Effects of Indoleamines and Short Photoperiods on the Encystment of Gonyaulax polyedra

At a temperature of 15^oC, Gonyaulax polyedra responds to short days (light ≤ 10 h) by transition to the stage of a resting cyst. At 20^oC, even an lightdark (LD) cycle of 6:18 is incapable of inducing this process. In otherwise cyst-inducing conditions (15^oC; 10 h of light per day), an interruption of the scotophase by 2 h of light (LDLD 8:2:2:12 or 2:2:8:12) prevented encystment. Cyst induction is, therefore, initiated by a photoperiodic mechanism rather than by light deficiency. In Gonyaulax, photoperiodism may be mediated by the action of indoleamines. Melatonin, which exhibits a circadian rhythmicity in this organism, leads to encystment when given 1 h before lights-off in LD 11:13 at 15^oC, i.e., under otherwise noninducing conditions. Again, at 20^oC, melatonin is inefficient. Some analogues of melatonin, in particular, 5-methoxytryptamine and N,N-dimethyl-5-methoxytryptamine, and, at high concentrations, their respective precursors, serotonin and bufotenin, are capable of inducing cyst formation at 20^oC and in LD 12:12, whereas A'-acetyl-serotonin does not show this effect.     

Unicellular organisms and genetics

Abstract

Formation of asexual cysts can be induced in Gonyaulax polyedra by melatonin at 15 °C, or by its metabolite, 5‐methoxytryptamine, at 15 or 20 °C. When cells were exposed to a temperature of 10 °C, which holds the circadian oscillator(s), they were almost insensitive to melatonin, and the cyst‐inducing capacity of 5‐methoxy‐tryptamine was reduced to about 1/3. This desensitization was seen in various lighting conditions (LL, LD 16:8; LD 12:12; LD 11:13; DD). The reduction of cyst formation by decreased temperature was not due to a general incompetence of cells to encyst, because (a) low temperatures can lead to non‐photoperiodic cyst induction, and (b) both the MAO inhibitor tranylcypromine and the electroneutral Na+/H+ antiporter monensin lead to encystment in almost the entire cell population. After a 22‐h passage through the aperiodic state with a subsequent step‐up to 15 or 20 °C, the responsiveness to melatonin or 5‐methoxytryptamine, respectively, was approximately normal. Our results indicate that cyst formation via the short‐day/melatonin pathway requires an operating circadian oscillator.     

Daily Rhythms of Metabolic Rate and Body Temperature of Two Murids from Extremely Different Habitats

Daily circadian rhythms of body temperature (T_b) and oxygen consumption (VO₂) were measured in two murid species, which occupy extremely different habitats in Israel. The golden spiny mouse (Acomys mssalus) is a diurnal murid distributed in arid and hot parts of the great Syrio-African Rift Valley, while the broad-toothed field mouse (Apodeinns mystacinus) is a nocturnal species that inhabits the Mediterranean woodlands. In both species, the daily rhythms of T_b and VO₂ are entrained by the photoperiod. Under laboratory experimental conditions (ambient temperature T_a = 33^oC and photoperiod regime of 12L: 12D), Acomys russatus exhibits a tendency towards a nocturnal activity pattern, compared to the diurnal activity displayed by this species under natural conditions. Under the same photoperiod regime and at T_a = 28^oC, Apodemus mystacinus displays nocturnal activity, as observed under natural conditions. The maximal values of T_b were recorded in Acomys russatus at midnight (23:50 h), while the maximal values of VO₂ were recorded at the beginning of the dark period (18:20 h). In Apodemus mystacinus, the maximal values of T_b and VO₂ were recorded at 23:40 and 20:00 h, respectively. The ecophysiological significance of these results is discussed further.     

Dramatic Rises of Melatonin and 5-Methoxytryptamine in Gonyaulax Exposed to Decreased Temperature

The dinoflagellate Gonyaulax polyedra was previously shown to undergo asexual encystment in response to decreased temperature (15° instead of 20°C rearing temperature) in combination with short-days, a response which can be mimicked by melatonin and, much more efficiently, by 5-methoxytryptamine (= 5-MT). We demonstrate that these cyst-inducing conditions lead to enormous accumulations of the two methoxyindoleamines. The circadian rhythmicity of melatonin is maintained for the two days usually preceding cyst formation, though, at an elevated level. Transiently, very high concentrations of melatonin can occur, eventually exceeding 1 millimolar. These extreme concentrations decay rapidly; during this decline, 5-MT and 5-methoxytryptophol appear in large amounts. The concentrations of 5-MT which are measured during this process are higher than those required for cyst induction by the exogenous indoleamine.     

Dramatic Rises of Melatonin and 5-Methoxytryptamine in Gonyaulax Exposed to Decreased Temperature

The dinoflagellate Gonyaulax polyedra was previously shown to undergo asexual encystment in response to decreased temperature (15° instead of 20°C rearing temperature) in combination with short-days, a response which can be mimicked by melatonin and, much more efficiently, by 5-methoxytryptamine (= 5-MT). We demonstrate that these cyst-inducing conditions lead to enormous accumulations of the two methoxyindoleamines. The circadian rhythmicity of melatonin is maintained for the two days usually preceding cyst formation, though, at an elevated level. Transiently, very high concentrations of melatonin can occur, eventually exceeding 1 millimolar. These extreme concentrations decay rapidly; during this decline, 5-MT and 5-methoxytryptophol appear in large amounts. The concentrations of 5-MT which are measured during this process are higher than those required for cyst induction by the exogenous indoleamine.     

Regulation by Light and Darkness of Melatonin Secretion from the Superfused Masu Salmon (Oncorhynchus masou) Pineal Organ

The pineal organ of masu salmon Oncorhynchus masou was maintained in a flow-through, whole-organ culture (superfusion) system and melatonin secretory profiles were determined at 15 °C under light-dark cycles of 12:12 h (LD 12:12) or the same in combination with constant darkness (DD) for 72 h. Under LD 12:12, superfused pineal organs showed a rhythmic melatonin secretion with high and low rates during the dark phase and the light phase, respectively. When the pineal organs maintained under LD 12:12 for 24 h were transferred to DD, melatonin secretion was consistently activated and no endogenous component was evident. When the pineal organs maintained under DD for 48 h were transferred to LD 12:12, melatonin secretion was reduced only during the light phase. These results indicate that melatonin secretion from the superfused pineal organ of masu salmon is regulated not by an intra-pineal circadian oscillator but by the environmental LD cycles, via local photoreceptors.     

Regulation by Light and Darkness of Melatonin Secretion from the Superfused Masu Salmon (Oncorhynchus masou) Pineal Organ

The pineal organ of masu salmon Oncorhynchus masou was maintained in a flow-through, whole-organ culture (superfusion) system and melatonin secretory profiles were determined at 15 °C under light-dark cycles of 12:12 h (LD 12:12) or the same in combination with constant darkness (DD) for 72 h. Under LD 12:12, superfused pineal organs showed a rhythmic melatonin secretion with high and low rates during the dark phase and the light phase, respectively. When the pineal organs maintained under LD 12:12 for 24 h were transferred to DD, melatonin secretion was consistently activated and no endogenous component was evident. When the pineal organs maintained under DD for 48 h were transferred to LD 12:12, melatonin secretion was reduced only during the light phase. These results indicate that melatonin secretion from the superfused pineal organ of masu salmon is regulated not by an intra-pineal circadian oscillator but by the environmental LD cycles, via local photoreceptors.     

Influence of light intensity on plasma melatonin and locomotor activity rhythms in tench

Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12:12 light-dark (LD) conditions with two different light intensities (58.6 and 1091 microW/cm2), and the effect of I h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1091.4 microW/cm2) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12:12 and high light conditions displayed rhythmic variation, where values at MD (255.8 +/- 65.9 pg/ml) were higher than at midlight (ML) (70.7 +/- 31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12: 12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6 +/- 3.2 pg/ml (in the 3.3-10.5 microW/cm2 range) and to 61.8 +/- 18.3 pg/ml (with the 1091.4 microW/cm2 light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).     

Effect of temperature and photoperiod on the calling behaviour of a migratory insect, the oriental armyworm Mythimna separata

Abstract The pre-calling period (PCP), between emergence and first release of pheromone, determines the number of nights over which female Mythimna separata (Walker) (Lepidoptera: Noctuidae) can express their migratory potential. In its seasonal migrations in eastern Asia, this armyworm penetrates to latitudes at which it is unable to over-winter but at which reliable photoperiodic and temperature cues anticipate the deterioration of conditions. The effect of these environmental factors on PCP was examined in moths descended from samples collected in Nanjing (32^oN) during the summer migration. Under LD 14:10h, a reduction in temperature, from 23^oC during larval development to 18^oC from 24 h after pupation, resulted in a twofold increase in PCP compared with insects held at 23^oC. At 23^oC, short days (LD 12:12 h) extended PCP but the magnitude of the response was increased under a regime of decreasing photoperiod, from LD 15:9h, to LD 12:12 h through larval, pupal and adult development. Further experiments demonstrated that the response depended on a decrease in photoperiod from pre-pupal stage to adult emergence (the pupal period is spent below ground). A reduction of 50–60min over the 13–15 day pupal period at 23^oC was effective. The significance of decreasing photoperiods, irrespective of absolute levels, as a cue for species whose migrations traverse substantial ranges of latitude is considered and the results are discussed in relation to the genetic determination of PCP in M.separata and to its seasonal migrations in eastern China.     

Effects of temperature, irradiance, and daylength on the marine diatom Leptocylindrus danicus Cleve. IV. Growth

Growth and dark respiration rates of the marine diatom Leptocylindrus danicus Cleve were measured in axenic batch culture under 49 combinations of temperature (5, 10, 15, 20°C), daylength(15:9, 12:12, 9:15 LD), and irradiance (at least four irradiances per daylength). Cell division rates exhibited a temperature-dependent daylength effect. Optimal temperatures occurred between 15 and 20°C. Both the initial slope () and the growth rate at light saturation (μ_max) were strongly influenced by temperature; increased five-fold and μ_max by an order of magnitude between 5 and 20°C. The compensation irradiance (I_c) was independent of temperature. μ_max was 2.7 div day⁻¹ at 20°C, 2.6 at 15°C, 1.1 at 10°C, and 0.3 at 5 °C. Cells grown under 15:9 and 12:12 LD exhibited similar growth-light curves at 20°C and at 15°C. μ_max of cells grown under 9:15 LD at these temperatures were substantially lower than μ_max under longer daylengths. Growth at 10 and 5°C was independent of daylength.

Dark respiration rates were a linear function of cell division rates at 10, 15, and 20°C, and support the concept that growth rate is dependent on dark respiration rate. These relationships were not influenced by daylength. A detectable relationship between dark respiration and growth at 5°C was not observed.

Photosynthesis and excretion showed temperature-dependent curvilinear relationships with growth rate, reflecting the lower saturation irradiance for growth compared to light saturation of photosynthesis and excretion. The relationship between Chl a-specific photosynthesis and growth was controlled by the C:Chl a ratio, which showed a positive correlation with cell division rate. At 15 and 20°C, light saturation of growth was associated with C:Chl a ratios of 40 to 60; at 5 and 10°C, cells growing at μ_max contained C:Chl a in ratios of 80 to 110.     

Aspects of the thermal biology of the six-lined racerunner, Cnemidophorus sexlineatus (Squamata:Teiidae) in West-Central Florida

(1) I measured the critical thermal maximum (CTM_ax), mean selected temperature (MST), and mean activity temperature (MAT) of six-lined racerunners, (Cnemidophorus sexlineatus) from Florida. (2) Mean CTM_ax and MST of Florida lizards (n=10) were 48.2±0.6 and 33.5±1.9^oC, respectively. MAT (n=22) at 36.6^oC was statistically higher than MST. (3) CTM_ax, MST, and MAT of Florida lizards were statistically lower than those published for a conspecific Oklahoma population.     

Encystment of Gonyaulax polyedra: Dependence on Light

The unicellular alga Gonyaulax polyedra reacts to short days and low temperatures by forming asexual cysts. Its photoperiodic response is elicited via the physiological mediation of melatonin. This indoleamine known as a dark signal in vertebrates is also synthetised by this dinophyt attaining concentrations as high as in the mammalian pineal gland. Its level varies in a circadian fashion, showing a steep increase after the onset of darkness, followed by a gradual decline towards the beginning of photophase. The critical photoperiod of the encystment response shows in Gonyaulax polyedra the remarkably high precision of about half an hour. Under otherwise non-inducing conditions, a single addition of 10 -4 M melatonin, given 1 h before the onset of darkness, elicits encystment as much, and with similar kinetics, as in short-days. The effect of melatonin action during long-day conditions (11:13) and low temperature (15°C) has been investigated. After addition of 10 -4 M or 7x10 -5 M melatonin each 3 h, the cyst-inducing capacity depends on the circadian phase of treatment. The differences in efficiency of melatonin observed are negatively correlated with the endogenous melatonin production of Gonyaulax polyedra which is higher at the begining of darkness. These results lead to novel consequences relating to the rapid catabolism of this substance and the dependence of its efficiency on light.     

Encystment of Gonyaulax polyedra: Dependence on Light

The unicellular alga Gonyaulax polyedra reacts to short days and low temperatures by forming asexual cysts. Its photoperiodic response is elicited via the physiological mediation of melatonin. This indoleamine known as a dark signal in vertebrates is also synthetised by this dinophyt attaining concentrations as high as in the mammalian pineal gland. Its level varies in a circadian fashion, showing a steep increase after the onset of darkness, followed by a gradual decline towards the beginning of photophase. The critical photoperiod of the encystment response shows in Gonyaulax polyedra the remarkably high precision of about half an hour. Under otherwise non-inducing conditions, a single addition of 10 –4 M melatonin, given 1 h before the onset of darkness, elicits encystment as much, and with similar kinetics, as in short-days. The effect of melatonin action during long-day conditions (11:13) and low temperature (15°C) has been investigated. After addition of 10 –4 M or 7x10 –5 M melatonin each 3 h, the cyst-inducing capacity depends on the circadian phase of treatment. The differences in efficiency of melatonin observed are negatively correlated with the endogenous melatonin production of Gonyaulax polyedra which is higher at the begining of darkness. These results lead to novel consequences relating to the rapid catabolism of this substance and the dependence of its efficiency on light.     

Effects of photoperiod and temperature on diapause induction and termination in the swallowtail,Sericinus montelus

Abstract Sericinus montelus overwinters as diapausing pupae. In the present study, the effects of photoperiod and temperature on diapause induction and termination of diapause are investigated. The results obtained demonstrate that high temperature can reverse the effect of short day‐lengths on diapause induction. Under an LD 12 : 12 h photoperiod, all pupae enter diapause at 15, 20 and 25 °C, whereas all pupae develop without diapause at 35 °C. No pupae enter diapause under an LD 14 : 10 h photoperiod when the temperature is above 20 °C. Photoperiodic response curves obtained at 25 and 30 °C indicate that S. montelus is a long‐day species and the critical day‐length is approximately 13 h at 25 °C. At 25 °C, the duration of diapause is shortest when the diapausing pupae are maintained under an LD 16 : 8 h photoperiod and increases under LD 14 : 10 h and LD 12 : 12 h photoperiods. Under an LD 16 : 8 h photoperiod, the duration of diapause is shortest when the diapausing pupae are maintained at 25 °C, followed by 20 and 30 °C, and then at 15 °C. These results suggest that a moderate temperature favours diapause development under a diapause‐averting photoperiod in this species. The duration of diapause induced by an LD 12 : 12 h photoperiod is significantly longer at 25 °C than those at 15, 20 and 30 °C, and is shortest at 15 °C. At 25 °C, the duration of diapause induced by LD 6 : 18, LD 12 : 12 and LD 13 : 11 h photoperiods is similar and longer than 90 days. Thus, the diapause‐inducing conditions may affect diapause intensity and a photoperiod close to the critical day‐length has significant influence on diapause intensity in S. montelus.     

Influence of Light Intensity on Plasma Melatonin and Locomotor Activity Rhythms in Tench

Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12∶12 light‐dark (LD) conditions with two different light intensities (58.6 and 1,091 µW/cm²), and the effect of 1 h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1,091.4 µW/cm²) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12∶12 and high light conditions displayed rhythmic variation, where values at MD (255.8±65.9 pg/ml) were higher than at midlight (ML) (70.7±31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12∶12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6±3.2 pg/ml (in the 3.3–10.5 µW/cm² range) and to 61.8±18.3 pg/ml (with the 1,091.4 µW/cm² light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).     

Changes in melatonin binding sites under artificial light-dark, constant light and constant dark conditions in the masu salmon brain

To test whether the affinity (Kd) and total binding capacity (Bmax) of melatonin receptors exhibit daily and circadian changes in teleost fish whose melatonin secretion is not regulated by intra-pineal clocks, we examined the changes in melatonin binding sites in the brains of underyearling masu salmon Oncorhynchus masou under artificial light-dark (LD), constant light (LL) and constant dark (DD) conditions. In Experiment 1, fish were reared under a long (LD 16:8) or short (LD 8:16) photoperiod for 69 days. Blood and brains were sampled eight times at 3 h intervals. Plasma melatonin levels were high during the dark phase and low during the light phase in both photoperiodic groups. The Bmax exhibited no daily variations. Although the Kd slightly, but significantly, changed under LD 8:16, this may be of little physiological significance. In Experiment 2, fish reared under LD 12:12 for 27 days were exposed to LL or DD from the onset of the dark phase under LD 12:12. Blood and brains were sampled 13 times at 4 h intervals for two complete 24 h cycles. Plasma melatonin levels were constantly high in the DD group and low in the LL group. No significant differences were observed in the Kd and the Bmax between the two groups, and the Kd and the Bmax exhibited no circadian variation either in the LL or DD groups. These results indicate that light conditions have little effect on melatonin binding sites in the masu salmon brain.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC₅₀ values of 0.8 n M for melatonin and 7.2 n M for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[¹²⁵I]iodomelatonin. Melatonin displaced specific 2-[¹²⁵I]iodomelatonin with a K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[¹²⁵I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.     

Photic entrainment of circadian activity patterns in the tropical labrid fish Halichoeres chrysus

Yellow wrasses (Halichoeres chrysus) show clear daily activity patterns. The fish hide in the substrate at (subjective) night, during the distinct rest phase. Initial entrainment in a 12h:12h light-dark (12:12 LD) cycle (mean period 24.02h, SD 0.27h, n = 16 was followed by a free run (mean period 24.42h, SD 1.33h) after transition into constant dim light conditions. Light pulses of a comparable intensity as used in the light part of the LD cycles did not result in significant phase shifts of the free-running rhythm in constant darkness. Application of much brighter 3h light pulses resulted in a phase-response curve (PRC) for a fish species, with pronounced phase advances during late subjective night. The PRCs differed from those mainly obtained in other vertebrate taxa by the absence of significant phase delays in the early subjective night. At that circadian phase, significant tonic effects of the light pulses caused a shortening of the circadian period length. Entrainment to skeleton photoperiods of 1:11 LD was observed in five of six wrasses exposed, also after a 3h phase advance of this LD cycle. Subsequently, a 1:11.25 LD cycle resulted in entrainment in four of the six fish. It is suggested that the expression of the circadian system in fish can be interpreted as a functional response to a weak natural zeitgeber, as present in the marine environment. This response allows photic entrainment as described here in the yellow wrasse. (Chronobiology International, 17(5), 613-622, 2000)     

Resistance, biguanide sorption and biguanide-induced pentose leakage during encystment of Acanthamoeba castellanii

AIMS: This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. METHODS AND RESULTS: A non-nutrient encystment system was used to investigate the changes in the levels of sorption (uptake) of three non-cysticidal concentrations (10, 20 and 50 microg ml(-1)) of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) as well as their effects on viability and leakage of pentose sugars during the first 36 h of encystment. Trophozoites treated with CHA or PHMB were more sensitive and generally sorbed more of each biocide than cysts. During encystment, the largest increases in resistance developed between 18 and 36 h for both biguanides with the resistance emerging to biguanide concentrations of 10 or 20 microg ml(-1) between 18 and 24 h. At 50 microg ml(-1) resistance emerged between 24 and 36 h. There was a general decrease in biocide sorption during encystment between 0-24 and 0-21 h for CHA and PHMB, respectively, at a concentration of 50 microg ml(-1). The greatest decline in biguanide-induced pentose leakage was between 0 and 12 h. CONCLUSIONS: The results suggest that during encystment some of the changes in the susceptibility to CHA or PHMB may be related to decreases in the levels of biocide sorption, which is limited by the developing cyst wall. SIGNIFICANCE AND IMPACT OF THE STUDY: During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.