 1965; Badham 1971). However, as Packard et ul. (1977) point out, it cannot be concluded that water absorbed by these eggs is not incorporated into forming tissue without taking into account both the oxidation of organic material by metabolism of the embryo. and the mass of the egg shell and other extraembryonic membranes. My observations in S. quoyii support the conclusion of Packard et ul. (1977) that water absorbed is incorporated into the developing embryo and is presumably essential in embryonic development. Dry wright unrl orgunic weight There was no increase
Final “;, Hz0
in either dry weight or or-
‘)
Author
Shine (1977) Clark & Sisken (1956) Hadley
& Christie
(1974)
ganic weight of the egg of S. yuoyii which, if present, would suggest uptake of nutrients from the mother. In fact, total dry weight decreased by about loo/ and organic weight by about 13”,;,. In egg-laying species, where the only sources of energy available are in the yolk, reported changes in dry weight are variable ranging from 14.5% (Cunningham & Hurtwitz, 1936) to 620,; (Rickiefs & Cullen, 1973). Reported changes for viviparous species, where there may be external sources of energy via the maternal circulation, are also variable. Clark et al. (1955) reported that in the viviparous snake Thamnophis &a/is, dry weight decreased by 187,: whilst organic weight decreased by 2Opb. However, in the same species, Clark & Sisken (1956) found no significant change in dry weight during development and since ash weight did not change signifi~ntiy there could be no change in organic weight either. In two other species of viviparous snakes, Natechis scututus and Psrudrchis parphyriacus, Shine (1977) found decreases in dry weight of 34 and 1% respectively. The organic weight in Psrudechis pffr~~~~~,riucus decreased by about 6%. Discussing the only information, to my knowledge, for a viviparous lizard, Avery (1975) states that the total dry weight of eggs in a clutch of Lticertu ciripara remains constant after transfer to the oviducts. However. there are no data on ash content of the eggs so the change in organic weight cannot be calculated. Hogarth (1976) estimates that during the development of embryos of viviparous fish, a decrease in organic weight of less than 20% would suggest some uptake of material from the mother. In the absence of any estimate for reptiles, this estimate can be taken as a guide. in S. quoyii, where organic weight decreased by 13x, additional sources of energy may be supplied by the mother. To determine whether this is true, I have drawn on two independent sources of information. First, the change in liver weight of females during gestation and, secondly, the predicted decline in organic weight which can be estimated knowing the principal sources of energy for development and the estimated metabolic rate of the embryos. In S. yuoq’ii, there was no significant change in the liver weight of females during pregnancy. This suggests that there is no significant depletion of the metabolic reserves of the female which might occur if nutrients were being supplied to the developing embryos (Hogarth, 1976). The principal organic constituents of the egg in S. quoyii are lipid and protein. Carbohydrate is probably a relatively insignificant source of energy. In S. yuoyii lipid content of combined yolk and embryo decreased
Nutrients for lizard embryos 20'C
20 6 9 160 F LI 1.2E Y gQ8-
z
04/J 0. 40
l
.
l* . 50
60
70
,,I 80
90
100
Age, days
30’C
6
.
Age,
days
Fig. 7. Estimated oxygen consumption (see Discussion for explanation) of S. quoyii embryos at 20-C and 30 ‘C as a function of age of the embryo.
by 56% while the protein content decreased only shghtly, showing that embryos derive energy principally from the combustion of fat. To determine theoretically whether the amounts of lipid metabolized would be sufficient for the embryo, an estimate of the oxygen consumption is required. A few metabolic measurements have been made on embryonic snakes (Zarrow & Pomerat, 1937; Clark, 1952, 1953a,b; Dmi’el, 1970) and turtles (Lynn & von Brand, 1945), but there are no data available for any embryonic lizards. Metabolic rates of reptiles vary with temperature (Bennett & Dawson, 1976) and there may be differences in metabolic rates of embryos at different stages of deveopment. These latter differences may be a reflection only of the increase in weight of the embryo (Dmi’el, 1969). In addition there may be daily fluctuations in metabolic rates in embryos (Dmi’el, 1969) as well as differences between species (Dmi’el, 1970; Lynn & von Brand, 1945). As an approximation, I have taken Bennett and Dawson’s (1976) equations for oxygen consumption of adult lizards at 20” and 3O’C which is within the range of temperatures to which embryos of S. quoyii are exposed (Veron & Heatwole, 1970). Their equations are valid for lizards weighing down to 1 gm, approx. the weight of hatchling S. quoyii. The calculation doesnot include the contribution ofextra-embryonit membranes or of very early stage embryos to the total rate of metabolism both of which are likely to be small (Lynn & von Brand, 1945; Clark, 1953~).
517
Predicted oxygen consumption of S. quo!,ii embryos of known weights and ages are plotted in Fig. 7 and the total oxygen consumption estimated at each temperature by calculating the area under each curve. At 30°C an estimated 175 ml O2 are consumed and at 2O”C, 57 ml. Assuming the metabolism of 1 mg fat uses 2.03 ml 0, (Prosser & Brown, 1961) and that all oxygen consumed is used for metabolism of lipid, at 3O’C the amount of fat consumed would be 84mg and, at 2O”C, 30mg. The actual amount of lipid consumed was between 35 and 40 mg which is within the range predicted. Assuming the estimate of oxygen consumption is reasonable, the metabolism of this quantity of lipid would therefore be adequate for the energetic requirements of the embryos. Therefore there is no need to postulate additional sources of energy such as protein. The small observed decline in total protein in the whole egg can be attributed to the fact that relatively little protein is metabolised rather than being the resultant of protein metabolised plus protein gained (as amino acids) from the mother. Despite the fact that the placenta in S. quoyii is permeable to amino acids (Thompson, 1977) the metabolic calculations above suggest that uptake of protein precursors may not be essential, at least for the energetic requirements of the embryo. This conclusion differs from that of Clark et ul. (1955) and Clark & Sisken (1956) who concluded there must be transfer of amino acids from the mother to the embryo in Thamnophis .sirtulis. However, in this species, they found a significant increase in protein content of the egg during development which was not found in S. quoyii. In summary, the results reported here suggest that there is sufficient protein and lipid in the yolk of S. quoyii for embryonic development. There is no evidence to suggest a significant gain of organic nutrients from the mother; however, there is definite evidence for uptake of water and inorganic ions from the maternal circulation. A detailed examination of mineral uptake will be reported elsewhere (Thompson, 1981). Acknowlrdyemmfs--I thank Drs Gordon Grigg, Richard Shine and Gillian Courtice and Associate Professor John Simons for their interest and advice during the project. The work was supported by a Commonwealth Postgraduate Research Scholariship.
REFERENCES AVERY R. A. (1975) Clutch
size and reproductive effort in the lizard L~crrtu ricipurtr (Jacquin). O~o/oc/iu 19 165-170. BADHAM J. A. (1971) Albumen formation in eggs of the agamid Amphibolurus hurbtms htrrhutu.s. Copricr 1971, 5433545. BAUCHOT, R. (1965) La placentation chez les reptiles. A~ln/.s Biol. 4, 547-574. BELLAIRS A. d’.&. (1970) The Lij> of’Rrptrlr.s. Weidenfeld & Nicolson, London. BELLAIRS R., GRIFF~THS I. & BELLAIRSA. d’A. (1955) Placentation in the adder Viperu hrrus. Nuturr 176, 657-658. BENNETT A. J. & DAWSON W. R. (1976) Metabolism. In Biology of‘ thr Reptilia. Vol. 5. (Edited by CANS. C.) pp. 127-223. Academic Press, New York. BLIGH E. G. & DYER W. J. (1959) A rapid method for total lipid extraction and purification. Can. J. Bioc,hrm. Phydol. 37, 91 l-917.
518
JANE TI
BRAND VON J. & LYNN W. G. (1947) Chemical changes in the turtle embryo. Proc. Sot. c\-p. Biol. Med. 64, 6lm 62. BRANSON B. A., 6i BAKER E. C. (1974) An ecological study of the Queen Snake, Rrqinu .srptemrittuttr (Say), in Kentuckv. T1~/~7neStud. Zoo/. Bat. 18, 153-171. BROW< E. E. (1956) Nests and young of the six-lined racerunner. Cnrmidopl2oru.s sc~.~linrtrtw~ (Linnaeus). J. E/i&r Mitc+rl/ scirnt. Sec. 72, 30 40. BUSTARD H. R. (1965) Observations on Australian geckos. flcrpctolnyicu 21, 294~302. BUSTARU H. R. (1966) Notes on the eggs. incubation and young of the bearded dragon. Anlphiholuruv hurhutus htrrhotus (Cuvier). Br. J. Hvrpet. 3, 252 259. BIISTARII H. R. (1967) Reproduction in the Australian gekkonid genus Oedwtr (Gray 1X42). Herputologicu 23, 27&2X4. BI;SI-ARDH. R. (197la) Temperature and water tolerances of incubating sea turtle eggs. Br. J. Herpet. 4, 196-198. B~ISTARDH. R. (197lb) Temperature and water tolerances of incubating crocodile eggs. Br. J. Hrrptr. 4. 198-200. CI.AKK H. (1952) Data on respiration of snake embryos. Anut. Rrc,. 113, 549. CLARK H. (1953a) Eggs. egg-laying and incubation of the snake Eluphe rwwr~i (Baird and Girard). Copriu 1953, YO 92. CLARK H. (19S3b) Metabolism of the black snake embryo. I. Nitrogen excretion. J. rlup. Biol. 30, 492-501. CI ARK H. (19.53~) Metabolism of the black snake embryo. II. Respiratory exchange. J. e.xp. Biol. 30, 502-505. CI.ARK H.. FIORIO B. & HURO~I~-z R. (1955) Embryonic growth of Thtrumophis s. sirtrrlis In relation to fertilization date and placental function. Cop&r 1955, 9-13. CI,ARK H. & SISKW B. F. (1956) Nitrogenous excretion by embryos of the viviparous snake Tlwtmophis s. \Irtu/is (L.). J. rzp. Eiol. 33, 384 393. CLARK H.. SISKEN B. & SHANNON J. E. (1957) Excretion of nitrogen by the alligator embryo. J. cc//. camp. Ph,nio/. 50, Con
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134.
E. E. & STUMPF P. K. (1973) Outlirlcs o~Bio~~hrr?tiatry. Wiley. New York. CLIUKINGHAM B. & HUENL E. (1938) Further studies on water absorption by reptile eggs. Am. Nut. 72, 3X0-385. CIJNNINGHAY B. & H~RWI~Z A. P. (1936) Water absorption by reptile eggs during incubation. Atn. :Vtrt. 70, 590 5Y5. DESSA~IRH. C. & Fox W. (1959) Changes in ovarian follicle composition with plasma levels of snakes during estrus. .4m. J. Physiol. 197, 36c-366. DMI’EL R. (1967) Studies on reproduction, growth and feeding in the snake Spulrrosophi.scljfordi (Colubridae) Cop&~ 1967, 332 346. DMI’EL, R. (1969) Circadian rhythm of oxygen consumption in snake embryos. L[je Sci. 8, 1333-I 341. D~l’tl.. R. (1970) Growth and metabolism in snake embryos. J. Emhr!o/. c\-p. Morph. 23, 761 -772. FIT(‘H H. S. & FITCH A. V. (1967) Preliminary experiments on physical tolerances of the eggs of lizards and snakes. E&o
R. E. (1960) The influence of moisture on sariation in the eggs and hatchlings of ilr~olrs (‘. wro/itwn.~i~. (Voigt.) Cllicn
GORDON
SHINE: R. (lY77) Reproduction in Australian elapid \nakeh. II. Female reproductive cycles. .Aust. J. Zoo/. 25, 655-666. THOMPSON J. (1977a) Embryo-maternal relatlonships in ;I viviparous skink. Sp/t~,lorilorphrc\ quoxii (Laccrtilia: Scincidae). In Reproductirm tmd Er~oilrtrorl. (Edited by C'ALAII~ J. H. & TYNIIAI F-BIS~OE C. H.) pp. 179- 280. Australian Academy of Science, 1977. THOMPSON J. (l977b) The transfer of ammo acids across the placenta of a viviparous lizard, Sp/tc~tl~Jmorphlr,s quorii. 7’11(,~io(/~no/oy!, 8, 15X. THOMPSON J. (1981) Uptake of inorganic ions from the maternal circulation during development of the embryo of a viviparous lizard, Sp/7cnon,o,p/tIr\ yuoxii. C~>rnp. Bioc,he01. P/t!,.siol. (In press). TOMITA M. (1929) Beitrage 7ur Embryochemle dcr Reptilien. I. J. Binchrm. (Tokyo) IO, 351~ 356. VERON J. E. N. & HEATWOL.E H. (1970) Temperature relatlons of the water skmk, Sp/tr,lomorp/tlc.\ yrcoj,ii. J. Hw pet. 4, 141~153. WCPKES H. C. (1927) Placentation and other phenomena in the scincid lizard L~~l0\001tr (Hirtulitr) qwrii. Proc. Lu7rr. sot,. 1v.s.I+: 52, 499 554. WEI:KES H. C. (lY35) A review of placentation among rcptiles with particular regard to the function and evolution of the placenta. Proc. Zoo/. SM., Low/. 2, 625 645. ZARROW M. X. & P~M~;RATC. M. (1037) Respiration of the egg and young of the Smooth Green Snake, I.iopc/tl.\ rrrrvdis (Harlan). Grontll I, IO3 I IO.
QUOYII
JANE THOMPSON* School of Biological
Sciences,
University of Sydney, N.S.W. 2006, Australia
(Recriced 7 May 1981) Abstract ‘-1. The Eastern water skink is a viviparous lizard with a simple placenta and a large yolk sac. 2. Changes in wet, dry and ash weights of developing follicles and of the egg and egg components during gestation were analysed. 3. The total wet weight of the whole egg (embryo, yolk plus embryonic fluids and extraembryonic membranes) increases significantly during development. 4. This increase is due to uptake of water and inorganic ions from the mother. 5. Both the water and inorganic ions are incorporated into the developing embryo. 6. Lipid is the principal organic material metabolized and it is estimated that there is sufficient lipid in the yolk for embryonic growth and metabolism. Relatively little protein is metabolized. 7. The results show that embryos take up water and some inorganic ions from the mother but there is no evidence to suggest a dependence on the mother for organic nutrients.
INTRODUCTION
egg increases during gestation for any viviparous reptile. Hall (1969) claimed that there was such an increase in the snake, Regina grahami, but his results are not statistically significant. In two species of viviparous snake, Thamnophis sirtulis and Psrudechis porphyriacus, dry weight does not change significantly during development (Clark & Sisken, 1956; Shine, 1977 respectiveIy). In these species there may be addition of nutrients from the mother since some reduction in (ash-free) dry weight would be expected during gestation as a result of metabolism. In P. porphyriacus there is also an increase in the ash content of eggs during development suggesting an uptake of minerals from the mother (Shine, 1977). Clark & Sisken (1956) made a similar claim for 7: sirtnlis but their conclusion is based on too few samples for statistical significance. In this study of S. quqvii changes in the chemical composition of pre-ovulatory follicles and in the eggs during the entire gestation period were examined. A preliminary account of some of these results has been reported elsewhere (Thompson, 1977a).
Pfacentation has been described in a number of species of reptile (Weekes. 1935; Bauchot, 1965). However, very little is known of the function of the placenta, in particular, whether it is a route for transfer of metabolites from the mother to the embryo. In general, the eggs of viviparous reptiles contain a large quantity of yolk, although there are some species where it appears to be reduced (Weekes, 1935; Bellairs, 1970) or where yolk sparing occurs (Bellairs et al., 1955). In any case, the yolk is probably the principal source of nutrients for the embryo but it is possible that there may, in addition, be some exchange of metabolites between the maternal and embryonic circulations. The Eastern water skink is a typical viviparous lizard with a simple placenta and large yolk sac (Harrison & Weekcs, 1925; Weekes, 1927). The aim of this study was to determine whether there was uptake of metabolites from the mother or whether the yolk contained suflicient nutrients for complete development of the embryo. Dessaur & Fox (1959) pointed out that in assessing the possible role of the reptilian placenta in exchange of metabolites it would be invaluable to compare the chemical composition of eggs at different stages of embryonic development. Few such studies have been reported in the literature. In the garter snake, T&PInopf1i.ssirtulis, there is sufficient lipid present in the yolk but there may be some transfer of amino acids across the placenta (Clark et al., 1955; Clark & Sisken, 1956). In some species, water is gained from the maternal circulation (Clark et al., 1955; Coin & Goin, 1971; Branson & Baker, 1974; Shine, 1977). However, there is no evidence that the total dry weight of the
MATERIALS
* Present address: Zoology Department, Australian National University, P.O. Box 4, Canberra, A.C.T. 2600, Australia.
AND
METHODS
Fifty-four pregnant females with a total of 309 embryos were collected from a study area about 30 km south of Sydney at regular intervals during one breeding season. Lizards were killed with an intra-peritoneal injection of Nembutal within four days of capture. The liver was removed and weighed. In non-pregnant females ovaries were removed and the developing follicles separated and weighed. Dry weight of follicles was determined by drying to constant weight in a vacuum drier at 25°C. Dried samples were then heated in crucibles in a mufBe furnace at 42O’C for 24 hr to determine the ash weight. In pregnant females. the oviducts were removed and the eggs released by teasing away the oviducts with a pair of fine forceps.
509
510
JAUE
. ‘t
THOMPSOI\
(c)
.
xl-
.
. .*
. .
.
. f
.
.
.o
.
e IO-
rn 100
G
.
‘.*
l
SO-
l. .
t
.*
.’
8
jo-
o-
0 +
I
I
I
. .
(d) 15-
. e ..*
. IO -
.
32 g 00 t
*’
2
0 =
.
.
. .
. .
.
8
.
5-
9 50
:
:
I-
8: OO
II I
,
I
100
Follicle
wet ?Gight,
mg
30.3
400
quoyii
I
I
I
100
200
300
Follicle
Follicle
Fig. 1. Changes
88z:
A
wet
weight,
wet
weight,
mg
mg
in dry weight (a), water content (b). organic weight (c) and ash weight (d) of follicles of S with growth of the follicles. The data are combined in Fig, I (e) for comparison.
Stages studied ranged from immediately after ovulation to full term embryos. The wet weights of the whole eggs, which included embryo, yolk sac and extra-embryonic membranes, were recorded. One or two whole eggs from each female were used for immediate determination of total
dry and ash weight as above. In females carrying early stage eggs, the remainder of the eggs were frozen at - 20 6 for later analysis. In later stage samples. after removing the extra-embryonic fluids and membranes. the embryo and yolk sac were separated, weighed and then stored as above.
4c
Nutrients Anu/ytical
for lizard embryos
procrdurrs
Total nitrogen in dried yolk sacs and embryos was determined using the micro Kjeldahl technique. Total protein was then estimated as total nitrogen x 6.25. This calculation assumes that the conversion factor 6.25 is appropriate for lizard protein and that all nitrogen is in the form of protein. In support of this assumption, Tomita (1929) estimated that 98.5”;, of the total nitrogen in the yolk of Thu~~~s.soc~/wlys sp. was in the form of protein and, further, excretory nitrogen probably represents only a small fraction of total nitrogen (Clark ef ul., 1957). Total lipid in dried samples of early stage eggs or in later stages in dried yolk sacs and embryos was determined uiing the diphasic extraction procedure of Bligh & Dyer (1959) modified for small samples. Dried samples were ground in a mortar and pestle and a known weight transferred to a 15 ml conical centrifuge tube to which 0.8 ml IN I\;aCI was added. mixed and left to stand overnight at 4 C. One ml chloroform and 2ml methanol (AR grade) were then added and mixed. Another 1 ml chloroform was added, mixed, followed by 1 ml distilled water and the mixing repeated. The resulting emulsion was then centrifuged at 4 C for IOmin after which it separated into an upper aqueous layer and a lower one containing lipids with a thick white layer. presumed to be mainly protein, at the interface. The top layer was removed and discarded and the interface was displaced gently so that the bottom layer could be removed and transferred to a pre-weighed small g&s drying vessel. The chloroform was then evaporated in a desiccator under vacuum and the weight of lipid determmed.
E.dimution of the uyr 0f embrps In captured adult females follicles developed approximately synchronously and, during the breeding season, at any one time. pregnant females carried embryos at approximately the same stage of gestation. Therefore the age of embryos was estimated by taking Day 1 as the day on which I first found females which had ovulated, assuming that fertilization took place at ovulation. RESULTS
Changes in composition of developing follicles Follicles ranged from very early stages where yolk deposition was just beginning (wet wt approx. 13 mg)
511
to fully developed follicles (wet wt approx. 380mg) near ovulation representing a period of approximately three weeks. Changes in all measured variables are shown in Fig. 1. Dry weight of the follicles increased significantly during this period and the rate of deposition of solids was constant. The water content of the follicles (wet wt-dry wt) also increased constantly and there was a significant and linear increase in organic weight (dry wt-ash wt) parallel with the increase in dry weight. The ash content of the follicle also increased linearly with growth of the whole follicle. Relatively, there was a decrease in the proportion of water in the follicle while the proportion of dry matter, organic matter and ash increased. Changes follicles
in lirer weight qf ftimales
with developing
The ratio of liver weight to body weight (corrected for weight of follicles) decreased with growth of the follicles (Fig. 2). Gross changes in the whole egg und egg components 1. The whole egg. Changes in the composition of the whole egg, which includes the embryo, yolk, extraembryonic fluids and membranes are shown in Fig. 3. Total wet weight increased approximately 4-fold during gestation. The pattern of growth was triphasic with the most rapid increase in weight in the middle of the gestation period. The increase in total wet weight is due mainly to uptake of water. The total water content increased in the same manner as the total wet weight, however, there was a significant decrease in total dry weight. Organic weight also decreased significantly and the amount by which it declined was greater than the decline in dry weight. This can be accounted for by the fact that the total ash weight increased by approximately 4 mg. Relatively, the proportion of water in the eggs changed more than any other measured variable. 2. The embryo and yolk sac’. Growth of the embryo proceeds at the expense of the yolk sac (Fig. 4). The wet weight of the embryo increased sigmoidally,
45-
30 0
100
I 200
1 300
400
Follicle wet weight, mg Fig. 2. Change in liver weight of S. quoyii females as a function of stage of development of the follicles.
JANE THOMPSC>N
512
. . .
.
l
* .
t .
.
.
350
.
l l
l
l
l
.
400-
00 I
,
20
1
40 , Estimated
60 age,
,
80
days
0
Solids
0
1 20
I 40
Estimated
60
4 OnpofllC So
age, days
Fig. 3. Changes in wet weight (a). dry weight (b), water content (c), organic weight (d) and ash weight (e) of whole eggs (embryo, yolk sac plus extra-embryonic fluids and membranes) of S. yuoyii with age of the embryo. The data are combined in Fig. 3 (f) for comparison. whereas
the weight of the yolk sac increased to about the time of n~id-gestation and then declined steadily. The wet weight of the embryo at term was greater than the initial wet weight of the yolk. However, the dry weight of the embryo did not exceed the initial dry weight of the yolk. Thus the gain in weight of the
embryo can be attributed, in part, to addition of water as can the increase in total wet weight of the yolk at mid-gestation. The change in organic matter in the yolk and embryo followed a similar pattern to the dry weight changes. However, there was an increase in the ash
.
Nutrients for lizard embryos
3X3-
.(d)
i
S
250-
200-
150-
o-
I
(e) . 30-
. 25-
.
0
. i
20! 15-
IO-
5-
OO
Estimated age, days
Estimated age, days Fig. 4. Changes in the wet weight (a), dry weight (b), water content (c), organic weight (d) and ash weight (e) of the yolk (a) and embryo (0) of S. quoyiiduring gestation.
content of the embryo above that initially in the yolk. The gain is approximately equal to the gain observed in the whole egg (Fig. 3). Protein and lipid composition of the embryo and yolk SUC
The amount of protein in the embryo at term was
similar to that initially in the yolk while the lipid content of the full term embryo was significantly less (Fig. 5). There was a decrease in the total amount of lipid in the yolk and embryo combined, the most ranid rate of decline being in the last third of Restatidn while the total proteyn content of the cornLined embryo and yolk sac decreased only slightly.
JANE THOMPSON
514
f50
IKJ-
90
60-
30-
0
eo”
4
(
60
40-
20 -
0;
.
.
150 7
.
Promn
‘
l
8 90
0 6Q-F
5x3 -i 0
[cl
I 20
I
40 Estimtrted age,
I
60 days
I
80
1
Fig. 5. Changes in the protein (a) and lipid content (b) of the yoIk (e) and embryo (0) of S. quoyii with age of the embryo. Fig. 5 (c) shows the total protein (II) and lipid (0) in the whole egg (yolk and embryo) as a function of age of the embryo.
Nutrients for lizard embryos
515
3
Days
since ovubtion
Fig. 6. Changes in the liver weight of pregnant females of S. quo~ii during gestation.
Chortges in iiwr
wright
qf j~mu1e.s during gestation
There was no significant change in the ratio of liver wsight to total body weight during gestation (Fig. 6). DlSCL’SSIOiV
Yolk substances are laid down in the ovarian follicle prior to ovulation. The pattern of growth of the ovarian follicles in S. quovii differs from that in the Ribbon Snake, T~*arnn~~h~.~ sauritus (Dessaur 8~ Fox, 1959). The measurements on folficies from s. 4~0~~~ correspond to the second phase described by Dessaur & Fox; no measurements were made on the very early white translucent follicles in my study. In S. quoyii, there was a steady decrease in percentage water in the follicles as solids accumulate whereas in Thrrmnopkis sauritus the percentage water increased rapidly at first and then at a lower rate during this phase of follicular growth. There was a significant decrease in the weight of the li\er in females with enlarging follicles. similar to that
observed in Thamnophis suuritus (Dessaur & Fox, 1959). Since S. yuqii has no obvious fat bodies which are a source of yolk material in some lizards and snakes (Dessaur & Fox, 1959: Hahn & Tinkle, 1965). the liver is probably the principal source of yolk substances in S. quoyii. Changes gestution
in chemicul
composition
“j, Lipid
the
rgy
dwiny
On a dry weight basis the egg of S. quoyii consists mainly of fat (2%;) and protein (SSq<>)at ovulation. These values are similar to those reported for other reptiles (Table I). Minerals account for 67% of the dry matter in the egg. Material unaccounted for may include carbohydrate although this is expected to be very low and probably less than 3”,, (Tomita, 1929; Fukuda, 1939). In addition, there may be some inaccuracies in measurements leading to less than lOO”/;, recovery. The most obvious change in the whole egg during gestation was an increase in the total amount and in the proportion of water. There are two possible
Table 1. Lipid and protein content of reptilian eggs Species
of
01,Protein
32 14.3 25.1
55.6
30
40
66
51
28 30
66 SO
39.2 36-43 36 30 28
48.3 50 55
Author Tomita (1929) Lynn & von Brand (1945) von Brand & Lynn (1947) Fukuda (1939) Clark & Sisken (1956) Clark et ul. (1955) Dessaur & Fox (1956) Hahn & Tinkle (1965) Ricklefs & Cullen (1973) Hadley & Christie (1974) Avery (1975)
JANE THOMPSON
516 Table 2. Water
content
of eggs of viviparous
squamates
‘
content at ovulation
51 54
84 81
40 40
80
sources of water: the maternal circulation and metabolic water. The amount by which water content increased is more than would be expected from the nletabolism of lipid. One mmoi of a typical fatty acid, paimitic acid {moi.wt 261) yields 288 mg Hz0 (Conn & Stumpf, 1973). The expected yield from 40mg of lipid would therefore be approximately 45 mg HZ0 which is much less than the measured increase of about 800 mg H,O. Thus, there is definite evidence in S. q~~~)~i~ for uptake of water from the mother. Similar increases in the water content of developing eggs have been reported in both oviparous and viviparous reptiles (Packard t’t al., 1977). Increase in water content of eggs of viviparous species are compared in Table 2. There is some disagreement in the literature regarding the adaptive significance of water absorption and storage in reptilian eggs. In egg laying species, accumulated water may act merely as a buffer against the dangers of potential desiccation (Fitch & Fitch, 1967; Badham, 1971: Bustard, 1971a,b) and water uptake and storage may not be essential for the normal development of the embryo (Bustard, 1966, 1971a; Dmi’el, 1967). However, in Anolis mralinmsis, absorption and storage of water is important for the deveioping embryo, since hatchiings are smaller when hatched from eggs deprived of water (Gordon, 1960) so water absorption may be a critical factor in the normal development of these and other reptilian eggs (Packard et ui.. 1977). The hatchiings of some oviparous lizards, snakes and turtles weigh more than freshly laid eggs (Cunningham & Hucne. 1938; Brown, 1956; Mendelssohn, 1963: Bustard, 1965) which is possible only if part of the water absorbed is incorporated into the embryo. On the other hand. hatchlings of some species weigh less than freshly laid eggs (Lynn & von Brand, 1945; Bustard. 1967; Dmi’ei, 1967 ; Mendelssohn, 1963, 1965; Badham 1971). However, as Packard et ul. (1977) point out, it cannot be concluded that water absorbed by these eggs is not incorporated into forming tissue without taking into account both the oxidation of organic material by metabolism of the embryo. and the mass of the egg shell and other extraembryonic membranes. My observations in S. quoyii support the conclusion of Packard et ul. (1977) that water absorbed is incorporated into the developing embryo and is presumably essential in embryonic development. Dry wright unrl orgunic weight There was no increase
Final “;, Hz0
in either dry weight or or-
‘)
Author
Shine (1977) Clark & Sisken (1956) Hadley
& Christie
(1974)
ganic weight of the egg of S. yuoyii which, if present, would suggest uptake of nutrients from the mother. In fact, total dry weight decreased by about loo/ and organic weight by about 13”,;,. In egg-laying species, where the only sources of energy available are in the yolk, reported changes in dry weight are variable ranging from 14.5% (Cunningham & Hurtwitz, 1936) to 620,; (Rickiefs & Cullen, 1973). Reported changes for viviparous species, where there may be external sources of energy via the maternal circulation, are also variable. Clark et al. (1955) reported that in the viviparous snake Thamnophis &a/is, dry weight decreased by 187,: whilst organic weight decreased by 2Opb. However, in the same species, Clark & Sisken (1956) found no significant change in dry weight during development and since ash weight did not change signifi~ntiy there could be no change in organic weight either. In two other species of viviparous snakes, Natechis scututus and Psrudrchis parphyriacus, Shine (1977) found decreases in dry weight of 34 and 1% respectively. The organic weight in Psrudechis pffr~~~~~,riucus decreased by about 6%. Discussing the only information, to my knowledge, for a viviparous lizard, Avery (1975) states that the total dry weight of eggs in a clutch of Lticertu ciripara remains constant after transfer to the oviducts. However. there are no data on ash content of the eggs so the change in organic weight cannot be calculated. Hogarth (1976) estimates that during the development of embryos of viviparous fish, a decrease in organic weight of less than 20% would suggest some uptake of material from the mother. In the absence of any estimate for reptiles, this estimate can be taken as a guide. in S. quoyii, where organic weight decreased by 13x, additional sources of energy may be supplied by the mother. To determine whether this is true, I have drawn on two independent sources of information. First, the change in liver weight of females during gestation and, secondly, the predicted decline in organic weight which can be estimated knowing the principal sources of energy for development and the estimated metabolic rate of the embryos. In S. yuoq’ii, there was no significant change in the liver weight of females during pregnancy. This suggests that there is no significant depletion of the metabolic reserves of the female which might occur if nutrients were being supplied to the developing embryos (Hogarth, 1976). The principal organic constituents of the egg in S. quoyii are lipid and protein. Carbohydrate is probably a relatively insignificant source of energy. In S. yuoyii lipid content of combined yolk and embryo decreased
Nutrients for lizard embryos 20'C
20 6 9 160 F LI 1.2E Y gQ8-
z
04/J 0. 40
l
.
l* . 50
60
70
,,I 80
90
100
Age, days
30’C
6
.
Age,
days
Fig. 7. Estimated oxygen consumption (see Discussion for explanation) of S. quoyii embryos at 20-C and 30 ‘C as a function of age of the embryo.
by 56% while the protein content decreased only shghtly, showing that embryos derive energy principally from the combustion of fat. To determine theoretically whether the amounts of lipid metabolized would be sufficient for the embryo, an estimate of the oxygen consumption is required. A few metabolic measurements have been made on embryonic snakes (Zarrow & Pomerat, 1937; Clark, 1952, 1953a,b; Dmi’el, 1970) and turtles (Lynn & von Brand, 1945), but there are no data available for any embryonic lizards. Metabolic rates of reptiles vary with temperature (Bennett & Dawson, 1976) and there may be differences in metabolic rates of embryos at different stages of deveopment. These latter differences may be a reflection only of the increase in weight of the embryo (Dmi’el, 1969). In addition there may be daily fluctuations in metabolic rates in embryos (Dmi’el, 1969) as well as differences between species (Dmi’el, 1970; Lynn & von Brand, 1945). As an approximation, I have taken Bennett and Dawson’s (1976) equations for oxygen consumption of adult lizards at 20” and 3O’C which is within the range of temperatures to which embryos of S. quoyii are exposed (Veron & Heatwole, 1970). Their equations are valid for lizards weighing down to 1 gm, approx. the weight of hatchling S. quoyii. The calculation doesnot include the contribution ofextra-embryonit membranes or of very early stage embryos to the total rate of metabolism both of which are likely to be small (Lynn & von Brand, 1945; Clark, 1953~).
517
Predicted oxygen consumption of S. quo!,ii embryos of known weights and ages are plotted in Fig. 7 and the total oxygen consumption estimated at each temperature by calculating the area under each curve. At 30°C an estimated 175 ml O2 are consumed and at 2O”C, 57 ml. Assuming the metabolism of 1 mg fat uses 2.03 ml 0, (Prosser & Brown, 1961) and that all oxygen consumed is used for metabolism of lipid, at 3O’C the amount of fat consumed would be 84mg and, at 2O”C, 30mg. The actual amount of lipid consumed was between 35 and 40 mg which is within the range predicted. Assuming the estimate of oxygen consumption is reasonable, the metabolism of this quantity of lipid would therefore be adequate for the energetic requirements of the embryos. Therefore there is no need to postulate additional sources of energy such as protein. The small observed decline in total protein in the whole egg can be attributed to the fact that relatively little protein is metabolised rather than being the resultant of protein metabolised plus protein gained (as amino acids) from the mother. Despite the fact that the placenta in S. quoyii is permeable to amino acids (Thompson, 1977) the metabolic calculations above suggest that uptake of protein precursors may not be essential, at least for the energetic requirements of the embryo. This conclusion differs from that of Clark et ul. (1955) and Clark & Sisken (1956) who concluded there must be transfer of amino acids from the mother to the embryo in Thamnophis .sirtulis. However, in this species, they found a significant increase in protein content of the egg during development which was not found in S. quoyii. In summary, the results reported here suggest that there is sufficient protein and lipid in the yolk of S. quoyii for embryonic development. There is no evidence to suggest a significant gain of organic nutrients from the mother; however, there is definite evidence for uptake of water and inorganic ions from the maternal circulation. A detailed examination of mineral uptake will be reported elsewhere (Thompson, 1981). Acknowlrdyemmfs--I thank Drs Gordon Grigg, Richard Shine and Gillian Courtice and Associate Professor John Simons for their interest and advice during the project. The work was supported by a Commonwealth Postgraduate Research Scholariship.
REFERENCES AVERY R. A. (1975) Clutch
size and reproductive effort in the lizard L~crrtu ricipurtr (Jacquin). O~o/oc/iu 19 165-170. BADHAM J. A. (1971) Albumen formation in eggs of the agamid Amphibolurus hurbtms htrrhutu.s. Copricr 1971, 5433545. BAUCHOT, R. (1965) La placentation chez les reptiles. A~ln/.s Biol. 4, 547-574. BELLAIRS A. d’.&. (1970) The Lij> of’Rrptrlr.s. Weidenfeld & Nicolson, London. BELLAIRS R., GRIFF~THS I. & BELLAIRSA. d’A. (1955) Placentation in the adder Viperu hrrus. Nuturr 176, 657-658. BENNETT A. J. & DAWSON W. R. (1976) Metabolism. In Biology of‘ thr Reptilia. Vol. 5. (Edited by CANS. C.) pp. 127-223. Academic Press, New York. BLIGH E. G. & DYER W. J. (1959) A rapid method for total lipid extraction and purification. Can. J. Bioc,hrm. Phydol. 37, 91 l-917.
518
JANE TI
BRAND VON J. & LYNN W. G. (1947) Chemical changes in the turtle embryo. Proc. Sot. c\-p. Biol. Med. 64, 6lm 62. BRANSON B. A., 6i BAKER E. C. (1974) An ecological study of the Queen Snake, Rrqinu .srptemrittuttr (Say), in Kentuckv. T1~/~7neStud. Zoo/. Bat. 18, 153-171. BROW< E. E. (1956) Nests and young of the six-lined racerunner. Cnrmidopl2oru.s sc~.~linrtrtw~ (Linnaeus). J. E/i&r Mitc+rl/ scirnt. Sec. 72, 30 40. BUSTARD H. R. (1965) Observations on Australian geckos. flcrpctolnyicu 21, 294~302. BUSTARU H. R. (1966) Notes on the eggs. incubation and young of the bearded dragon. Anlphiholuruv hurhutus htrrhotus (Cuvier). Br. J. Hvrpet. 3, 252 259. BIISTARII H. R. (1967) Reproduction in the Australian gekkonid genus Oedwtr (Gray 1X42). Herputologicu 23, 27&2X4. BI;SI-ARDH. R. (197la) Temperature and water tolerances of incubating sea turtle eggs. Br. J. Herpet. 4, 196-198. B~ISTARDH. R. (197lb) Temperature and water tolerances of incubating crocodile eggs. Br. J. Hrrptr. 4. 198-200. CI.AKK H. (1952) Data on respiration of snake embryos. Anut. Rrc,. 113, 549. CLARK H. (1953a) Eggs. egg-laying and incubation of the snake Eluphe rwwr~i (Baird and Girard). Copriu 1953, YO 92. CLARK H. (19S3b) Metabolism of the black snake embryo. I. Nitrogen excretion. J. rlup. Biol. 30, 492-501. CI ARK H. (19.53~) Metabolism of the black snake embryo. II. Respiratory exchange. J. e.xp. Biol. 30, 502-505. CI.ARK H.. FIORIO B. & HURO~I~-z R. (1955) Embryonic growth of Thtrumophis s. sirtrrlis In relation to fertilization date and placental function. Cop&r 1955, 9-13. CI,ARK H. & SISKW B. F. (1956) Nitrogenous excretion by embryos of the viviparous snake Tlwtmophis s. \Irtu/is (L.). J. rzp. Eiol. 33, 384 393. CLARK H.. SISKEN B. & SHANNON J. E. (1957) Excretion of nitrogen by the alligator embryo. J. cc//. camp. Ph,nio/. 50, Con
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