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Melatonin and photoperiod alter growth and larval development in Xenopus laevis tadpoles
~ELAT~NIN AND P~OT~PERI~R ALTER GROWTH AND LARVAL ~EVEL~P~E~ IN ~~~U~~~ LAE&-‘kS TADPOLES MA&A JF&JS DELGADO,* PATRICIA GUT~BRREZand MERCEDES ALONSO-BEDATE Departamento de Fisiologia Animal, Facultad de Ciencias Biologicas, Universidad Complutense, 28040, Madrid, Spatn
Abstract-l. The role of environmental Iighting and melatonin administration on Xenopus 1~16 larval growth and development has been studied. 2. Melatonin treatment retarded the larval growth and development, regardless of the light regime. 3. Extreme lighting conditions (continuous darkness and continuous h&t) retarded the larval growth and also delayed the metamorphic development. 4. The uossible influence of environmental conditions through the pineal complex on amphibian iarvat
factors that influence Amphibian larvae development and metamo~hosis have been the subject of very few studies. Among these factors, light is one of the most difficult ones ta study. Up to date, most of the available data are incansistent and contradictory. Doetsch (1949) and Miline (1950) found that tadpoles of Ranu esculenta and &ma remporaria did not metamorphose when kept in the dark. On the contrary, complete darkness did not alter the abihty of Rana pipiens tadpoles to metamorphose (Eichler, 1976). Continuous light stimulated metamorphosis in Rana temporaria (Guy&ant, 1964) and Alytes obstetricans (Disclos, 1959). We have found that premetamorphic ~~&og~#s~~ pictus larvae exposed to continuous light were si~i~~~t~y smaller than those maintained in continuous darkness, and also reached more delayed stages of development (Gutitrrez et ai., 1984). The pineal eye of Xenopus laevis arises embryologically as a single dorsal vesicular evagination of the dieneephalon and anatomically is very similar to that of other developing amphibians; the pineai eye photoreceptors in the embryos and larvae are most sensitive to light of a wavelength around 520 nm (Foster and Roberts, 1982). This probably ensures maximum sensitivity to the wavelengths of light that penetrate the freshwater environment. A long time ago, Axelrod er al. (1965) demonstrated that the enzyme that forms the amphibian skin-lightening agent, mebtonin, was present in the brain and pineal area of Xenopus Iaevis. Later, Baker (1969) realized several studies in order to determine the melatonin levels in developing Xenopus luevis and its localization in the eyes of larval Xenclpus (Baker and Hoff, 1971). More recent work has demonstrated that the pineaf of Xenoplsf has a direct excitatory effect on behaviour (Foster and Roberts, 1982).
The ~~~r~~rn~tal
*To whom all correspondence should be addressed. 417
XII view of the possible influence of the pineal compiex on larval growth and development of Xenopus larvae, we have carried out the present study. We have tested the e&et of~ont~nuous melatonin administration and three different photoregimens on growth and development in this species. MATERIALS
AND METHODS
Adult Xpnopus faevis were obtained from a commercial supplier (Fanjab, S.A.) in Barcelona (Spain). They were maintained in aerated. dechlorinated water at 20 i 1°C. All the larvae of Xenon& laevis used in this investigation were obtained through matings induced with human chorionic gonadotropin. The tadpoles were reared In glass aquaria with dechlorinated tap water at a temperature of 20 & 1°C. They were fed a boiled spinach suspension made by means of’a liquidizer. They were staged according to the table of Nieuwkoop and Faber (1956). The tadpoles were distributed in severat groups (16 tadpoles per group) according to the photoperiodic condition in which they were maintained and the hormonal treatment they were subjected to. Three different lighting conditions were used in this experiment: (If Constant lighting (24 L): constant light was achieved by a single 20 watt Auomscent tube (Sylvania F 20 T 12/D). (2) Constant darkness (24 D): animais were maintained in complete darkness throughout the experiment except for the time needed for feeding or manipulation of the animals every day. (3) Diurnal lighting (12L: 12D): tadpoles were subjected to this photoperiod in a light controlled room with white light (~uo~nt tubes) on from OWOhr to 19OOhr. Two groups of tadpoles were placed In each light condition: a “control group” and a “melatonin group”; the latter consisted of 16 tadpoles that were continuously immersed in a melatonin solution (5 x 10-4M). This solution was prepared by dissolving the melatonin (Sigma) in a few drops of absolute ethanol and adding water to obtain the desired final concentration. Control groups received the same amount of ethanol. The experiment was initiated when animals reached stage 26 of development and continued over 60 days. Total length and tail length of all the tadpoles were measured at the beginning of the experiment. All tadpoles were of similar size and weight at the onset of the experiment.
MARIA
418
Jnsijs
AnimaIs were measured four times during the course of the experiment (25,40, 53 and 60 days after the experiment was initiated). Tadpoles were anesthesized with MS-222 (1: 10,000) during the time they had to be measured. On day 60, wet and dry weights were also taken from each tadpole. Statistical comparisons between meiatonin-treated and control tadpoles were carried out by use of Student’s r-test. The P values < 0.05 were considered statistically significant. In the case of tadpoles exposed to different lighting conditions and not melatonin-treated, comparisons were carried out using a one-way analysis of variance followed by
DELGADO et
40 36 32
al.
iZL.lZD
the Scheffe test for differences among several means. RESULTS
The effects of melatonin on tadpole growth are represented in Figs 1 and 2: a retardation of growth in the melatonin-treated larvae can be seen. By day 25, significant differences were already found in total length and tail lengths of the melatonin-immersed tadpoles as compared to control tadpoles. When comparisons between melatonin and control tadpoles were made on days 40, 53 and 60, this significant retardation of growth also existed and was even more obvious. Thus, melatonin-treated tadpoles always showed significantly smaller values for total length and tail length. This response was found in all melatonin-treaty animals regardless of the lighting condition in which they were maintained during the experiment (12L: 12D, 24L, 24D). A significant difference (P < 0.01) was noted between the wet weight and dry weight of the animals immersed in the melatonin medium and those raised without melatonin. These data are represented in Fig. 3. Wet and dry weights were taken at the end of the experiment (on day 60). Also in this case, this effect of melatonin was observed in all melatonin-treated
Fig. 2. Tail length of Xenopus laeuis larvae maintained under different lighting conditions: diurnal lighting (12L: 12D), constant darkness (24D) and constant lighting (24L). --: controls ----: melatonin-treaty larvae. Data are expressed as mean + SEM. Statistical differences: ****: P < 0.001.
tadpoles, in spite of the light regime to which the animals were exposed. If we take into account the influence of the lighting conditions (Table 1) on tadpole growth, it can be seen that by day 25, no significance was noted in total and tail lengths of animals raised in continuous darkness and day lighting 12L: 12D; however, tadpoles raised in continuous lighting were significantly smaller than
! I;, n-
,
I_
I 1
I 25
I 40 Days
I
I
53
60
Fig. 1. Total length of Xenopus IueGs larvae maintained under different lighting conditions: diurnal lighting (12L: 12D), constant darkness (24D) and constant lighting (24L). -: controls, ------I melatonin-treated larvae. Data are expressed as mean k SEM. Statistical differences: ****: P < 0.001 I
~ 12L:lZD
I, II 240
.L
Fig. 3. Dry weight and wet weight of Xenopus faeuis larvae maintained under different lighting conditions (12L: 12D, 24D and 24L) over 60 days. Vertical bars represent SE of the mean. Statistical differences: ****: P < 0.001.
Lighting conditions and melatonin on amphibian growth
419
Table 1. Statisticaldifferences between total and tail lengths of tadpoles maintained under a 12L: 12D photoperiod and those maintained under extreme lighting conditions (24L and 24D)
-,
SC
4 0
ThIL LENGTH
52 00
**p
< 0.01;
***p
< 0.005.
those in constant darkness (24D) and 12L: 12D. Similar results were obtained on days 40 and 53. On day 60, significant differences were found between the lengths of tadpoles maintained under continuous darkness and those under diurnal lighting, those raised in 24D being the smaller of the two groups. During the course of the experiment a difference in the rate of development and the spread of stages was observed when comparing the three control groups and their corresponding melatonin-treated groups. On day 25 most of the control tadpoles in the three light regimens had reached a more advanced stage of development (stages 5&5 l), while melatonin-treated
larvae were delayed relative to their controls (most of them being at 47, 48 and 49 stages). These results are represented in Fig. 4 as the percentage of tadpoles raised in the three different lighting conditions and/or not melatonin-treated in each developmental stage. As the experiment continued (on days 40, 53 and 60) this difference was found to be more drastic. Thus, on day 60 (Fig. 5), the highest percentage of control tadpoles with no melatonin treatment had reached stages 57-60, whereas melatonin-treated animals were delayed in their larval development usually reaching stages 51-54.
: I 25 dlas
ii n
P
24D
24L
24D
12L 12D
"r
60 dfas
70
60 50 % 40 30
% 40 30
20
20
10
10
12L:lZD
24D
24L 12L 12D
Fig. 4. Per cent of tadpoles that reach each developmental stage in the three different lighting conditions (12L: 12D, 24D and 24L) after 25 and 40 days of treatment: 0 control tadpoles q melatonin-treated tadpoles.
24D
24L
Fig. 5. Percent of tadpoles that reach each developmental stage in the three lighting conditions (12L: 12D, 24D and 24L) after 53 and 60 days of treatment: 0 control tadpoles m melatonin-treated tadpoles.
MARIAJES~~SDELGADOet al.
420 DISCUSSION
In general, there is very little information on the role of the pineal complex on growth and development in amphibians. As far as we know, the first work is that of McCord and Allen (19 17); they found that a bovine pineal extract devoid of melatonin activity had a stimulatory effect on growth, but not on development, of Rana pipiens larvae. Some years later, Addair and Chidester (1928) observed that Bufo americanus tadpoles fed with mammalian pineal showed a marked acceleration of metamorphosis. More recent studies about the relationship between the pineal and growth and metamorphosis of amphibians also show apparent contradictions. Thus, while Kelly (1958) found that pinealectomy failed to influence metamorphosis of Taricha torosa, Remy and Disclos (1970) found a marked acceleration of metamorphosis after pinealectomy of A/_vtes obstetricans larvae. With respect to the influence of light on growth and development of anuran amphibians we have made several studies using Rana ridibunda and Discoglossus pictus tadpoles (Delgado et al., 1984; Gutierrez et al., 1984). Premetamorphic Rana ridibunda tadpoles maintained under a 6L: 18D photoregime grow more than those maintained under continuous darkness. In Discoglossus pictus we have found that, during premetamorphosis as well as prometamorphosis and climax, continuous darkness accelerated growth and development in comparison with tadpoles reared in constant lighting conditions. In the same way, the results obtained in the present work show that X. laeuis tadpoles develop more rapidly when they are maintained under a I2L : 12D photoperiodic condition; this lighting condition is demonstrated to be the optimal for development. Continuous light retarded this process, and tadpoles raised in this photoregime were significantly smaller in size than those raised in 12L : 12D. We did not find significant differences in the lengths of tadpoles from groups 12L: 12D and 24D throughout the experiment, except for the measures taken on day 60, when the experiment ended. On this day, tadpoles maintained under continuous darkness presented significantly lower values (total and tail lengths) than the animals from the 12L: 12D group. Similar results were obtained by Eichler and Gray (1976) in Rana pipiens. Animals held in constant light were consistently smaller than those held in 12L: 12D and continuous darkness. Our results show that melatonin retarded growth and development of the larvae regardless of the lighting conditions. Melatonin acted as a “brake” for metamorphic development. In the same way, J. E. Platt (unpublished) found that melatonin retarded TSH-induced and spontaneous metamorphosis of Ambystoma tigrinum. Contradictorily, Norris and Platt (1973) observed that neither purified melatonin nor commercial bovine pineal powder influenced iodide uptake of intact Ambystoma tigrinum larvae. In contrast, pinealectomized larval salamanders (A. tigrinum) exhibited decreased thyroidal uptake of injected radioiodide (“‘I) (Gern and Boer, 1973; Platt and Norris, 1973). De Vlaming (1980) has suggested a functional relationship between the pineal organ and growth in
Carassius auratus. He found that the effects of pinealectomy and melatonin administration on growth and body weight gain in this species were photoperiod-dependent. Pinealectomy decreased the rate of linear growth in fishes exposed to a short but not a long photoperiod. Melatonin treatment accelerated growth and weight gain in goldfish maintained on a short but not a long photoperiod. In the present work, melatonin was administered in such away that the hormone was continuously present in the water in which the tadpoles were maintained. When melatonin was administered by acute injections, similar results were found with respect to its effect on growth and development, although more drastic effects seemed to appear when melatonin was continuously available. There is much data for mammals that demonstrate different effects of melatonin depending on its mode of administration. How can melatonin influence the growth and metamorphosis of the tadpoles? The importance of prolactin in amphibians development is well known. Among the fractions separated from pituitary glands of larval Rana catesbeiana by disc gel electrophoresis, the fraction with a pronounced prolactin activity had both a stimulating effect on the collagen synthesis in the tail fin of bullfrog tadpoles and a supressive effect on T,-induced resorption of Bufo bufo japonicus tadpole tails in oitro (Kikuyama et al., 1980). In previous work we have found that exogenous prolactin exerts both growth-promoting and antimetamorphic influences on D. pictus larvae (AlonsoBedate and Delgado, 1983). Bromocryptine (a dopamine agonist) administration during larval development of the same species has a contrary effect on growth and larval structures. In most vertebrates, including amphibians, prolactin release is under inhibitory hypothalamic control (Bern, 1983). Dopamine is the principal candidate as a prolactin inhibitory factor, and at least pharmacologically, dopamine and other catecholamines are effective inhibitors in amphibians (Ball, 1981). In bullfrogs, dopamine directly inhibits prolactin release, and serotonin directly stimulates prolactin synthesis and secretion (Seki and Kikuyama, 1981. A very attractive hypothesis is that melatonin might regulate the larval growth by altering hypothalamic dopamine or serotonin levels. There are no published data concerning the relationship between melatonin and prolactin in anurans. However, in mammals there are many studies on the regulation of prolactin release mediated by the different compounds produced by the pineal. Richardson et al. (1981) has found that brain indoleamines, serotonin and melatonin, are involved in the modulation of GH secretion in rats. We could postulate that, in our experiment, in those tadpoles maintained under extreme photoregimes (24L, 24D) the retinal photoreceptors and/or extraretinal photoreceptors (such as the pineal complex) would “translate” the environmental information in a hormonal response, altering the melatonin levels and rhythms; this response could regulate the process of growth and metamorphosis by altering the endocrine state of the animal.
Lighting conditions and melanonin on amphibian growth
421
~ck~on~~edge~enr~-This study was supported by Grants in Aid for Developmental Scientific Research from the Ministry of Education and Science to Maria Jesus Delgado and Patricia Gutierrez and is part of the project No. 1450 from CAICYT (Comision Asesora de Investigation Cientifica y T&mica).
Foster R. G. and Roberts A. (1982) The pineal eye in Xenopus laevis embryos and larvae: a photoreceptor with a direct excitatory effect on behaviour. J. romp. Physiol. 145,413-419. Gern W. A. and De Boer K. F. (1973) Pineal thyroid relationships in neoteny. J. Co/o.-Fro. Acad. Sci. 7,
REFERENCES
Gutierrez P. Delgado M. J. and Alonso-Bedate M. (1984) Influence of photoperiod and melatonin administration on growth and metamorphosis on Discoglossus pictus larvae. Comp. Biochem. Physiol. 79A, 2555260. Guyetant R. (1964) Actions comparees de la lumiere et de l’obscurite sur’la croissance at la metamorphose de titards de Rana temporaria. L. Ann. Sci. Univ. Besanqon-
Addair .I. and Chidester F. E. (1928) Pineal and metamorphosis: the influence of pineal feeding upon the rate of metamorphosis in frogs. Endocrinology 12, 791-796. Alonso-Bedate M. and Delgado M. J. (1983) Effects of prolactin and bromocryptine in Discoglossus pictus (Anuran Amphibian, Otth) tadpoles. Comp. Biochem. Physiol. %A, 7655772. Axelrod J., Quay W. B. and Baker I?. C. (1965) Enzymatic synthesis of the skin-lightening agent, melatonin, in Amohibians. Nature. Lond. 23, 203, 386. Baker P. C. (1969) Melatonin levels in developing Xenopus laevts. Comp. Biochem. Physiol. 28, 1387-1393.
Baker P. C. and Hoff K. M. (1971) Melatonin localization in the eyes of larval Xenopus. Comp. Biochem. Physiol. 39A, 879-881. Ball J. N. (1981) Hypothalamic control of the pars distalis in fishes, amphibians, and reptiles. Cm. Comp. Endocr. 44, 135-170.
Bern H. A. (1983) Functional evolution of prolactin and growth hormone in lower vertebrates. Am. Zool. 23, 663-67 1.
Delgado M. J., Gutierrez P. and Alonso-Bedate M. (1984) Growth response of premetamo~hic Runa rjd~bund~ and ~jscog~ussus picrn.s tadpoles to melatonin injections and photo~riod. Acta Embryol. Morphof. Exper. n. s., 5, 23339. De Vlaming V. (t980) Effects of pinealectomy and melatonin treatment on growth in the goldfish, Carassius auratus. Gen. camp. Endocr. 40, 245-250.
Disclos P. (1959) Influence regulatrice de la lumiere sur le developpement de titards d’Alytes obstetricans Laur. soumis a un traitement thyroxinien. Cr. Acad. Set., Paris 249, 1277-1279. Doetsch H. 11949) Exoerimentelle Untersuchungen iiber den Einfluss des L&e und der ultravioletten-strahlen auf Wachstum mit Entwicklung von Amphibien-larven. Wi~heim Roux Archs Ent. Organ. 144, 2530.
Eichler V. B. and Gray H. S., Jr (1976) The inguence of environmental lighting on the growth and prometamorphic development of larval f&ma pipiens. Becelop. Growth DzjY 18, 177-182
32-33.
Zoil. Physioi. 19, 77-98.
Kelly D. E. (1958) Embryonic and larval epiphysectomy in the salamander, Taricha rorosa, and observation on scoliosis. J. Morph. 103, 503-538. Kikuyama S., Yamamoto K. and Mayumi M. (1980) Growth-promoting and antimetamorphic hormone in pituitary glands of bullfrogs. Gen. romp. Endocr. 41, 212-216.
McCord C. P. and Allen F. P. (1917) Evidences associating pineal gland function with alteration in pigmentation. J. exp. Zool. 23, 207-224. Miline R. (1950) Influence de la lumitre et de l’obscurite sur la morphogenese. Med. Arch. 25, 34. Nieuwkoop P. D. and Faber J. (1956) Normal Tat&s of Xenopus laevis (Daudin). North Holland, Amsterdam. Norris D. 0. and Platt J. E. (1973) Effects of pituitary hormones, melatonin, and thyroid inhibitors on radioiodide uptake by the thyroid glands of larvae and adult tiger salamanders, An~bys~om~ t~grinum. Fen. romp. Endocr. 21, 368-376.
Platt J. E. and Norris D. 0. (1973). The effects of melatonin, bovine pineal extract and pinealectomy on spontaneous and induced metamorphosis and thyroidal uptake of “‘I in larval Ambystoma tigrinum. J. Cola.- Wyo. Acad. Sci. 7, 40.
Remy C. and Disclos P. (1970) Influence de l’epiphysectomie sur le developpement de la thyroide et des gonades chez les t@tards d’dlytes obstetricans. C.R. Sot. Biol. 164, 1989-1993.
Richardson S., Hollander C., Prasad J. and Hirookay Y. (1981) Somatostatin release from rat hypothalamus in vitro: Effects of melatonin and serotonin. Endocrtnofogy 109, 2, 6022606. Seki T. and Kikuyama S. (1982) fn vitro studies on the regulation of prolactin secretion in the bullfrog pituitary gland. Gen. camp. endocr. 46, 473-379.
Abstract-l. The role of environmental Iighting and melatonin administration on Xenopus 1~16 larval growth and development has been studied. 2. Melatonin treatment retarded the larval growth and development, regardless of the light regime. 3. Extreme lighting conditions (continuous darkness and continuous h&t) retarded the larval growth and also delayed the metamorphic development. 4. The uossible influence of environmental conditions through the pineal complex on amphibian iarvat
factors that influence Amphibian larvae development and metamo~hosis have been the subject of very few studies. Among these factors, light is one of the most difficult ones ta study. Up to date, most of the available data are incansistent and contradictory. Doetsch (1949) and Miline (1950) found that tadpoles of Ranu esculenta and &ma remporaria did not metamorphose when kept in the dark. On the contrary, complete darkness did not alter the abihty of Rana pipiens tadpoles to metamorphose (Eichler, 1976). Continuous light stimulated metamorphosis in Rana temporaria (Guy&ant, 1964) and Alytes obstetricans (Disclos, 1959). We have found that premetamorphic ~~&og~#s~~ pictus larvae exposed to continuous light were si~i~~~t~y smaller than those maintained in continuous darkness, and also reached more delayed stages of development (Gutitrrez et ai., 1984). The pineal eye of Xenopus laevis arises embryologically as a single dorsal vesicular evagination of the dieneephalon and anatomically is very similar to that of other developing amphibians; the pineai eye photoreceptors in the embryos and larvae are most sensitive to light of a wavelength around 520 nm (Foster and Roberts, 1982). This probably ensures maximum sensitivity to the wavelengths of light that penetrate the freshwater environment. A long time ago, Axelrod er al. (1965) demonstrated that the enzyme that forms the amphibian skin-lightening agent, mebtonin, was present in the brain and pineal area of Xenopus Iaevis. Later, Baker (1969) realized several studies in order to determine the melatonin levels in developing Xenopus luevis and its localization in the eyes of larval Xenclpus (Baker and Hoff, 1971). More recent work has demonstrated that the pineaf of Xenoplsf has a direct excitatory effect on behaviour (Foster and Roberts, 1982).
The ~~~r~~rn~tal
*To whom all correspondence should be addressed. 417
XII view of the possible influence of the pineal compiex on larval growth and development of Xenopus larvae, we have carried out the present study. We have tested the e&et of~ont~nuous melatonin administration and three different photoregimens on growth and development in this species. MATERIALS
AND METHODS
Adult Xpnopus faevis were obtained from a commercial supplier (Fanjab, S.A.) in Barcelona (Spain). They were maintained in aerated. dechlorinated water at 20 i 1°C. All the larvae of Xenon& laevis used in this investigation were obtained through matings induced with human chorionic gonadotropin. The tadpoles were reared In glass aquaria with dechlorinated tap water at a temperature of 20 & 1°C. They were fed a boiled spinach suspension made by means of’a liquidizer. They were staged according to the table of Nieuwkoop and Faber (1956). The tadpoles were distributed in severat groups (16 tadpoles per group) according to the photoperiodic condition in which they were maintained and the hormonal treatment they were subjected to. Three different lighting conditions were used in this experiment: (If Constant lighting (24 L): constant light was achieved by a single 20 watt Auomscent tube (Sylvania F 20 T 12/D). (2) Constant darkness (24 D): animais were maintained in complete darkness throughout the experiment except for the time needed for feeding or manipulation of the animals every day. (3) Diurnal lighting (12L: 12D): tadpoles were subjected to this photoperiod in a light controlled room with white light (~uo~nt tubes) on from OWOhr to 19OOhr. Two groups of tadpoles were placed In each light condition: a “control group” and a “melatonin group”; the latter consisted of 16 tadpoles that were continuously immersed in a melatonin solution (5 x 10-4M). This solution was prepared by dissolving the melatonin (Sigma) in a few drops of absolute ethanol and adding water to obtain the desired final concentration. Control groups received the same amount of ethanol. The experiment was initiated when animals reached stage 26 of development and continued over 60 days. Total length and tail length of all the tadpoles were measured at the beginning of the experiment. All tadpoles were of similar size and weight at the onset of the experiment.
MARIA
418
Jnsijs
AnimaIs were measured four times during the course of the experiment (25,40, 53 and 60 days after the experiment was initiated). Tadpoles were anesthesized with MS-222 (1: 10,000) during the time they had to be measured. On day 60, wet and dry weights were also taken from each tadpole. Statistical comparisons between meiatonin-treated and control tadpoles were carried out by use of Student’s r-test. The P values < 0.05 were considered statistically significant. In the case of tadpoles exposed to different lighting conditions and not melatonin-treated, comparisons were carried out using a one-way analysis of variance followed by
DELGADO et
40 36 32
al.
iZL.lZD
the Scheffe test for differences among several means. RESULTS
The effects of melatonin on tadpole growth are represented in Figs 1 and 2: a retardation of growth in the melatonin-treated larvae can be seen. By day 25, significant differences were already found in total length and tail lengths of the melatonin-immersed tadpoles as compared to control tadpoles. When comparisons between melatonin and control tadpoles were made on days 40, 53 and 60, this significant retardation of growth also existed and was even more obvious. Thus, melatonin-treated tadpoles always showed significantly smaller values for total length and tail length. This response was found in all melatonin-treaty animals regardless of the lighting condition in which they were maintained during the experiment (12L: 12D, 24L, 24D). A significant difference (P < 0.01) was noted between the wet weight and dry weight of the animals immersed in the melatonin medium and those raised without melatonin. These data are represented in Fig. 3. Wet and dry weights were taken at the end of the experiment (on day 60). Also in this case, this effect of melatonin was observed in all melatonin-treated
Fig. 2. Tail length of Xenopus laeuis larvae maintained under different lighting conditions: diurnal lighting (12L: 12D), constant darkness (24D) and constant lighting (24L). --: controls ----: melatonin-treaty larvae. Data are expressed as mean + SEM. Statistical differences: ****: P < 0.001.
tadpoles, in spite of the light regime to which the animals were exposed. If we take into account the influence of the lighting conditions (Table 1) on tadpole growth, it can be seen that by day 25, no significance was noted in total and tail lengths of animals raised in continuous darkness and day lighting 12L: 12D; however, tadpoles raised in continuous lighting were significantly smaller than
! I;, n-
,
I_
I 1
I 25
I 40 Days
I
I
53
60
Fig. 1. Total length of Xenopus IueGs larvae maintained under different lighting conditions: diurnal lighting (12L: 12D), constant darkness (24D) and constant lighting (24L). -: controls, ------I melatonin-treated larvae. Data are expressed as mean k SEM. Statistical differences: ****: P < 0.001 I
~ 12L:lZD
I, II 240
.L
Fig. 3. Dry weight and wet weight of Xenopus faeuis larvae maintained under different lighting conditions (12L: 12D, 24D and 24L) over 60 days. Vertical bars represent SE of the mean. Statistical differences: ****: P < 0.001.
Lighting conditions and melatonin on amphibian growth
419
Table 1. Statisticaldifferences between total and tail lengths of tadpoles maintained under a 12L: 12D photoperiod and those maintained under extreme lighting conditions (24L and 24D)
-,
SC
4 0
ThIL LENGTH
52 00
**p
< 0.01;
***p
< 0.005.
those in constant darkness (24D) and 12L: 12D. Similar results were obtained on days 40 and 53. On day 60, significant differences were found between the lengths of tadpoles maintained under continuous darkness and those under diurnal lighting, those raised in 24D being the smaller of the two groups. During the course of the experiment a difference in the rate of development and the spread of stages was observed when comparing the three control groups and their corresponding melatonin-treated groups. On day 25 most of the control tadpoles in the three light regimens had reached a more advanced stage of development (stages 5&5 l), while melatonin-treated
larvae were delayed relative to their controls (most of them being at 47, 48 and 49 stages). These results are represented in Fig. 4 as the percentage of tadpoles raised in the three different lighting conditions and/or not melatonin-treated in each developmental stage. As the experiment continued (on days 40, 53 and 60) this difference was found to be more drastic. Thus, on day 60 (Fig. 5), the highest percentage of control tadpoles with no melatonin treatment had reached stages 57-60, whereas melatonin-treated animals were delayed in their larval development usually reaching stages 51-54.
: I 25 dlas
ii n
P
24D
24L
24D
12L 12D
"r
60 dfas
70
60 50 % 40 30
% 40 30
20
20
10
10
12L:lZD
24D
24L 12L 12D
Fig. 4. Per cent of tadpoles that reach each developmental stage in the three different lighting conditions (12L: 12D, 24D and 24L) after 25 and 40 days of treatment: 0 control tadpoles q melatonin-treated tadpoles.
24D
24L
Fig. 5. Percent of tadpoles that reach each developmental stage in the three lighting conditions (12L: 12D, 24D and 24L) after 53 and 60 days of treatment: 0 control tadpoles m melatonin-treated tadpoles.
MARIAJES~~SDELGADOet al.
420 DISCUSSION
In general, there is very little information on the role of the pineal complex on growth and development in amphibians. As far as we know, the first work is that of McCord and Allen (19 17); they found that a bovine pineal extract devoid of melatonin activity had a stimulatory effect on growth, but not on development, of Rana pipiens larvae. Some years later, Addair and Chidester (1928) observed that Bufo americanus tadpoles fed with mammalian pineal showed a marked acceleration of metamorphosis. More recent studies about the relationship between the pineal and growth and metamorphosis of amphibians also show apparent contradictions. Thus, while Kelly (1958) found that pinealectomy failed to influence metamorphosis of Taricha torosa, Remy and Disclos (1970) found a marked acceleration of metamorphosis after pinealectomy of A/_vtes obstetricans larvae. With respect to the influence of light on growth and development of anuran amphibians we have made several studies using Rana ridibunda and Discoglossus pictus tadpoles (Delgado et al., 1984; Gutierrez et al., 1984). Premetamorphic Rana ridibunda tadpoles maintained under a 6L: 18D photoregime grow more than those maintained under continuous darkness. In Discoglossus pictus we have found that, during premetamorphosis as well as prometamorphosis and climax, continuous darkness accelerated growth and development in comparison with tadpoles reared in constant lighting conditions. In the same way, the results obtained in the present work show that X. laeuis tadpoles develop more rapidly when they are maintained under a I2L : 12D photoperiodic condition; this lighting condition is demonstrated to be the optimal for development. Continuous light retarded this process, and tadpoles raised in this photoregime were significantly smaller in size than those raised in 12L : 12D. We did not find significant differences in the lengths of tadpoles from groups 12L: 12D and 24D throughout the experiment, except for the measures taken on day 60, when the experiment ended. On this day, tadpoles maintained under continuous darkness presented significantly lower values (total and tail lengths) than the animals from the 12L: 12D group. Similar results were obtained by Eichler and Gray (1976) in Rana pipiens. Animals held in constant light were consistently smaller than those held in 12L: 12D and continuous darkness. Our results show that melatonin retarded growth and development of the larvae regardless of the lighting conditions. Melatonin acted as a “brake” for metamorphic development. In the same way, J. E. Platt (unpublished) found that melatonin retarded TSH-induced and spontaneous metamorphosis of Ambystoma tigrinum. Contradictorily, Norris and Platt (1973) observed that neither purified melatonin nor commercial bovine pineal powder influenced iodide uptake of intact Ambystoma tigrinum larvae. In contrast, pinealectomized larval salamanders (A. tigrinum) exhibited decreased thyroidal uptake of injected radioiodide (“‘I) (Gern and Boer, 1973; Platt and Norris, 1973). De Vlaming (1980) has suggested a functional relationship between the pineal organ and growth in
Carassius auratus. He found that the effects of pinealectomy and melatonin administration on growth and body weight gain in this species were photoperiod-dependent. Pinealectomy decreased the rate of linear growth in fishes exposed to a short but not a long photoperiod. Melatonin treatment accelerated growth and weight gain in goldfish maintained on a short but not a long photoperiod. In the present work, melatonin was administered in such away that the hormone was continuously present in the water in which the tadpoles were maintained. When melatonin was administered by acute injections, similar results were found with respect to its effect on growth and development, although more drastic effects seemed to appear when melatonin was continuously available. There is much data for mammals that demonstrate different effects of melatonin depending on its mode of administration. How can melatonin influence the growth and metamorphosis of the tadpoles? The importance of prolactin in amphibians development is well known. Among the fractions separated from pituitary glands of larval Rana catesbeiana by disc gel electrophoresis, the fraction with a pronounced prolactin activity had both a stimulating effect on the collagen synthesis in the tail fin of bullfrog tadpoles and a supressive effect on T,-induced resorption of Bufo bufo japonicus tadpole tails in oitro (Kikuyama et al., 1980). In previous work we have found that exogenous prolactin exerts both growth-promoting and antimetamorphic influences on D. pictus larvae (AlonsoBedate and Delgado, 1983). Bromocryptine (a dopamine agonist) administration during larval development of the same species has a contrary effect on growth and larval structures. In most vertebrates, including amphibians, prolactin release is under inhibitory hypothalamic control (Bern, 1983). Dopamine is the principal candidate as a prolactin inhibitory factor, and at least pharmacologically, dopamine and other catecholamines are effective inhibitors in amphibians (Ball, 1981). In bullfrogs, dopamine directly inhibits prolactin release, and serotonin directly stimulates prolactin synthesis and secretion (Seki and Kikuyama, 1981. A very attractive hypothesis is that melatonin might regulate the larval growth by altering hypothalamic dopamine or serotonin levels. There are no published data concerning the relationship between melatonin and prolactin in anurans. However, in mammals there are many studies on the regulation of prolactin release mediated by the different compounds produced by the pineal. Richardson et al. (1981) has found that brain indoleamines, serotonin and melatonin, are involved in the modulation of GH secretion in rats. We could postulate that, in our experiment, in those tadpoles maintained under extreme photoregimes (24L, 24D) the retinal photoreceptors and/or extraretinal photoreceptors (such as the pineal complex) would “translate” the environmental information in a hormonal response, altering the melatonin levels and rhythms; this response could regulate the process of growth and metamorphosis by altering the endocrine state of the animal.
Lighting conditions and melanonin on amphibian growth
421
~ck~on~~edge~enr~-This study was supported by Grants in Aid for Developmental Scientific Research from the Ministry of Education and Science to Maria Jesus Delgado and Patricia Gutierrez and is part of the project No. 1450 from CAICYT (Comision Asesora de Investigation Cientifica y T&mica).
Foster R. G. and Roberts A. (1982) The pineal eye in Xenopus laevis embryos and larvae: a photoreceptor with a direct excitatory effect on behaviour. J. romp. Physiol. 145,413-419. Gern W. A. and De Boer K. F. (1973) Pineal thyroid relationships in neoteny. J. Co/o.-Fro. Acad. Sci. 7,
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