Lipid-reduced evaporative water loss in two arboreal hylid frogs

Camp.

Pergamon 0300-%29(94)00213-4

Lipid-reduced hylid frogs

Biocbem.

Ph_wioi.

Vol. I I I A, No. 2, pp. 283-291. 1995 Elsevier Science Ltd Printed in Great Britain 0300-96?9/95 $9.50 + 0.00

evaporative water loss in two arboreal

Andrew P. Amey and Gordon C. Grigg Department

of Zoology, The University of Queensland, Qld 4072, Australia

Rates of evaporative water loss were measured in seven species of Australian frogs from different habitats and compared with agar replicas. The two arboreal species, Litoria fallax and Litoria peroni, showed a marked resistance to water loss, and each has a cutaneous lipid layer which appears to provide the waterproofing mechanism. Rates of water loss were compared between Litoria peroni and one of the other species, Limnodynastesfletcheri, at 20, 25, 35 and 40°C. Even at the highest temperatures, Litoria peroni maintained its resistance to water loss, implying no attempt at thermoregulation by evaporative cooling. Key words: Anura; Litoriu; Skin morphology;

Lipids; Cutaneous water loss; Thermoregulation;

Arboreal frogs; Water loss resistance. Comp. Biochem. Physiol. I I IA, 283-291,

1995.

Introduction 1984) Hyperolius (Geise and Linsenmair, 1986; Kobelt and Linsenmair, 1986; Withers et al., 1982a,b) and Phyllomedusa (McClanahan et al., 1978; Shoemaker et al., 1972; Shoemaker and McClanahan, 1975), the rate of water loss is very low, equivalent to that of desert reptiles. These species have been termed the “waterproof” frogs (Withers et al., 1984). In contrast to other frogs, they do not actively avoid the hot, dry conditions of their habitats. Consequently, there is considerable potential for overheating, which appears to be offset either by an increase in the rate of cutaneous water loss at higher temperatures, by ‘sweating’ from mucous glands (Shoemaker et al., 1987) or as a consequence of the ‘melting’ of waterproofing skin lipids (Blaylock et al., 1976), or reflectance of heat by guanine rich iridophores (Kobelt and Linsenmair, 1986; Schmuck et al., 1988; Schmuck & Linsenmair, 1988). Despite the interesting implications of ‘waterproofing’, relatively little work has been done on the mechanism/s. Phyllomedusa sauvageii (Shoemaker et al., 1987) and Litoria caerulea (Christian et al., 1988) have been reported to wipe the secretions of specialized lipid glands over their bodies to retard water loss, but no other species has had its mechanism so fully

Most frogs lose water from their skin at the same rate as water is lost from a free water surface (Rey, 1937). Despite this, frogs live in a surprising diversity of terrestrial habitats, including deserts (Duellmen and Trueb, 1986). There have been many studies of the ways in which frogs are able to survive quite inhospitable environments (for reviews, see Deyrup, 1964; Bentley, 1966; Heatwole, 1984; Toledo and Jared, 1993). Most frogs have not developed a resistance to cutaneous water loss (Bentley, 1966; Shoemaker and Nagy, 1977; Toledo and Jared, 1993), probably because of the importance of a moist skin in respiration (Chew, 1961). There are, however, important exceptions. Many authors have identified species of Anura in which water loss across the skin is less than that from a free water surface (see review by Toledo and Jared, 1993). In nearly every case, the species is arboreal. In some xeric, arboreal species, such as those of the genera Chiromantis (Drewes et al., 1977; Withers et al., Correspondence 10: A. P. Amey, Dept. of Anatomical Sciences, The University of Queensland, Qld 4072. Australia. Received 16 August 1994; revised 16 November 1994; accepted 24 November 1994. 283

284

A. P. Amey and G. C. Grigg

elucidated. Suggestions have included a mucopolysaccharide skin layer reportedly found in terrestrial Anura only (Elkan, 1976) specialized guanine-rich chromatophores found in species of Chiromantis and Hyperolius (Kobelt and Linsenmair, 1986) and intercellular epidermal lipids (Withers et al., 1984). Bearing in mind the arid nature of the Australian continent and the diversity of its frogs, including many arboreal species (Barker and Grigg, 1977; Cogger, 1992) a study of Australian frogs seemed likely to provide further examples of ‘waterproofing’ and opportunities for further investigation of the phenomenon and its mechanism/s. Accordingly, rates of water loss were measured in a number of species from a diversity of habitats to compare living individuals of these species with their agar replicas. The replicas lost water at the same rate as a free water surface and therefore had rates of water loss which would be expected from a frog whose skin has no resistance to evaporative water loss. This was with a view to correlating the different habitats/lifestyles of the species of frogs with any cutaneous resistance to water loss. The effect of temperature on the resistance to water loss, and therefore evaporation rate, was tested and differences between the skin structure of resistant and non-resistant species were compared histologically.

Material and Methods Animals Adults of seven species were studied, as follows. The habitat descriptions are from Cogger (1992). Limnodynastes fletcheri Aquatic species: (n = 5) mean body mass _+ S.E. 4.8 _+ 0.9g, always found in water or sheltering in moist places such as under logs, yabbie (Decapoda) burrows, etc. Litoria latopalmata Terrestrial species: (n = 7) 3.4 + 0.2g, often found foraging in forests and floodplains well away from water, although it returns to breed. Uperokia fusca (n = lo), I. 1 +_ 0.1 g, a burrowing species, foraging in forests and breeding in water-filled grassland depressions. 1.1 + 0.1 g, Uperoleia rugosa (n = 10). another burrower, found in dry sclerophyll forests and breeding in flooded grasslands. Cyclorana novaehollandiae (n = 4), 34.8 + 8.9 g, primarily an arid zone species which aestivates underground probably in a cocoon like its close relative Cyclorana platycephaia (van Beurden, 1977) and emerging to breed in temporarily inundated areas. Arboreal species: Litoria fallax (n = lo), 0.9 + 0.1 g, found in reeds and other vegetation

around creeks and dams as well as away from water. Litoria peroni (n = I5), 9.3 _+ 1.3 g, often found long distances from water in a wide variety of habitats, breeding in low-lying, inundated areas. All individuals were collected from various sites around Queensland during the breeding season (February-April, 1992) and were of adult size. Since they were caught by tracking calls, most are male. Some were located by sight, and their sex is unknown (C. novaehollandiae, Limnodynastes ,fletcheri and some Litoria fallax ). Animals were kept in terraria with open water available at 25°C and fed ad lihitum on cockroaches and mealworms. Measurement

of rates of cutaneous

water loss

The method used is similar to that described by Buttemer (1990), in which a test subject’s weight loss in a dry air stream is monitored continuously. Each subject was first placed in water at the test temperature for half an hour to ensure full hydration. It was then towelled lightly and placed in a small plexiglass chamber which was open at both ends and surrounded by a larger chamber through which the air flowed. The inner chamber stood free on an electronic balance reading out directly to a computer. A slow (0.92 set/cm) but constant stream of dry air (< 100 ppm water) flowed through the outer chamber. supplied from a gas cylinder (Commonwealth Industrial Gases). The entire set-up was placed in a room temperature controlled to & 1°C. Isothermal conditions within the chamber were maintained by a space within the walls of the outer chamber through which water circulated from a large bath, heated independently in the same room. Temperatures were monitored in the chamber and in the water bath by LM35 monolithic temperature sensors, and the humidity in the air downstream of the frog was monitored using an RH-CAP humidity sensor (+ 1% R,H,). The average weight of the frog was read automatically every minute, using the AD-DATA program. The rate of water loss (mg/hr) was taken as the slope of the regression line of weight over time, as calculated by the Lotus l-2-3 program, divided by the surface area (cm?. for calculation of surface area, see later). Trials were of 2 hr duration. The frogs were not anaesthetized or restrained. Because movement, as in attempts to escape, would affect rates of water loss (see Heatwole et a/., 1969), a record of behaviour was made every half hour during a trial. We wished to keep disturbance of the animal down to a minimum and

Lipid-reduced

water

rigorous quantitative behavioural data was not desired, only an impression of overall response, so continuous observation was unnecessary. The amount of weight loss was also monitored during these checks, and trials were terminated whenever weight loss exceeded 30%. This occurred only in some of the smaller species (U. fusca, U. rugosa and a single L. fallax which was excluded from the analysis.) Tests commenced a few weeks after capture and continued for some months. Initially, each individual of each species was tested at 30°C. Subsequently, L. peroni and Limnodynastes fletcheri were tested at 20, 25, 35 and 40°C also. L. jetcheri was selected as the best comparison with Litoria peroni because of similarity in size and its fully aquatic habitat. It was thought to be the least likely to demonstrate any adaptation to limit cutaneous water loss. The order of species and temperatures was randomized. Agar replicas

To compare rates of evoporative water loss of the living frogs with that of a free water surface, agar replicas of the animals were created as per the methods of Spotila and Berman (1976). A frog was first anaesthetized with MS-222 (Sigma A5040) and then embedded in Kromapan alginate (American Dental Distributors), a non-toxic dental casting glue. Within minutes, the glue had set and the frog could be removed safely, rinsed with water and allowed to recover. A 3% agar solution was then poured into the mould to produce a replica of the living frog. Tests of 12.57 cm* lids filled with either water of agar demonstrated that there was no significant difference between the rate of evaporative water loss of an agar surface and a free water surface. When more than one replica was required, more permanent latex moulds were produced by pouring Plaster of Paris into the dental mould, allowing it to set and then immersing it in latex. Comparison of the masses of replicas produced from the Kromapan moulds and the latex moulds indicated that there was little difference between them. The agar replicas were tested in the same way as their living counterparts, except that the trials continued for only 1.5 hr. Su+ce

area

To obtain a measure of evaporative water loss per unit of surface area, an equation formulated by McClanahan and Baldwin (1969) to derive surface area from the body mass of an anuran was used. as modified by Withers et al. (1982a) to give the actual surface area exposed to the air by an anuran in a water-conserving posture by

285

loss in frogs

multiplying Surface

by two-thirds: area = 2/3(9.9(body

mass)“.56)

= 6.6(body

mass)“.56,

(1)

where body mass is in g, and surface area is in cm’. Regression exponents calculated for each species from the relationship between body mass and evaporative water loss (gHZO/hr) were usually not significant and varied greatly between species. The general equation was therefore the best compromise. Calculation

qf resistance

to water loss

Towards the end of the experiments, an Omega 8 17A electronic thermocouple became available. This was used to measure the body temperatures of frogs and replicas directly by cloaca1 insertion before and after trials. This allowed the resistance to water loss to be calculated, according to the equation: r=

(sd, - rh sd,) E

’

(2)

where sd, = the saturation vapour density of water at the skin temperature (g/cm3), sd, = the saturation vapour density of water at the ambient temperature (also g/cm3), E = evaporative water loss in g/cm*/sec, rh = relative humidity (fraction of saturation) and r = resistance in set/cm The total resistance of a surface calculated in this way is made up of two factors, boundary layer resistance and internal resistance. Boundary layer resistance is the resistance generated by the still boundary layer of air surrounding an object. Water must diffuse through this layer to reach the moving air which carries it away from the object. The thickness of the layer, and therefore the diffusion time, are determined by the velocity of the air relative to the object. Internal resistance is dependent on the waterretaining capacity of the surface. Free water (and agar) has no capacity to limit the evaporation of water from its surface. The resistance of the agar replicas should therefore be due entirely to the boundary layer. Since the boundary layer will be the same for the replicas and the live frogs (same physical conditions, same aerodynamic properties), the internal resistance of the frog can be determined by subtracting the resistance of its replica from its total resistance i.e. r, = r - rb,

(3)

where r, = internal resistance of the frog, r = total resistance of the frog and r,, = total resistance of the agar replica.

286

A. P. Amey and G. C. Grigg

U.fUS.

U.rug.

L.fal.

L.lat.

L.fle.

1

Live

q

Agar

L.per.

C.nov.

Species Fig. I. Rates of evaporative

water loss in seven species of Australian frogs and their agar replicas at 30 C Species are ranked in order of mean body mass.

These calculations require complete data from both the agar replica and the frog. This was available for only the following: all L. Jletcheri at 25°C. all Litoria peroni at 25“C, 40°C and some at 30°C. It was assumed that skin temperature was not significantly different to core temperature (Wygoda, 1984). Treatment

with lipid solvents

Five L. peroni were swabbed on a small area of their backs with cotton wads soaked in acetone immediately before being tested exactly as described previously. Five others were swabbed in a similar fashion with a 2: 1 mixture of chloroform : methanol. These treatments produced no apparent discomfort or permanent injury and the frogs made no attempts to rid themselves of any irritation. Five other individuals were not swabbed as controls. Histolog? One individual of each species was killed with an overdose of Nembutal. 1 cm’ samples of mid-dorsal and mid-ventral skin were taken immediately and either embedded in wax or cut in the cryostat. Wax sections, cut at 8 pm, were stained as follows: haematoxylm and eosin for general morphology, von Kossa’s and Alizarin Red to show the presence of calcium, Lillie’s allochrome and Hale’s colloidal iron to demonstrate mucopolysaccharides. Staining for lipids required preparation of sections using a cryostat. Individuals of the following species only were sampled: Cj~clorana novaehollandiae, Limnodynastesfletcheri, Litoria fa1la.q L. latopalmata and L. peroni. Fixation was by Milloniq’s solution and the tissue was embedded in gelatine. Sixteen-micrometre sections were cut with a Wild Leitz freezing microtome and stained with Sudan III.

Detailed histological in Luna (1968).

methods

may be found

Results Rates of water loss The two arboreal species Litoria fallax and Litoria peroni lost water significantly slower than their agar replicas (Fig. 1; P < 0.01, Student’s t-test). Of the other species, all lost water significantly faster than their replicas (P < 0.01). Losses greater than that of a free water surface, or an agar replica, may be explained by an individual being active and exposing a greater surface area for water loss. Behavioural data (Table 1) suggest that L. peroni, L. fallax and Cj~clorana novaehollandiae moved little during the trials, and generally assumed the water conserving position, described in other species, in which a frog sits with its ventral surface and throat pressed tightly to the surface, its limbs Table

I. Average percentage

of observations species was in each position

in which each

Species

%S

%T

%E

%P

C’. notuehollandiae L. fletcheri L. fa1lu.Y L. Inlopulnlutu L. peroni U. .fu.rcu u. rldgo.sa

0 50 14 17 3 3 20

0 50 IO 4 0 32 12

0 0 0 19 0 57 33

88 0 52 14 24 0 17

% W %M 12 0 ‘4 46 73 8 18

33 60 36 64 15 4.5 58

S = sitting in typical anuran position. head up above floor. T = standing, body supported by limbs above floor, e.g. forelimbs against side, or all limbs spread out from body. E = escaping, frog moving about, trying to escape chamber. P = parallel water conserving posture, facing into air flow or away from it. W = water conserving posture side on to air flow. %A4 = number of changes of position compared with total possible. e.g. if four observations made, frog changed position twice = 66% (two changes out of possible three).

Lipid-reduced

10

water

Temperature, l

Fig. 2. Rates

L. peroni, live L. peroni, agar

of evaporative

50

40

30

20

q

287

loss in frogs

“C

y = - 2.8127 + 0.32717x R’2 = 0.878 y= - 8.4340 + 0.65220~ IV2 = 0.996

water loss in ten Liroria peroni and their agar temperatures (mean and standard errors).

drawn into the body and the eyes closed (Campbell and Davis, 1971; Heatwole et al., 1969; Ralin, 1981). In contrast, Limnodynastesfletcheri never assumed the water conserving posture, moved about a great deal and had rates of cutaneous water loss much higher than its replicas. The other species (Litoria Iatopalmata, Uperoleia fusca and Uperoleia rugosa), were intermediate in their behaviour, sometimes assuming the water conserving position and sometimes moving around. Water loss at d@erent temperatures Rates of water loss were compared over a range of temperatures between L. peroni, a species with a resistance to water loss, and Limnodynastes fletcheri which loses water at least as fast as a free water surface. This difference persisted at all of the temperatures tested

replicas

over a range

of

(Figs 2 and 3), and there was no sign of any tendency towards increased rates of evaporative water loss by Litoria peroni at high temperatures. Measurements of core temperature showed that both species are able to tolerate surprisingly high body temperatures. L. peroni, when measured at the end of a trial at 40°C averaged a cloaca1 temperature of 38.1 “C. Limnodynastes jletcheri was not measured at this temperature, but, following the pattern of other temperatures, it would have been only a few degrees lower, presumably because of its higher rate of evaporative water loss. Neither species suffered any visible ill effects after 2 hr at this temperature. Calculated resistance values (mean + SE) were 1.46 + 0.27 secjcm in L. fletcheri at 25°C and, in Litoria peroni, 8.72 + 1.O secjcm at 25”C,

T

Temperature, 0 l

L.fletclW~ L.f. agar

Fig. 3. Rates of evaporative

(live)

y = - 4.7720 - 9.5440

Y=

“C + 0.91240x + 0.03090x

water loss in five LimnocrIvnasfesJlercheri of temperatures (means and standard

W2 = 0.491 R”2 = 0.934

and their agar replicas errors).

over a range

288

A. P. Amey and G. C. Grigg

8.15 + 2.86 secjcm at 30°C and 9.71 f 0.61 set/ cm at 40°C. There was no significant effect of temperature on the resistance of L. peroni over the range of measurement. Treatment with lipid solvents Swabbing with chloroform, but not acetone, increased evaporative water loss in L. peroni. There was no difference between the evaporative water loss of control L. peroni and those swabbed with acetone (mean rate +_ SE control = 6.6 f 0.7 g/cm’/hr, n = 5, cf. 5.9 + 0.6 g/cm’/ hr, n = 5, acetone treatment, P = 0.494). The average rate for the individuals swabbed with chloroform, however, was 8.6 +_0.2 mg/cm’/hr (n = 5), significantly higher than the control group (P = 0.038). The controls in this set of tests were not significantly different from the 10 L. peroni tested previously at 30°C (0.2 > P > 0.1). Histology General morphology. Staining with haematoxylin and eosin revealed no immediately apparent differences between the two water loss resistant species and the others, all showing a structure typical of frog skin (Duellmen and Trueb, 1986). In all, the topmost layer was the stratum corneum, a thin layer of keratinized cells attached only loosely to the epithelium. Below the epithelium, the dermis consists of the stratum spongiosum, the layer containing the mucous and poison glands, nerves, smooth muscle and chromatophores. Below this, the stratum compacturn is composed of thick collagen fibres. Finally, the thin tela subcutanea formed the outer lining of the subcutaneous space. On closer inspection, it was noted that the t. subcutanea in the dorsal skin of L. peroni was often separated from the s. compactum, being connected only by thin membranes. In L. fallax. the dorsal skin was often split between the s. spongiosum and the epithelium during preparation. The significance of these observations will become clear below. Calcium lmucopolysaccharide layer. All species, whether ‘waterproof’ or not, were found to possess a well-developed calcium/mucopolysaccharide layer. Von Kossa’s and Alizarin stains clearly showed a calcium layer in the dorsal s. spongiosum below the other structures of this layer, adjacent to the s. compactum. It was thickest dorsally in all species, usually being discontinuous and patchy ventrally, though it was entirely absent ventrally in both C. novaehollandiae and L. fallax. Hale’s colloidal iron technique showed this layer to be composed of mucopolysaccharides also in all

species, but Lillie’s allochrome method did not stain this layer well. Lipid staining. The skin of C. noraehollandiae and Limnodynastes jetcheri both stained negatively for lipids. Litoria latopalmata had a few, very isolated lipid droplets located in the t. subcutanea. The two arboreal and water resistant species, L. peroni and L. fallax, were found to have continuous layers of lipids in the dorsal skin, but in different regions (Fig. 4). In L. peroni, there was a relatively thick layer (I 9% of total skin thickness) in the dorsal t. subcutanea, explaining the enlarged appearance of this layer when mounted in wax (see above). In L. fallax, there is an indistinct lipid layer in the topmost region of the dorsal s. spongiosum, just below the epithelium, making up approximately 9% of the skin thickness. It was often obscured by the thick chromatophore layer, making exact measurement difficult. Its position explains the separation between layers seen in wax mounted specimens, where the lipids have been washed away during preparation (see above). Histological sections of skin samples treated with acetone and chloroform yielded interesting results. The lipid layer of the specimen treated with acetone appeared to have ‘congealed’ into discrete droplets. However, these droplets were still in contact and formed a more or less continuous layer. The skin treated with chloroform, however. was quite different. Its lipid layer was greatly disturbed, consisting of a few remnant droplets, discrete and isolated in a manner quite reminiscent of the skin of L. latopalmata.

Discussion Of all the species in this study, the two arboreal species, although occurring in mesic habitats and frequently around water, would be most likely to encounter potentially desiccating conditions. The others, though representing a range of habitats from xeric (Cyclorana nocuehollandiae), through mesic (Litoria latopalmata, Uperoleia fkca and Uperoleia rugosa) to aquatic (Limnodynastesfletcheri) are all ground dwelling and emerge from moist retreats only at night or during rain. C. novaehollandiue, like Cyclorana platycephala (van Beurden, 1977). probably secretes a waterproof, extra-cutaneous cocoon when aestivating. Arboreal species, in general, are exposed to a lesser availability of shelter and free water, and to stronger air movement (Yorio and Bentley, 1977). While an impermeable skin could be of use to nonarboreal species in certain situations, the advantages of a freely permeable skin (rapid rehydration and cutaneous respiration) are probably greater (Chew, 1961).

Lipid-reduced

water

loss in frogs

Stratum

Chromatophores /

compactum

(a

rela subcutanea

(b)

/

Stratum

s&ngiosum \ Epithelium

Chromatophores

/ Stratum Fig. 4. Dorsal

skin of Liroria

peroni

(a) and Litoria,/u/lus

Litoria peroni does not increase evaporative water loss in response to higher temperatures. This lack of response may be because it does not normally experience high temperatures. Unlike Chiromantis and Phyllomedusa, it is not exposed to the full desert sun during the day, but shelters under bark in mild conditions. The tolerance of all species to harsh conditions is noteworthy. All were maintained at 25’C for 9 months and tolerated an experimental temperature of 30°C for 2 hr without

compactum (b) stained

with Sudan

III

difficulty. L. peroni and Limnodynastes,fletcheri tolerated 40 ‘C for 2 hr without sign of injury. Northern temperate species do not survive such temperatures, even when acclimated to 30 C (Duellmen and Trueb, 1986). The critical thermal maximum (CTM), the point at which 50% of a group die, ranged from 31.3 to 4O.tYC in a range of northern species acclimated to 23 or 26°C in a study by Brattstrom (1968). Additionally. U. fisca and U. rugosa, routinely suffered water losses of 30% of their body weight without mortality.

290

A. P. Amey and G. C. Grigg

The frogs were kept under constant ‘summer’ conditions (warm and moist) throughout the study which continued for some months. There was no evidence of seasonal change in resistance to water loss over this time period, however, as the Litoriu peroni controls of the lipid solvents test, performed last, were not significantly different to the individuals of this species tested first in the species comparison trials. Although it is still possible that the water resistant species alter their resistance to water loss in response to the changing environment of the seasons, we contend that waterproofing is of benefit to arboreal frogs all year round. Only those species with a resistance to water loss possessed a cutaneous layer of lipids. They were otherwise indistinguishable in their skin morphology from the other species. In contrast, all species were well endowed with a calcium mucopolysaccharide layer. Thus, the physiological barrier used by the arboreal species is unlikely to be the calcium/mucopolysaccharide layer, as proposed by Elkan (1976). No function can be assigned to this layer, although we suggest it may provide structural support for the integument. Morphologically, the lipid layer in Litoria fallax would seem to be more effective as a barrier to water loss than that of L. peroni. It forms the top layer of the stratum spongiosum and thus covers the blood vessels of the skin. The lipid layer of L. peroni, however, is in the tela subcutanea and, presumably, functions only to protect the cutaneous lymphatic system. This notion is supported by the measured rates of evaporative water loss, for the difference between live L. fallax and their agar replicas is substantially greater than the difference between that of L. peroni and its replicas (Fig. 1). Wygoda et al. (1987) reported that some South American frogs possessed a lipid layer in a similar region of the skin as L. peroni. These species are apparently not water loss resistant. However, their layer was present both dorsally and ventrally and was thickest just before winter torpor. The most likely function is that of energy storage. L. peroni’s layer, in contrast, is only present dorsally. We cannot comment on seasonal variation, since the study animals were kept under constant conditions. These conditions mimicked that of summer so would not stimulate animals to lay down fat stores. Furthermore, the other species, which are not active in winter, did not have lipid layers, in particular, the xeric Cyclorana novaehollandiae which aestivates for most of the year. The cutaneous circulation of L. peroni, since it is above the lipid layer, could conceivably play a role in regulating cutaneous water loss. However, this role would be minor at most

compared with the effect of the lipid layer. Otherwise, we might expect cutaneous circulation to play a more active role in thermoregulation by increasing flow through the skin at higher temperatures, thereby increasing evaporation. Such an effect was not observed. An arboreal congener of L. fallax and L. peroni, Litoria caerulea, has been reported to have an entirely different means of waterproofing itself, by spreading lipids over the surface of its skin with its limbs, in a similar fashion to Phyllomedusa (Christian et al., 1988). Apparently, three different methods of waterproofing may have evolved in three different arboreal species of a single genus. This suggests that a resistance to water loss can be produced in a number of different ways relatively easily when selection favours it. Acknowledgements-We wish to thank L. Beard, C. E. Franklin, C. Smith and W. Stablum for invaluable assistance. The research was funded from a University of Queensland Research Grant and undertaken with the approval of the University’s Animal Ethics and Experimentation Committee.

References Barker J. and Grigg G. C. (1977) A Field Guide to Australian Frogs. Rigby Ltd., Adelaide. Bentley P. J. (1966) Adaptations of Amphibia to arid environments, Science 152, 619623. Beurden E. K. van (1977) Water and energy relations of the ‘Australian water holding frog’ Cyclorana platycephalus. PhD. Thesis, University of Sydney. Blaylock L. A., Ruibal R. and Platt-Aloia K. (1976) Skin structure and wiping behaviour of phyllomedusine frogs. Copeia 1976, 283-295. Brattstrom B. H. (1968) Thermal acclimation in anuran amphibians as a function of latitude and altitude. Camp. Biochem. Physiol 24, 93-l I 1. Buttemer W. A. (1990) Effect of temperature on evaporative water loss of the Australian tree frogs Litoria caerulea and Liforia chloris. Phvsiol. Zool. 63, 1043-1057. Campbell P. M. and Davis W. K. (1971) The effects of various combinations of temperature and relative humidity on the evaporative water loss of Bufo r’alliceps. Texas J. Sci. 22, 389402. Chew R. M. (1961) Water metabolism of desert inhabiting vertebrates. Biol. Ret). 36, l-3 1. Christian K. A.. Parry D. and Green B. (1988) Water loss and an extraepidermal lipid barrier in the Australian treefrog Litoria caerulea. Am. Zool. 28, 63. Cogger H. G. ( 1992) Reptiles and Amphibians of Australia. 5th ed. Reed Books, Sydney. Deyrup J. J. (1964) Water balance and the kidney. in Phvsiology of the Amphibia (Edited by Moore J. A.), pp. 251-328. Academic Press, New York. Drewes R. C., Hillman S. S., Putnam R. W. and Sokol 0. M. (1977) Water. nitrogen and ion balance in the African tree frog, Chiromantis petersi Boulenger (Anura: Phacophoridae), with comments on the structure of the integument. J. camp. Physiol. 116B, 257-267. Duellmen W. E. and Trueb L. (1986) Biology ofAmphibians. McGraw-Hill, New York. Elkan E. (1976) Ground substance: An anuran defence against desiccation. In Physiology of the Amphibia (Edited

Lipid-reduced

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