ORIGINAL PAPER A. I. Valenciano · A. L. Alonso-Go ´mez M. Alonso-Bedate · M. J. Delgado Effect of constant and fluctuating temperature on daily melatonin production by eyecups from Rana perezi Accepted: 22 June 1995 Abstract We analysed the effect of daily temperature cycles in relation to constant temperature on day/night melatonin synthesis in frog eyecups in culture. Eyecups were cultured for 24 h under 12L:12D photoperiod and two thermal regimes, constant temperature (25, 15 and 5 °C) and thermoperiod (WL/CD, thermophase coin- ciding with photophase and cryophase coinciding with scotophase; and CL/WD, cryophase coinciding with photophase and thermophase coinciding with scoto- phase). A negative correlation between ocular serotonin N-acetyltransferase activity and culture temperature for both diurnal and nocturnal activities has been observed. This effect of increased ocular activity at low tempera- ture is more pronounced than the well-known stimula- tory effect of darkness, and it does not depend on the photoperiod phase. The lack of interactions between the phase of photoperiod and culture temperature indicates that the effects of both factors are independent. Night- time temperature is the key factor in determining the amplitude of the melatonin rhythm in the Rana perezi retina. However, daytime temperature can not counter- act the inhibitory effect of light on ocular melatonin synthesis. Abbreviations BSA bovine serum albumine · MEL melatonin · NAT Serotonin N-acetyltransferase Introduction MEL (N-acetyl-5-methoxytryptamine) acts as a neuro- chemical transducer of environmental photoperiod in homeotherms (Reiter 1991). NAT is considered to be the rate-limiting enzyme of the MEL biosynthetic pathway. However, during the last 10 years some interesting dif- ferences have been observed concerning MEL bio- synthesis and regulation in ectothermic vertebrates compared to homeotherms. For instance, whereas the pineal gland is the main source of MEL in mammalian and avian species, in several ectotherms, particularly amphibians, the retina takes on this role (Pang and Allen 1986; Delgado and Vivien-Roels 1989; Skene et al. 1991). The MEL biosynthetic pathway is common to all the vertebrates so far studied and most species show a daily MEL rhythm with high levels during the scoto- phase of the photoperiod. The daily rhythm of pineal MEL secretion is mainly controlled by photoperiod in homeothermic species; however, temperature also plays a key role in the regulation of MEL synthesis in ec- totherms (Gern et al. 1983; Underwood 1985; Vivien- Roels et al. 1988; Firth and Kennaway 1989; Delgado and Vivien-Roels 1989; Rawding and Hutchison 1992; Valenciano et al. 1994). Vivien-Roels et al. (1979) sug- gested for the first time that temperature regulates the amplitude of the nighttime increase in MEL in turtles. Later, this hypothesis was corroborated for pineal and plasma MEL in other ectotherms: reptiles (Menaker and Wisner 1983; Underwood 1985; Firth et al. 1991; Tilden and Hutchison 1993) and fishes (Zachmann et al. 1991). In amphibians, the effect of temperature on MEL rhythms has been studied in the lateral eyes of the green frog, Rana perezi (Delgado and Vivien-Roels 1989) and in the plasma from both the neotenic tiger salamander, Ambystoma tigrinum (Gern et al. 1983) and the mud- puppy, Necturus maculosus (Rawding and Hutchison 1992). In all these studies, nighttime temperature mod- ified the amplitude of the MEL pulse. Subsequent stu- dies based on acclimation experiments in frogs showed that exposure to the appropriate temperature conditions in the laboratory reproduces the seasonal oscillations in both ocular NAT activity and MEL production ob- served in nature (Delgado et al. 1993; Valenciano et al. 1994). The effect of daily fluctuations of temperature on MEL synthesis has been little studied. In Tiliqua rugosa, J Comp Physiol B (1997) 167: 221–228  Springer-Verlag 1997 A.I. Valenciano · A.L. Alonso-Go ´mez · M. Alonso-Bedate · M.J. Delgado (&) Departamento de Biologı ´a Animal II (Fisiologı ´a Animal), Facultad de Biologı ´a, Universidad Complutense, E-28040 Madrid, Spain