JournaIof J Comp Physiol B (1988) 158:537-546 srst_ Comparative ....and!rmron- - PhysioIogy B ~ © Springer-Verlag 1988

Adaptations of the reed frog Hyperolius viridiflavus (Amphibia, Anura, Hyperoliidae) to its arid environment V. Iridopbores and nitrogen metabolism

R. Schmuck, F. Kobelt, and K.E. Linsenmair Zoologisches Institut 111, Röntgenring 10, D-8700 Würzburg, Federal Republic of Gennany

Accepted August 29, 1988

Summary. Ofall amphibians living in arid habitats, radiation refleetance, synthesis of iridopbore pig­ reed frogs (belonging to the super species Hypero­ ments does not eease. Rather, this pathway is fur­ lius viridiflavus) are the most peculiar. Froglets are ther used during the remaining dry season for solv­ able to tolerate dry periods of up to 35 days or ing osmotie problems eaused by accumulation of longer immediately after metamorphosis, in cli­ nitrogenous wastes. During prolonged water depri­ matically exposed positions. They face similar vation, in spite of reduced metabolic rates, purine problems to estivating juveniles, i.e. enduranee of pigments are produced at the same rate as in wet long periods of high temperature and low RH with season eonditions. This leads to a higher relative rather limited energy and water reserves. In addi­ proportion of nitrogen end products being stored tion, they must have had to develop meehanisms in skin pigments under dry season conditions. At to prevent poisoning by nitrogenous wastes that the end of an experimental dry season lasting rapidly accumulate during dry periods as a meta­ 35 days, up to 38% of the aecrued nitrogen is bolie consequenee of maintaining a non-torpid stored in the form of osmotically inactive purines state. in thc skin. Thus the osmotie problems caused by During dry periods, plasma osmolarity of H. evaporative water loss and urea production are v. taeniatus froglets strongly increased, mainly greatly reduced. through urea accumulation. Urea accumulation was also observed during metamorphic cJimax. During postmetamorphic growth, chromato­ phores develop with the density and morphology typical of the adult pigmentary pattern. The der­ Introduction mal iridophore layer, which is still incomplete at this time, is fully developed within 4-8 days after Members of the super species Hyperolius viridifla­ metamorphosis, irrespective of maintenance condi­ vus (Schi0tZ 1971) inhabit seasonally very hot, dry tions. These iridophores mainly contain the pu­ African savannas. The cJimatic conditions at meta­ rines guanine and hypoxanthine. The ability of morphosis are unpredietable espeeially towards the these purines to reflect light provides an excellent end of the rainy season when froglets leave their basis for the role of iridophores in temperature breeding site, and during the transitional period regulation. In individuals experiencing dehydra­ between wet and proper dry seasons. Immediately tion stress, the initial rate of purine synthesis is after metamorphosis, the froglets, weighing be­ doubled in eomparison to specimens continuously tween 200 and 300 mg, must be able to survive maintained under wet season conditions. This in­ intermediate dry periods of several days and, only crease in synthesis rate leads to a rapid increase a few weeks later (weighing between 300 and in the thiekness of the iridophore layer, thereby 700 mg), must be prepared for proper estivation. cffectively reducing radiation absorption. Thus, Neither newly metamorphosed froglets nor estivat­ thc danger of overheating is diminished during pe­ ing juveniles try to avoid the harsh climate prevail­ riods of water shortage when evaporative eooling ing above ground during dry periods by withdraw­ must be avoided. After the development of an iri­ ing into microclimatically favorable crevices in the dophore layer of sufficient thiekness for effective ground. Rather, they endure the extremely unfa- 538 R, Schmuck et al.: Adaptations of H. viridiflavus to its arid environment vorable conditions in exposed positIOns, where nahan 1982). Additional advantages ofuricotelism they must contend with high solar radiation load in anurans are: (1) such species can feed during (SRL) (Kobelt and Linsenmair 1986) and steep dry seasons, and (2) they might be able to selective­ water vapor gradients (Geise and Linsenmair ly bind cations to insoluble urate-salts at high elec­ 1986). Water reserves are very limited (Schmuck trolyte concentrations (Shoemaker and McClana­ and Linsenmair 1988); therefore, evaporative cool­ han 1975). Because the economical detoxification ing is not an appropriate means of avoiding criti­ of nitrogen end products by converting them to cally high body temperatures. Temperature regula­ urea engenders several problems, uricotelism at tion must therefore be achieved by other means. first glance would seem to be the optimal solution Reducing SRL by improved skin reflectance could for H. viridiflavus spp. These reed frogs, however, be one essential device for diminishing the danger are ureotelic (Withers et al. 1982b; Geise ami of overheating. This could .be achieved by increas­ LinsenJ1lai:rt986; &chmuck,ap:d .Linsenmair 19ß8). ingth~J;ilimbetof skin iridophqn;s filled with'pu­ Is this eiue to a poor degiee· of adaptation' to' rine plateiets which very effectively reflect incom­ a xeric envi~onment,or' did H .. viridijlavus spp. ing radiation in the visible and the illfrared range find another solution of how to cin::umvent the (Withers et al. 1982a; Kobelt and Linsenmair problems caus.ed by acc~ulation of nitrogen enQ 1986). Froglets also have todeal with the problem products? - .. of accumulating nitrogenous wastes. In contrast During dry season conditiöns, the number of to fQssoria~ amphibians, since reed frogs do .not chromatophores with light-teUectihg platelets (irj~ assume a.torpor-like state, reduction in metabo- . dophores) in the~kin of H. 'v .• taeniatus great1Y. lism is rather, limited. Cqnsequently,. they'accutilu­ increa.ses.These platelets mainly consist of thep.u­ late more nitrogen end products per unit of time rines guanine' (85-92%) and hypoxanthine than fossorial amphibians. (8-15%). As already stressed, iridophores act as Investigations of those anurans which remllin radiation reflect.ors that can considerably reduce above ground during ~ry periods show that eva po­ high SRL. (Kobelt and Linsen1l1air 1986). In tbe .rätive water ··loss (EWL) js considerably higher initial phase· ö['iridophore, synthesis, s~inreiJec7 thartlu amphibians. which withdraw into:!he . ta~cestrongly increases. However,afterth~ syn-' ground (Bentley 1966 ; McClanahan 1967; May­ thesis. of 2~31a:yers which rriay alrf:ady be pres~p.t hew 1968; Ruibal et al. 1969; Warburg 1972), even in juvenjle frogs prior to the onset of the dry peri­ though these anurans possess highly effective skin od, further production of iridophores, which al­ mechanisms to reduce EWL. This is due to the ways takes place under dry season conditions, only much lower average humidity of the surrounding marginally increases reflectance. This finding sug­ air above ground compared to the more favorable gests that iridophores have additional functions. microc1imates in burrows. Both a higher rate of Being a store for nitrogen end propucts, besides nitrogen accumulation as wen as a higher water allowing color changes and reducing SRL, might loss severely aggravate the osmotic problems that be a third function of the iridophores in H.v.spp. all amphibians face during prolonged periods of (Schmuck and Linsetmlair 1988) and was further water shortage. Therefore, red frogs must have de­ investigated in this study. veloped especially effective mechanisms to solve In freshly transformed froglets, only one in­ the problems of dealing with catabolic nitrogen complete layer of iridophores is present in the dor­ end products in a water-conserving and osmoti­ sal skin and only a few iridophores are found in cally harmless way. In many terrestrial amphibi­ the ventral skin. In addition, there are only small ans, urea is the main end product of nitrogen me­ differences among individuals in the amount of tabolism and is accumulated during dehydration skin purines at the time ofmetamorphosis. Injuve­ stress. However, its high solubility strongly in­ nUe frogs living for only a few weeks in wet season creases the osmolarity of the body fluids and there­ terraria, however, amounts of skin purines show fore causes osmotic problems when critical concen­ considerable variation between individual frogs in trations are reached. regard to their quantity (Schmuck and Linsenmair Only a few anurans of the genera Chiromantis 1988). This variation is probably caused by micro­ and Phyllomedusa have convergently evolved the c1imatic heterogeneity within the terraria. In some mechanism of excreting nitrogen end products as places conditions prevail that induce changes to osmotically inactive urate, which is also found in dry season physiology Uust below the hot 125 W many reptiles and in birds (Loveridge 1970; Shoe­ lamp). Therefore, freshly transformed froglets maker et al. 1972; Shoemaker 1975; Balinsky et al. seem to be more suitable for the study of (1) tbe 1976; Drewes et al. 1977; Shoemaker and McCla- differences in rates of skin purine synthesis under R. Schmuck et al.: Adaptations of H. viridiflavus to its arid environment 539 different climatic conditions and (2) the correlation graded serics of acetone and flat-embedded in Durcupan between purine synthesis and skin reflectance. (F1uka) plastic resin. For light mieroscopy, sections were cut with a uItramicrotome (Reichert-Jung Ultracut) at 211m and stained with methylene blue/azure II (Richardson et al. 1960). Material and methods Purine (l1Ialysis. The remaining parts of the removed dorsal Treatment offrogs. Specimens of H.v. taeniatus were kept and and ventral skin were dried at 60°C, homogenized with 10% bred in the laboratory at 28 °C/24 °C (day/night) and about phosphoric acid (2 washings) and kept at room temperature 70%/100% RH. Freshly transformed froglets (stages 64-{j6 of for 24 h. Sampies were then exposed to ultrasound for 60 min the normal table of Nieuwkoop and Faber (1956), or stages and centrifuged for 10 min at 10000 RPM. The supernatant 45 and 46 according to Gosner (1960» weighing 200-300 mg was buffered with NaHl P04 to pH2 • Analysis was performed were kept in plastic tubes for 2-3 days on wet filter paper at with a HPLC-analyser (Kontron). A sulfopropyl-daltosil 100 20-23°C, 90-100% RH and 13.5/10.5 h day/night cycle for column (Serva) served as the stationary phase, and a triethyla­ acelimation. mine buffer (pH 7.5) as the mobile phase. The flow rate was After fuH hydration, 80 froglets were divided into two 1.8 mI/min. Synthetic standards were used as identification groups and kept separately in small plastic tubes for 35 days markers. without feeding at 30 °/20 °C and 25-35%/55-{)5% RH (day/ night cyele). Frogs were not fed during this time since under Urea (l1Ialysis. Plasma was obtained by centrifugation of blood dry season conditions they never try to catch prey. Foraging and/or Iymph sampIes. Tbe amount of urea in these sampies was observed only when RH exceeded 90%. was photometricalIy deterrnined with diagnostic kits (Sigma Group 1, the wet-adapted controls, were watered daily for No. 535, colorimetric determination). Since thc amount of am­ 60 min to the fuH hydration level by placing them in 500 111 monia in the urine of all the tested sam pIes (71 = 20) was only double-distilled water. 2% ofthe total amount ofnitrogen, it was not caJculated sepa­ Group 2 frogs were placed in 500 j.ll double distilled water rately. (15 min) only when water losses exceeded 30-35% of body To measure urea excretion, frogs sitting in their tubes re­ mass; they were considered as dry-adapted. All froglets were ceived 500 ~I double distilled wateT for 15 min (group 2) or rehydrated to the full hydration level at day 4. Afterwards, 60 min (group 1). The remaining water (i.e. not absorbed) was they needed a first water supply between day 4 and day 12, removed with a pipette and analyzed for its urea content. and a second between day 12 and day 24. The limes at which Total body protein analysis. Total body protein was measured a third supply was needed showed considerable individual vari­ by the method ofvan Beurden (1980), using the dried carcasses. ation; in 90% of the froglets it was necessary between day 20 The initial body protein content was determined using a calibra­ and day 35, and only about 10% received a third supply before tion curve representing pro tein content in relation to body size day 20. in 30 freshly metamorphosed froglets of the same stock. At Reflect(l1lce measurements. Skin reflcctance was measured wilh each measurement interval, protein content of test froglels was a pyroelectric radiometer (Molectron PR 200, spectral sensitivi­ determined and subtracted from the initial values in order to ty 0.3-40.0 11m) with an integrating sphere (Earling). To simu­ obtain protein consumption. Since the liver and skin were used laIe a natural spectrum, a 30 W tungsten quartz halogen lamp for other analyses, the data do not give a quantitative measure­ (color temperature 3150 K) was run at about 37 W (Willmer ment of the total protein catabolism rate. We assumed, how­ and Unwin 1981). The light was focused to a smaIl spot and ever, that these changes reliably reflect the decrease of total broadband filtered by quartz lenses reducing the spectrum to body protein. 300-3500 nm. To determine the maximal reflectance, froglets sitting on Fat-pad analysis. The fat-pads of the dissccted animals were black metal plates were placed on a dish heated with a thermo­ removed and dried for 2-3 days at 50 oe. The weight of the stat to 40°C. At this body temperature most froglets became fat pads was expressed as a percentage of the body weight white (displaying high reflectance values) and remained almost of the anima! at the fuH hydration level. motionless. Thus, when handled carefully, anaesthesia was not Nitrogen balance. In order to analyse the compartmentalisation necessary. After 45 min of temperature and color adaptation, ofthe metabolized nitrogen, a nitrogen balance was determined. a white frog on its metal plate was transferred to a small mov­ Three compartrnents were considered: body fluids, excreted able dish also heated to 40°C. Thc focused beam of white urine, and the iridophores. The amount of nitrogen stored in light was directed onto the back of the frog and the dish was the body fluids was calculated from the urea concentration then placed elose to the measuring hole of the sphere. Skin of the plasma sampie. It was assumed that, as in the case of remittances were compared to that of a white standard, a frog­ juvenile H. v. taeniatus (Schmuck and Linsenmair 1988), urea shaped pIaster-cast covered with a 2 mm thick layer of Eastman is evenly distributed in the body fluids. The percent values 6080 paint. shown in Fig. 6 were calculated from the nitrogen difTerence Frog preparations. To take blood and/or lymph sampies, frog­ between two mcasurements; the nitrogen content ofthe various lets were first precooled and double pithed. The fluids were compartments are presented in relative amounts. then drawn into heparinized miero glass tubes (Fa. Serva-Prax). The dorsal and ventral areas of the skin were removed and Results prepared for further analysis. The remaining carcasses were dried at 60°C to constant weight. Light microscopy 2 Light microscopy. Small pieces of skin (2-3 mm ) were fixed At the end of metamorphosis the dermal irido­ in 6.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 30 min, then washed briefly in phosphate buffer and post­ phore layer in the skin of the frogIcts is still incom­ tixed for 1 h in 1% osmium tetroxide/potassium dichromate plete. lridophores are still absent in approximately (pH 7.3) (Wohlfahrt 1957). The tissuc was dchydrated in a 20--50% of the dorsal skin. In both groups the 540 R. Schmuck et al.: Adaptations of H. viridiflavus to its arid environment

o d-y odapted 50 • wet odapted Dorsal skin o dry adapted

QJ • wet adapted u C 200 c:l 1;j 40 1" ~ " c §'150 '';: Vl 30 11 E o ~100

20r-~~---r--~----r---1~--~~ 1c o 10 20 30 40 :i: Time[dl (J) 50 Fig. 1. lncrease in dorsal skin reflectance during the postmeta­ morphie .sta,ge in Hyperoliusviridiflavus taeniatus.· The skin reflcctance of wet-adapted froglets increases in a logarithmic pattern. Dry-adapted froglets show a faster increase. Values represent means of 10. specimens; vertical lines represent stan­ a dard deviations. Stars indicate significant differences between wet" and dry-adapted frags (P < 0.05; t-test) Ventral skin o dry adapted • wet adapt~d layer becomes completed within 4-8 days after metamorphosis. In group 1 the synthesis of a sec­ ond layei was not cOl11pleted within the 35 days of the experiment The thlckness of the layer wa$ 20 ± 6 1J.1l1 (n= 10). Grol,lp 2 had synthesized a sec- ' ond layer after 20 days. At day 35 the iridophore layer was 31 ± 5 IJ.m (significantly thicker than that of group 1, PsO.01, t-test; n= 10). 10 20 30 40 Timeldl Reflectance measurements Fig. 2. lncrease in the amount of purines (mg/g skin) in dorsal (a) and ventral (b) skin of Hyperolius viridiflavus taeniatus frog­ Just after transformation, the average dorsal skin lets during the course of their postmetamorphie development. reflectance of froglets is about 28%, which in­ Froglets were either watered daily (wet-adapted) or when water creases to 32% by the first day of measurement losses exceeded 30-35% of body mass, on average three times under acclimation conditions (Fig. 1). The reflec­ within 35 days (dry-adapted). Values represent means of 3 specimens; vertical lines represent standard deviations. Stars tance of wet-adapted froglets increases from 32% indicate significant differcnccs bctween wet- and dry-adapted to 41 % within 28 days in a logarithmic pattern. froglets (P< 0.05; confidencc interval) The reflectance of dry-adapted froglets increases more rapidly from 32% to 45%, and reaches a distinctly higher level than in group 1. The curve shows a conspicuous plateau between the 8th and Group 2: R=5.55 In t+26.023 the 12th day. No comparable plateau could be de­ r=O.77; n=77; P

o ~ 50 GI U o c: .... "U GI 't o ~ 40 :x (/J o Q) ;

30~~~--,----r--~--~--~ 10 20 JO o 100 200 300 a TIIIIl!!dl Skin purine content [mg/g J Fig. 3. Relationship between skin purine content and skin re­ flectance in dry-adapted Hypero/ius viridiflavus taeniatus frog­ lets [J] ay odopted o wet adapted

250 increasing amount of purines in the dorsal skin are similar in both groups (Fig. 2a). A phase of increased purine synthesis in group 2, however, can be recognized between day 8 and day 16. In this phase the amounl of purine increases in group 2 at approximately twice the rate of group 1. After­ wards, purine synthesis continues at about the same rate in both groups for the remainder of the test time of 35 days. Comparable events are found in the ventral skin (Fig. 2b). A phase of increased synthesis in group 2 also starts at day 8, but thc rise amounts to only approximately half the highest level in the dorsal skin. This phase lasts about 8 days then the rate of synthesis decreases. The increase of purines (P) in the dorsal skin of the dry-adapted froglets b affects skin remittance (R) according to tbe follow­ Fig. 4. Changes in plasma urea coneentration (a) and urine ing exponential function: excretion (b) in Hyperolius viridiflavus taeniatus froglets during their postmetamorphic development, dependent upon water R=0.171 pO.192 (mg/g) (see Fig. 3) availability. Wet-adapted froglets were watered daily for 60 min to tbe full hydration level; dry-adapted specimens received water for 15 min, only ifwater losses exceeded 3G-35% ofinitial body weight. Columns indicate the average rate of nitrogen Additional purine storage sites during release in the eonsidered time interval. Since the time of water long lasting dry periods supply in group 2 strongly va ried between individuals, the mean values of sub-groups watered at a speeifie time were also plot· H. v. spp. also use other connective tissues for stor­ ted. Numerals indieate the number of individuals per measure­ ing large quantities of purine crystals. In 2- ment 4 month old H. v. taeniatus, which were main­ tained under dry season conditions for periods of 2-3 months and supplied on average 3 times with Changes in the amount blood urea nitrogen water, the heart and liver epithelia served as depo­ 0/ sition sites. The liver epithelium of about 5- Blood urea nitrogen (BUN) increases in late meta­ 8 month old H. v. nitidulus, taken from the field morphic climax (day 0-2). It then decreases to a 4 weeks after the first rainfall, was tightly packed constant level in group 1 (Fig. 4a), whereas in with iridophores. The thickness of the layer was group 2 only a slight decrease, caused by the first about 60 Jlm approaching that of the iridophore water supply, could be observed. The BUN then layer in the stratum spongiosum of ventral skin rises until day 12 at about the same rate as in meta­ (about 75 ~m). morphic climax (166 ~g urea-N/g/d). This is fol- 542 R. Schmuck et al.: Adaptations of H. viridiflavus to its arid environment

In order to obtain the mean value ofN excreted

1.5 o dry adapted (n~5) per individual at tbe time of each measurement, ewet adapted (n~5) it was necessary to combine single values over spe­ cific time intervals (Fig. 4 b). The intervals were chosen so tbat tbey reflected the time frame in which water (n = 3) was administered. Tbe mean values given are calculated from the amount of urea excreted at the time of the watering, relative to the length of the respective time interval. Fi­ gure 4 b summarizes these results. U rination was detectedexc~usiyely during watenQg .. F{owever, if a frog had urinated betweenanygfthe water,sain~ pIe intervals, its voided urea \Yould have been col­ 5 10 15 20 2530 35 lected and measured with the next water sampie. a Time Idl Therefore, the total amount of urea excreted by both groups could be quantitatively measured by analyzing these water aliquots. .' o dry adapted (n=5) ewet adapted (n=5) Changes in the amount 01 body protein and fat-pads Until day 12' bqth groups meta\;lolize protein at almost the same rate. After day 12 a considerable reduction of protein catabolism is seen in group 2 (Fig. 5a). In contrast, in group 1 the rate. ofprotein catabolism dQes not decrease after day 12at;ld, ex­ cept 'between: day30 and 35; never .reachessuch low values'as ingroup 2. T4er~f

Z100 a b 10 o dry aOOpted ~100 o dry adapted ~100 c ~ :z ~ • wet adapted 2.:- • wet odapted I- C1I r------. 1 ~ I oe Q) oE BO ~ 80 ~80 ~ / VI i VI ;E I ~ ~ ~ 0 .2 .0= Q) I 1}60 60 ~ 'E:::l ]'60 .!: d ~ ~ ';> ~ ""@ &i ~ §40 §40 ~ 40 '-' > c: QJ !ii ai 15' I E 1:: :~ 20 ~20 :~ 20 QJ / :g l}i ~ o dryadapted ~ al • we t adapted u '- .~ b .J; 0 .E - .J; O~O~~~--r-~~,-~ 0 0 10 20 30 40 0 10 20 30 40 10 20 30 40 Timeldl Time[dJ Time[dJ Fig. 6. Compartmentalisation of nitrogenous end products in Hyperolius viridij1avus taeniatus froglets, dependent upon water availability. Nitrogen stored in body fluids (a) and iridophores (b) or voided via urine (e) is expressed as percentage of the increase in total nitrogen (TN) between tbe corresponding measurement and the one preceeding, during 35 days following metamor­ phosis

1975). Accumulation of urea, however, is not only eranee is vital for ureotelie anurans if they are to seen during water-deprivation but also in late successfully inhabit seasonally arid habitats. metamorphie elimax. After the transition from am­ monotelism to ureotelism during metamorphosis, Differentiation of pigmentation pattern the aetivity of the ornitbine-urea eycle enzymes is in postme/amorphie stages markedly inereased (e.g., Dolphin and Frieden 1955; Dodd and Dodd 1976; Fox 1984). In meta­ The morphological changes in pigmentation pat­ morphic climax of anurans, the larval mouthparts tern during metamorphosis include, among others, degenerate and the adult jaws develop. During this the differentiation and the numerical increase of period, amphibians are unable to feed. By tbis the chromatophores (Smith-Gill and Carver 1981), time, larval growth has ceased and all the energy, as weIl as the produetion of new pigments and/or as weIl as the metabolites needed for differentiation the development of new organelles (Bagnara 1976; and nutrition, are provided mainly by the resorp­ Bagnara et al. 1978). The metamorphie and post­ tion of the tail and by stored fat. This proeess metamorphie morphological color changes in wet­ is associated with protein eatabolism and anabol­ adapted H. v. taeniatus are caused by a rapid in­ ism leading to a high output of urea (Dolphin and erease in the number of iridophores in the dorsal Frieden 1955). After transition to terrestrial habi­ dermis. This inerease almost doubles under dehy­ tats, the eontinuous water influx ceases although dration stress, leading to the synthesis of an irido­ metamorphosis is still ineomplete. When water re­ phore layer within 10 to 12 days, which in group 2 serves are limited urine produetion should be re­ is about twiee as thiek (+ 12.1 ~m) as in wet­ duced. This may have resulted in a primary selee­ adapted froglets (+ 6. 7 ~m). The increase in the tion pressure towards developing tolerance for thiekness of the iridophore layer is accompanied high urea eoneentrations in the body fluids during by a rapid increase in skin reflectance. Between this ontogenetic phase. the 8th and the 12th day, however, no increase Under dehydration stress, the amount of urea in skin refleetance of dry-adapted froglets could in the body fluids rises strongly to levels more than be deteeted. Tbis possibly results from proeesses three times as high as in metamorphie elimax, eonnected to tbe changeover to dry season physiol­ probably requiring a high degree of tolerance ogy that mayaiso affect the hormonal state of which might have developed as mentioned above. the frogs, and therewith their ability to physiologi­ Thus, a tolerance to urea accumulation, primarily cally change color. Such an assumption seems jus­ developed to avoid urea toxicity during metamor­ tified, since no comparable plateau could be de­ phie climax, may represent the evolutionary basis tected by investigating the inerease in total purine for the selection of a secondary tolerance to high content of the skin. After the initial increase in urea eoncentrations oecurring later in life. This tol- purine synthesis, the rate of production during the 544 R. Schmuck et al.: Adaptations of H. viridijlavus to its arid environment

following three weeks in dehydrated frogs is re­ portion of nitrogen (up to 38%) that is stored as duced to values similar to wet-adapted animals. non-toxic osmotically inactive purines is strongly This latter rate seems to be a basic level at which increased by reducing protein catabolism and guanine synthesis is maintained before and after maintaining a high rate of purine synthesis. Thus, the transition phase. guanine (and toa far lesser extent hypoxantlllne) Under dehydration stress, the initially in­ now serves as an outlet, that helps to regulate the creased purine synthesis greatly reduces the high urea level in the body. Our conclusion that guan­ SRL prevailing in tropical arid habitats. During ine, besides its role in physiological color change the wet season, this type of protection from SRL and in reduction of SRL, also serves as a store is not needed to such a great degree as in dry sea­ for nitrogen end products, is supportyd by the fact son, because sufficient water. is available and EWL that the endothelia of the heart and the liver of cal). be used for cooling. During this time, the main . frogsmay beco~e hel;lvily filled ~ith guanine crys~ function of the iridophores might be seen in the tals during. estivation. 'This phenomenon is context of color change (Bagnara 1976) and possi" especially apparent in animals that have survived ~ly . UV radiation absorption:. . several month~under dry season conditipns in the field. The.storage of. guanine in.these.. tis,slie~ dO,es ,.,,' not reduce,high SRt. The only Plau~i~le explaria~ Changes in pro tein ! cataboiism during. dry season stale '. tion we ca!l s,ee is that of nitrogen storage. The climatespace diagram of H; v. nitidulus' indi~ates At day 12, after a . water restriction lasting Q78 that in West African habitats skin 'niflectance has days, pro tein catabolism is remarkably reduced. , to be above 0.6 to avoid overheatingd.~ri~g the Thus the output of nitrogen end products is lnini­ hottest times (Kobelt, unpublished).' Giventhat mized. This. reduction in protein catabolism most such a high skin reflectance is a vital need for sur­ probably marks tlie p<:>int at which· thefrog has vival during the dry season, then gu~niIle is, be­ completed thetransitiqIl.to ciry season physlqlogy. ca4se ofit~ qig!1,er refractive Jnde~~n9 b~tt~r crys­ Stärva.tiohaJorte.d.o~sp.öt exett such,~ti yrf~ct.: .' talline.. qualiti~s;more 'effe~tive inraisi~g skin' re" flectancethanudcacid. Also due to jts beUer N: C •. "J>, ratio, 'guanin~is more effectivetl1an uric 'acid, in Nitrogen balance during the dry season state eliminating nitrogen end products in an. osmoti­ During the dry season state, nitrogenous wastes, cally inactive form. Thus, guanine' probably has unavoidably resulting from protein catabolism and a higher adaptive value for H. viridiflavus than uric protein turnover, are predominantly stored as urea acid, wruch is produced by other anurans which in the body fluids. Although water is occasionally end ure dry, periods above ground (Shoemaker present durülg the dry season for a limited period, et a1. 1972; .Balinsky et al. 1976). urea excretion is nevertheless suppressed during The storag~ of nitrogen end products in' the the first phase of adaptation. to dry. seäson phys­ form of sltill pigments does not 'n!quire the acquisi- , iology; the' stored urea most probably serves to tion of new enzymatic pathways or qualltatively accelerate water Uptake along a steep osmotic gra­ new storage sites. Iridophores are supposed to have dient (Schmuck and Linsenmair 1988). Most frogs been primarily developed for color change (Bag­ can survive a long-lasting dry season only by using nara 1966). Their ability to reflect light provides every chance to replenish lost water (Geise and an excellent basis for their use in temperature regu­ Linsenmair 1986). Water is available during the lation (Withers et a1. 1982a; Kobelt and Linsen­ transition from wet to dry season mainly as dew, mair 1986). Because the on set of a dry period can and during the dry season water is only occasional­ never be accurately predicted, it is rughly advanta­ Iy available as poor rains that are very short and geous to build up and maintain an effective antira­ which evaporate very quickly. Therefore, dry sea­ diation safeguard, and this requires synthesis of son frogs must minimize EWL and maximize up­ purines at a rate sufficient to counteract the de­ take of available water. However, as soon as the crease in the thickness of the iridophore layer disadvantages of nitrogen accumulation exceed the caused by body growth. Therefore, it comes as no advantages of increasing rehydration rates, urea surprise that a continuous synthesis of purines is excretion via the urine should be used to avoid also found in wet-adapted froglets. Within certain dangerously high osmotic concentrations. Before limits, however, purine synthesis rate can be con­ the frog fully changes to dry season physiology, siderably raised when early onset of dry season only about 12% of the released nitrogen is stored conditions necessitates a rapid increase in skin rc­ in iridophores. In dry-adapted frogs, the relative flectance. Besides its function in color change and R. Schmuck el al.: Adaptations of H. viridijlavus to its arid environment 545 temperature regulation, the iridophore system has Bagnara JT, Frost SK, Matsumoto J (1978) On the deveIop­ gained an additional and very essential importance ment of pigment patterns in amphibians. Am Zool in reducing osmotic problems by storage of nitro­ 18:301-312 Balinsky JB, Chemaly SM, Currin AE, Lee AR, Thompson gen end products during times of prolonged water RL, Westhuizen DR van der (1976) A comparative study deprivation. The stratum spongiosum of the dorsal of enzymes of urea and urie acid metabolism in different and ventral skin of H. v. taeniatus, which were species of Amphibia, and the adaptation 10 the environment exposed to dry season conditions over a long peri­ of the tree frog Chiromantis xerampelina Peters. Comp Bio­ chem Physiol 54(B): 549-555 od of time (> 3 month), is complete1y filled with Bentley PJ (1966) Adaptations of amphibia to arid environ­ iridophores. ments. Science 152: 619--623 Geise (unpublished) demonstrated that survival Beurden EK van (1980) Energy metabolism of dormant Austra­ time during dry periods is highly correlated with lian water-holding frogs (Cycloranaplatycephalus). Copeia: body size and stored fat reserves. Only frogs reach­ 787-799 Dodd MHJ, Dodd JM (1976) Tbe biology of metamorphosis. ing a body length of more than 1.6 cm and storing In: Lofts B (ed) Physiology of the Amphibia. Academic about 14% of body dry weight as fat (subcutane­ Press, New York San Francisco London ous fat deposits, fat pads and body lipids com­ Dolphin JL, Frieden E (1955) Biochemistry ofamphibian meta­ bined) can successfu11y survive the following dry morphosis. J Biol Chem 217: 735-744 Drewes RC, Hillman SS, Putnam RW, Soko10M (1977) season. Because adult H. viridiflavus spp. spawn Water, nitrogen and ion balance in the African tree frog throughout the wet season some froglets transform Chiromantis petersi Boulanger (Anura: Rhacophoridae) at the end of the rainy season, or even later during with comments on the structure of the integument. J Comp the transition period to true dry season (Linsen­ Physiol 116: 257-267 mair, unpublished field observations). Therefore, Fox H (1984) Amphibian morphogenesis. Humana Pre~s, Clif­ ton, New Jersey freshly transformed froglets also have to be able Geise W, Linsenmair KE (1986) Adaptations of the reed frog to survive intermediate dry periods that frequently Hyperolius viridijlavus (Amphibia, Anura, Hyperoliidae) to and unpredictably occur during the transition peri­ its arid environment: H. Some aspects of the water encon­ od. Dur results indicate that froglets are able to omy of Hyperolius viridijlavus nitidulus under wet and dry season conditions. Oecologia 68: 542-548 tolerate such intermediate periods even immediate­ Gosner KL (1960) A simplified table for staging anuran em­ ly after metamorphosis by increasing their skin re­ bryos and larvae with notes on identification. Herpetologica flectance and, subsequently, by reducing their ener­ 16: 183--190 getic demands. On average, however, these froglets Kobelt F, Linsenmair KE (1986) Adaptations of the reed frog are too sm all to survive a prolonged dry season Hyperolills t'iridijlavus (Amphibia, Anura, Hyperoliidae) to its arid environment: I. The skin of Hyperolius viridijlavus and must therefore use every opportunity for ener­ nitidulus in wet and dry season conditions. Oeeologia gy accumulation and growth during the transition 68:533--541 period between rainy and dry season. After rainfall Loveridge JP (1970) Observations on nitrogenous excretion and or dew, froglets are able to feed immediately, there­ water relations of Chiromantis xerampe/ina (Amphibia, Anura). Arnoldia 5: 1-6 by rapidly accumulating energy reserves through Mayhew WW (1968) Biology of desert amphibians and reptiles. a high net production rate (Schmuck, unpub­ In: Brown GW (ed) Desert biology: special topic on the lishcd). The rise in skin reflectance by purine depo­ physical and biologieal aspects of arid regions. Academic sition and the use of purine as a store for nitrogen Press, New York end products during intermediate dry periods pro­ McClanahan L (1967) Adaptations of the spadefoot toad, Sca­ phiopus couchi, to desert environments. Comp Biochem vides an important adaptation for survival under Physiol 20: 73-99 fluctuating and temporarily extreme environmen­ McClanahan LL (1975) Nitrogen excretion in arid-adapted am­ tal conditions. phibians. In: Hadley NF (ed) Environmental physiology of desert organisms. Halsted Press, Stroudsburg, Pa, Dow­ den Acknowledgements. This study was supported by the Deutsche Nieuwkoop PD, Faber J (1956) Normal Table of Xenopus laevis Forschungsgemeinschaft Research Grant Li 150/11-1/2. We (Daudin), 2nd edn. North Holland, Amsterdam thank two anonymous referees fOT valuable criticism and Mrs. Richardson KC, Jarett L, Finke EH (1960) Embedding in L. Rott for checking our English. epoxy resins for ultrathin sectioning in electron microscopy. Stain Tech 35:313-323 Ruihal R, Tevis L, Roig V (1969) Tbe terrestrial ecology of the spadefoot toad Scaphiopus hammondii. Copeia: 571- References 584 Sachs L (1978) Angewandte Statistik. Springer, Berlin Heidel­ Bagnara JT (1966) Cytology and cytophysiology of non­ berg New Y ork melanophore pigment cells. Rev Cytol 20: 173-205 Sehietz A (1971) The superspecies Hyperolius viridijlavus Bagnara JT (1976) Color Change. In: Lofts B (ed) Physiology (Anura). Vidensk Meddr Dansk Naturh Foren 134:21-76 of the Amphibia IH. Academic Press, New York San Fran­ Schmuck R, Linsenmair KE (1988) Adaptations of the reed cisco London frog Hyperolills viridijlavus (Amphibia, Anura, Hyperolii- 546 R. Schmuck et al.: Adaptations of H. viridiflavus to its arid environment

dae) to its arid environment. IH. Aspects of nitrogen Frieden E (eds) Metamorphosis. A problem in developrnen­ metabolism and osmoregulation in the reed frog, Hyperolius tal biology 2nd edn, Plenum Press, New York London viridiflavus taeniatus, with special reference to the role of Warburg MR (1972) Water economy and thermal balance of iridophores. Oecologia 75: 354-361 Israeli and Australian amphibia from xeric habitats. Syrnp Shoemaker VH (1975) Adaptations to aridity in amphibians Zool Soc London 31 : 79--111 and reptiles. In: Vernberg I (00) Physiological adaptations Willmer PG, Unwin DM (1981) Field analysis of insect heat to the environment. Intext Educational Publishers, New budgets: reflectance, size and heat rates. Oecologia York 50:250--255 Shoemaker VH, McClanahan LL (1975) Evaporative water Withers PC, Louw G, Nicolson S (1982a) Water loss, oxygen loss, nitrogen excretion and osmoregulation in phyllomedu­ consurnption and colour change in 'waterproof' reed frogs sine frogs. J Comp Physiol100:331-345 (Hyperolius). S Afr I Sei 78: 30--32 Shoemaker VH, McClanahan LL (1982) Enzymatic correlates Withers PC, Hillman SS, Drewes RC, Soko10M (1982b) Water and ontogeny of uricotelism in tree frags of the genus Phyl- loss an<) nitrogell eX'iretion in sharp-nosed reed frogs,(Hy­ , lomedusa.·J Exp ZpoI22Q:'163~169, " .' c" • pero/ius n.tmi.tu.~:; Antira, Hype~?liidae). J Exp" Biol Shoemaker VH, Balding D, RuiJ:>al R,'McClanahanLL (1972) 97:335--343 '" 'i" ,,' , ", , Uricotelism and low evaporativewaterloss'in a South Wohlfahrt KE (1957)Di~ 'Kontrastierung tierischer Zellen und American frog. Science 175:'1018-1020, ' Gewebe im Rahmen,ihrer elektronenmikroskopischen Un­ Srnith-Gill SI, Carver V,(1981) Biochernicai characterization tersuchung an ultra-dünnen Schnitten. Naturwissenschaften of org~n differentiatiori and maturation. In: Gilbert LI, 44:287-288 '

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