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Nocturnal Activity in the Green Sea Turtle Alters Daily Profiles of Melatonin and Corticosterone
Hormones and Behavior 41, 357–365 (2002) doi:10.1006/hbeh.2002.1775, available online at http://www.idealibrary.com on
Nocturnal Activity in the Green Sea Turtle Alters Daily Profiles of Melatonin and Corticosterone Tim S. Jessop,* ,† ,1 Colin J. Limpus,‡ and Joan M. Whittier† *Department of Zoology and Entomology, University of Queensland, Brisbane Q4072, Australia; †Department of Anatomy, and Developmental Biology, School of Biomedical Sciences, University of Queensland, Brisbane Q4072, Australia; and ‡Department of Environment, P.O. Box 155, Brisbane Q4002, Australia Received June 15, 2001; accepted October 1, 2001
In nature, green turtles (Chelonia mydas) can exhibit nocturnal activity in addition to their typically diurnal activity cycle. We examined whether nocturnal activity in captive and free-living green turtles altered daily plasma profiles of melatonin (MEL) and corticosterone (CORT). In captivity, diurnally active green turtles expressed distinct diel cycles in MEL and CORT; a nocturnal rise was observed in MEL and a diurnal rise was observed in CORT. However, when induced to perform both low- and high-intensity nocturnal activity, captive green turtles exhibited a significant decrease in MEL, compared to inactive controls. In contrast, plasma CORT increased significantly with nocturnal activity, and further, the relative increase in CORT was correlated with the intensity of the nocturnal behavior. In free-living green turtles that performed nocturnal activity including: nesting, mate searching, and feeding/swimming behaviors, plasma profiles in MEL and CORT exhibited relatively little, or no, daily fluctuation. Our findings demonstrate that nocturnal activity in green turtles is often associated with MEL and CORT profiles that resemble those measured during the day. We speculate that these conspicuous changes in MEL and CORT during nocturnal activity could either support or promote behaviors that enable acquisition of transient resources important to the survival and reproductive success of green turtles. © 2002 Elsevier Science (USA) Key Words: daily hormone cycles; melatonin; corticosterone; daily resource availability; nocturnal activity; green turtle; Chelonia mydas.
INTRODUCTION Animals often exhibit regular patterns of daily activity. These patterns are thought to have evolved in 1 To whom correspondence and reprint requests should be addressed. Present address: Center for Reproduction of Endangered Species, Zoological Society of San Diego, California 92112. E-mail: [email protected].
0018-506X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
response to maximizing daily resource acquisition and/or by limiting an individual’s exposure to negative environmental fluctuations (Daan, 1981; Pittendrigh, 1981). However, animals may also dramatically alter their daily or seasonal activity patterns in response to transient ecological factors that could influence their survival or reproductive success (Daan, 1981; Cannicci, Fratini, and Vannini, M. 1999; Cotton and Parker, 2000). While animals may change daily patterns of activity, we could find no published evidence that such temporal shifts in nature are supported by the appropriate phases of endocrine cycles set by endogenous clocks that are entrained to physical cues (Aschoff, 1979). For example, in animals with diurnal activity patterns, daily cycles in corticosterone (CORT) and melatonin (MEL) can exhibit opposing phases entrained by photoperiod and/or temperature (Joseph and Mier, 1973; Reiter, 1981; Underwood, 1990). A peak in basal CORT is often present during the day, coinciding with activity (but see Breuner, Wingfield, and Romero, 1999), and is suspected to support heightened metabolism and energy demands (Pancak and Taylor, 1983; Summers and Norman, 1988; Dallman, Strack, Akana, Bradbury, Hanson, Scribner, and Smith, 1993). In contrast, a peak in MEL is maximal at night during the rest or sleep phase in diurnally active animals (Reiter, 1981; Underwood, 1990). In some diurnally active vertebrates, there is evidence that MEL has an important role in setting the phase of the daily activity cycle as well as mediating inactivity or sleepiness (Hendel and Turek, 1978; Turek, 1989; Underwood, 1990; Hyde and Underwood, 2000). The capacity of animals to exhibit temporal shifts in daily activity patterns raises the obvious question of what might happen to underlying patterns in daily endocrine cycles. For example, do endocrine cycles
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remain coupled to the normal phase, set by environmental zeitgeibers, independent of phase changes in the daily activity cycle? Alternatively, when animals exhibit temporal shifts in activity to what is normally their sleep or inactive phase, are daily cycles in MEL or CORT modulated? Perhaps as an acclimation response to ensure that the appropriate phases of daily endocrine cycles concur with activity? Studies on humans, investigating the effects of night-shift work, nocturnal exercise, or jetlag on the daily MEL and CORT cycles suggest that individuals have a variable capacity to compensate endocrine cycles to temporal shifts in daily activity (Montelone, Maj, Fusco, Orazzo, and Kemali, 1990; Weibel, Spiegel, Follenius, Erhart, and Brandenberger, 1996; Weibel, Spiegel, Gronfier, Follenius, and Brandenberger, 1997). However, to our knowledge, these hypotheses remain to be tested on other species, specifically within a broader ecological framework under natural conditions. This study attempts to address these issues by examining if the green turtle (Chelonia mydas), a marine reptile, modulates endocrine cycles of MEL and CORT during periods of nocturnal activity. Sea turtles typically exhibit diurnal activity patterns (Odgen, Robinson, Whitlock, Daganhardt, and Cebula, 1983; Van Dam and Diez, 1996; Hays, Adams, Broderick, Godley, Lucas, Metcalf, and Prior, 2000), however, they may also exhibit several distinct behaviors that result in intermittent bouts of nocturnal activity. During the nesting season, breeding female green turtles will occupy an internesting habitat and periodically emerge at night from the ocean to nest on a beach. During courtship, male green turtles often rely on scramble competition and will search actively throughout the night to locate receptive females (Jessop, FitzSimmons, Limpus, and Whittier, 1999b). Further, we have observed green turtles feeding or swimming at night within the coral reefs of the Great Barrier Reef. These three examples illustrate the ability of sea turtles to use nocturnal activity in addition to a diurnal activity pattern to acquire transient resources present at night, including a cooler nesting habitat, receptive females, and forage, respectively. In an earlier study on a captive population of green turtles, Owens, Gern, and Ralph (1980) described a typical MEL rhythm with a substantial nocturnal peak. However, when nesting at night, captive female green turtles exhibited low concentrations of plasma MEL similar to those recorded during the day (Owens et al., 1980). These authors attributed this decreased nocturnal MEL to possible stress or heightened metabolism during the physically intense nesting process
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in this species. Although some phases of nesting behaviour are metabolically demanding, it is possible that this result also provides evidence that nocturnal activity, or simply, nocturnal arousal per se, was responsible for depression of the nocturnal MEL peak. In our study, we conducted research under laboratory and field conditions to test interactions between nocturnal activity and daily cycles of MEL and CORT in green turtles. We predicted that green sea turtles showing nocturnal activity could modify daily plasma MEL or CORT cycles by decreasing MEL and increasing CORT levels at night, and thus effectively reduce daily variation in these hormone cycles. For this study, we addressed three aims relating to the possible influence of nocturnal activity upon the daily cycles of MEL and CORT in the green turtle. First, we reconfirmed whether captive-reared green turtles have a typical nocturnal increase in MEL and a diurnal CORT increase. Second, we examined if varying intensities of nocturnal activity in captive turtles were correlated with changes in nocturnal plasma concentrations of MEL and CORT. In this second study, we also sought to identify if there was some threshold of intensity at which activity changed nocturnal concentrations of MEL and CORT. Our third study, undertaken in the field, compared the interactions between three different nocturnal behaviors and endocrine profiles of MEL and CORT in the green turtle.
METHODS Captive Studies Six captive-reared juvenile green turtles, housed at the Department of Zoology and Entomology, at the University of Queensland in Brisbane, Australia were utilized for our two initial studies. Turtles were kept in two 500 l saltwater tanks and fed daily (07:00 –09:00) with a commercial diet of dry trout pellets. Tanks were housed in a semi-enclosed building that provided direct overhead cover but ambient light was introduced through open-sided walls. The water was maintained at a constant temperature of 27 ⫾ 2°C using submersible heaters. In January when these experiments were conducted, turtles were exposed to an ambient summer photoperiod with sunrise and sunset recorded at 6:06 ⫾ 03 and 19:17 ⫾ 03, respectively. To determine the presence of MEL and CORT cycles, turtles were bled twice daily, at two times, selected from 10 time intervals, every three days for a 15-day
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Daily Profiles of Melatonin and Corticosterone
period. Blood samples were collected at 3:00, 6:00, 9:00, 12:00, 15:00, 18:00, 19:30, 21:00, 22:30, and 24:00. We staggered our bleeding protocol to minimize the possible effects of cumulative daily disturbance (through repetitive capture, handling, and blood sampling) on profiles of plasma CORT and MEL. As a general aside, we do not refer to the daily MEL and CORT cycles as circadian rhythms (although they may well be) as we did not establish if these hormone cycles persisted in captive sea turtles under constant conditions.
The Effect of Nocturnal Activity on Hormone Profiles in Captive Green Turtles To determine if nocturnal activity altered daily hormone profiles in diurnally active green turtles, we compared MEL and CORT profiles in response to three different levels of activity: (1) Inactive (Control). Turtles were allowed to rest (akin to sleep) undisturbed during their normal inactive period and blood samples were taken at 22:00 to get reference values for nocturnal levels of plasma MEL and CORT. (2) Low-intensity activity. Turtles were subjected to a passive disturbance administered at night from 21:00 –22:00 by repetitively and gently moving the mesh dividers partitioning turtles in their tanks. This low-intensity activity stimulus was designed to arouse turtles. It induced the turtles to float in the water column or to gently swim, and it prevented inactivity. At 22:00, this low-intensity arousal stimulus was stopped and blood samples were collected. (3) High-intensity activity. Turtles were subjected to a high-intensity activity stimulus by exposing them to a circular water current (⬎1 m/s, created by bilge pumps) in their tanks. The current forced turtles to exhibit bursts of very active swimming. At 22:00, this high-intensity stimulus was ceased and blood samples were collected. Each activity treatment was conducted independently and separated by a three-day interval. Again, we decided against using a simultaneous comparison of treatments (repeated over several nights) as the disturbance of the bilge pumps and water flow was enough to induce nocturnal arousal in turtles in the adjacent tank.
Field Studies A total of 531 green sea turtles were captured and blood sampled in the field. Field studies were conducted in waters adjacent to, or on, the beaches of Heron (23° 26⬘S; 151°57⬘E) and Raine Islands (11° 37⬘S; 144°01⬘E), located within the Great Barrier Reef, off the coast of Queensland, Australia. Our field studies took place during late spring and early summer (mid October to mid December). The mean sunrise and sunset were recorded at 5:15 ⫾ 02 h and 18:05 ⫾ 01 h for Heron Island, and 5:51 ⫾ 01 h and 18:37 ⫾ 02 h for Raine Island. The mean daily air and ocean temperature on Heron and Raine islands during this study were 25.6 ⫾ 0.16°C and 25.25 ⫾ 1.02°C and 32.4 ⫾ 0.12°C and 29.2 ⫾ 0.01°C, respectively. At both islands, blood samples were collected from sea turtles that were hand captured in water using the rodeo method (Limpus and Reed, 1985), or on land during the day and night while displaying one of three behaviors: (1) Female nesting behavior. Individual breeding females were captured and blood sampled throughout the day and night. On Raine Island, females were sampled while exhibiting nesting behavior on the beach throughout the entire day. These blood samples were only analyzed for MEL, as diurnal nesting on this island resulted in heat-stress by mid-afternoon which significantly increased CORT levels (Jessop, Hamann, Read, and Limpus, 2000). To measure the influence of nesting behavior on the daily cycle of plasma CORT, nesting and internesting females (females captured in water during the day when no nesting activity takes place) were sampled from Heron Island. (2) Male courtship behavior. During courtship, male green turtles exhibit various reproductive behaviors (i.e., mate searching and copulation) that persist throughout the night. We compared plasma hormone profiles of MEL and CORT in male turtles captured when exhibiting reproductive behavior during the day (between 9:00 and 15:00) against profiles of males captured at night (21:30 –1:00). (3) General activity behavior. During October fieldwork, swimming or feeding behavior was observed in green turtles around Heron Island throughout the day and night, albeit more commonly during the day. For green turtles in this category, we focused our sampling on juvenile green turtles (less than 65 cm in
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curved carapace length), as overall these turtles were the most similar in size and reproductive status to captive animals. Again, turtles were captured and blood sampled during the day (9:00 –13:00) and night (21:00 –1:00). Blood Sampling Protocols Blood samples (1–2 ml) were taken from the dorsal cervical sinus of sea turtles using a 21gauge needle and 5-ml syringe. All blood samples were transferred to lithium heparin vials and placed in iceboxes or refrigerators for up to 6 h, then centrifuged at 4000 rpm for 5 min. Plasma was decanted into cryotubes and frozen in liquid nitrogen and then transferred to a ⫺ 20°C refrigerator for storage. Extended nocturnal exposure (⬎1–2 h) to bright white light has been demonstrated to suppress nocturnal MEL synthesis in sea turtles, however, shorter periods, up to several minutes, are not thought to suppress plasma MEL levels (Owens et al., 1980; Owens and Gern, 1985; Owens, personal communication). In our captive studies, we took measures to limit any light effects on plasma MEL by capturing and blood sampling turtles under red light, a method widely used to eliminated any inhibitory effects of white light on MEL. In the field, our precautions included limiting the use of small, head-mounted lights to brief periods (⬍2 min for turtle captured in water, ⬍1 min for nesting females) during the final phase of the turtle’s capture. Furthermore, during blood sampling the turtles eyes were covered with our hands to prevent exposure to dull torchlight. Radioimmunoassay Plasma MEL was analyzed by a specific radioimmunoassay (RIA) slightly modified from that used for humans (Fraser, Cowen, Franklin, Francey, and Arendt, 1983). Plasma CORT was analyzed by a direct RIA procedure similar to that previously validated for green turtles (Jessop, Limpus, and Whittier, 1999a). A 100 –300 l aliquot of plasma was extracted twice with 3 ml of diethyl ether, vortexed for 1 min, and centrifuged at 2500 rpm for 5 min. The organic phase was pipetted into a 16 ⫻ 100 mm disposable glass culture tube and placed into a 37°C dry bath, then evaporated to dryness before 0.5 ml Tris buffered saline, pH 7.0, was added. Duplicated samples were incubated overnight at 4°C with tritiated labeled MEL (Amersham, Australia) and MEL specific antibody (Stockgrand Ltd., Surrey, UK), or with tritiated labeled CORT
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(NET-399, Dupont Inc., Sydney, Australia) and CORT specific antibody (Endocrine Sciences, Tarzana, CA). Unbound antigen was separated from free antigen using a charcoal– dextran solution (1.25%/0.125%). The bound fraction was decanted into poly-Q vials (Beckman Scientific) containing 4 ml aqueous based scintillant (BCS, Amersham) and counted on a Beckman LS6000 scintillation counter. Mean extraction efficiencies were 88.6% and 88.5% for MEL and CORT as determined by recovery of tritiated label added to samples prior to adding diethyl ether. The average intraassay coefficient of variation was 8.7 and 12.2%, for MEL and CORT respectively. The interassay coefficient of variation was 7.6 and 11.2%, for MEL and CORT, respectively. Statistical Analysis All hormone data was log transformed (log ⫻ ⫹ 1) prior to analysis to correct for heteroscedasticity. To prevent violation of the assumption of independence, a single blood sample was taken from each individual captured in the field. Repeated measures ANOVA were used to analyze differences in plasma concentrations of MEL and CORT in captive green turtles. A post hoc multiple comparison test was performed using Tukey’s HSD. For data collected from breeding males and juvenile green turtles in the field, a twosample t test or the nonparametric Mann–Whitney test was used to detect differences between day and night levels of MEL and CORT. ANOVA or nonlinear regressions were used to identify significant correlation’s between MEL and CORT and the time of day in nesting green turtles. All differences were considered significant at P ⬍ 0.05.
RESULTS Captive Green Turtles In diurnally active captive green turtles, large daily variation was observed in the daily cycle of plasma MEL (Fig. 1). As expected, MEL increased during the night from 19:30 onward before declining from its peak at 12:00 and reaching basal levels at 9:00 (repeatedmeasures ANOVA, F 9,50 ⫽ 10.62, P ⬍ 0.001). In contrast, CORT displayed a diurnal profile in which plasma levels were significantly elevated during the day from 6:00 to 15:00 before declining to basal levels during the night coinciding with inactivity (repeatedmeasures ANOVA, F 9,50 ⫽ 11.76, P ⬍ 0.001).
Daily Profiles of Melatonin and Corticosterone
FIG. 1. The daily profiles of plasma melatonin (closed circles; mean ⫾ SEM) and corticosterone (open circles; mean ⫾ SEM) in captive juvenile green turtles maintained at 27 ⫾ 5°C under an ambient summer photoperiod. There was significant variation in both hormones with time of day (P ⬍ 0.01). Six turtles were sampled at each time period, the downward pointing arrows represent the onset of sunrise and sunset, respectively, and the horizontal black bars denote the scotophase.
Nocturnal activity in captive green turtles during their inactive rest phase had conspicuous effects on the nocturnal profiles of plasma MEL and CORT. Plasma MEL decreased significantly in response to both low- and high- intensity nocturnal activity compared to levels in inactive turtles sampled at 22:00 (repeated-measures ANOVA, F 2,13 ⫽ 6.19, P ⫽ 0.011; Fig. 2). Post hoc comparisons indicated that MEL levels in the high activity (Tukey HSD, q ⫽ 4.98, P ⬍ 0.05) and low activity treatments (Tukey HSD, q ⫽ 4.26, P ⬍ 0.05) differed significantly from the inactive control group, but not from one another (Tukey HSD, q ⫽ 2.35, P ⬎ 0.05). In contrast, plasma CORT increased significantly and in step with the different intensities of nocturnal activity compared to inactive controls (repeated measures ANOVA F 2,13 ⫽ 23.35, P ⬍ 0.001; Fig. 2). Post hoc comparisons indicated that CORT levels for all treatments were significantly different from each other (Tukey HSD’s all P ⬍ 0.05).
361 F 1,203 ⫽ 7.69, P ⬍ 0.001, Fig. 3a). Despite this general pattern, 40% of nocturnal nesting females had plasma MEL levels that were the equal to or below the mean of 3.69 ⫾ 0.71 pg/ml measured for nesting females sampled throughout the day (6:00 –14:00). Nesting females showed no relationship between plasma levels of CORT and time of day (Polynomial Function: Plasma CORT ⫽ 2.751 ⫹ ⫺ 0.356*Time of Day 2, ⫺ 0.01*Time of Day 2, R 2 ⫽ 0.028, N ⫽ 119; ANOVA: F 1,118 ⫽ 1.33; P ⫽ 0.34, Fig. 3b). CORT levels remained relatively uniform throughout the day in nesting females and overall, levels of plasma CORT were greater in nesting females compared to captive juveniles. Unlike the pronounced daily endocrine cycles of their captive diurnal counterparts, free-living nocturnally active breeding male and juvenile green turtles had either minimal or reversed daily variation in profiles of MEL and CORT. Again, the relative concentrations of plasma MEL and CORT were less and greater, respectively, than that measured for captive turtles. In breeding male green turtles exhibiting nocturnal mate searching we found no difference between day and night levels of plasma MEL (Mann–Whitney Rank Sum Test: T ⫽ 1830, N (Day) ⫽ 24, N (Night) ⫽ 112, P ⫽ 0.29, Fig. 4A) or CORT (T test: t 1,137 ⫽ 1.26, P ⫽ 0.21). Likewise, juvenile green turtles exhibiting swimming or foraging activity had levels of plasma MEL that were similar between day and night sam-
Free-Living Sea Turtles Nesting females showed small daily variation in plasma MEL compared to juveniles in captivity. However, despite this decreased daily variation, plasma MEL was still significantly correlated with time of day, with an overall increase at night (Quadratic Function: Plasma MEL ⫽ 18.08 ⫹ ⫺ 2.23 * Time of Day ⫹ 0.08 * Time of Day 2, R 2 ⫽ 0.071, N ⫽ 204; ANOVA:
FIG. 2. A comparison of plasma melatonin (mean ⫾ SEM) and corticosterone (mean ⫾ SEM) concentrations measured for captive green turtles (at 22:00) which were inactive or that had just performed 1 h of low or high intensity activity prior to blood sampling. Nocturnal activity induced a significant decrease in plasma melatonin (P ⬍ 0.01), but significantly increased plasma corticosterone (P ⱕ 0.01) compared to inactive turtles. Significant post hoc (Tukey’s HSD) differences in plasma hormone levels are denoted by different superscripts.
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free-living sea turtles undertook a range of different nocturnal activities, there was either no or only a small overall increase in nocturnal plasma MEL. Even in nesting turtles where nocturnal levels of MEL were slightly, but significantly, greater in magnitude than daytime values, many females still had plasma MEL levels within the range of those measured during the day. In other diurnal species, including humans and a captive bird, nocturnal exercise or activity may also be associated with decreased levels of plasma MEL (Montelone et al., 1989; Van-Reeth, Sturis, Byrne, Blackman, L’Hermite-Baleriaux, Leproult, Oliner, Refetoff, Turek, and Van-Cauter, 1994; Gwinner, 1996; Buxton, Frank, L’Hermite-Baleriaux, Leproult, Turek, and Van Cauter, 1997). However, in comparison to humans and birds, the intensity of nocturnal activity required by sea turtles to suppress MEL is much less. Relatively mild but sustained nocturnal arousal was
FIG. 3. The daily profiles of (A) plasma melatonin and (B) corticosterone from nesting green turtles sampled at Raine Island (N ⫽ 204) and Heron Island (N ⫽ 119), respectively. Nonlinear regression indicated a significant relationship between time of day and plasma Melatonin. Downward pointing arrows represent the onset of sunrise and sunset, the horizontal black bars denote the scotophase.
pling periods (T test: t 1,35 ⫽ 0.01, P ⫽ 1.00; Fig. 4B). However, juvenile turtles active at night had plasma CORT levels that were significantly greater than those measured in juvenile turtles active during the day (T test: t 1,35 ⫽ 3.07, P ⫽ 0.003; Fig. 4B).
DISCUSSION A typical nocturnal MEL cycle was observed in captive green turtles exhibiting a diurnal activity cycle. This result is consistent with findings in many animals, including a previous study addressing the presence of a daily MEL cycle in green turtles (Owens et al., 1980; Reiter, 1981; Delgado and Vivien-Roels, 1989; Gwinner, 1996). However, when captive and
FIG. 4. Comparison of day (turtles sampled between 9:00 and 15:00) and night (turtles sampled between 21:30 and 1:00) levels of melatonin (mean ⫾ SEM) and corticosterone (mean ⫾ SEM) in breeding male green turtles involved in (A) reproductive behaviors and (B) juvenile green turtles exhibiting feeding/swimming behavior at Heron Island. Nocturnal levels of plasma corticosterone were significantly elevated (P ⬍ 0.05 denoted by asterix) compared to day levels in juvenile turtles captured while swimming or feeding. Sample sizes for each category are numerically represented within the bars.
Daily Profiles of Melatonin and Corticosterone
enough to decrease plasma MEL compared to levels obtained from inactive turtles in captivity. Captive green turtles possessed a broad day time peak in plasma CORT coinciding with daily activity; this pattern was similar to some other diurnal species (Joseph and Mier, 1973; Summers and Norman, 1988). This diurnal peak in the daily CORT cycle was obscured by nocturnal activity that increased plasma CORT concentrations during the night and thus reduced daily variation in this endocrine cycle. Further, it was evident that nocturnal levels of CORT could exceed daytime levels if the intensity of nocturnal activity was great enough, as observed in swimming green turtles. Exercise-induced stress may have also played a factor in increasing plasma CORT in captive turtles exhibiting intense bouts of swimming. This general tendency for increased CORT levels with nocturnal activity is likely to reflect increased metabolism. During greater aerobic demands, catabolic hormones such as CORT serve to catabolize endogenous fuel reserves to maintain the expression of activity (Norris, 1997). Therefore, behavior per se may not actually alter the endogenous basal cycle, instead the presumed metabolic effects of nocturnal behaviors would seem to increase CORT and thus mask any inherent daily variation in this endocrine cycle (Jessop, 2000). Sea turtles exhibiting nocturnal activity typically reduced their daily variation in hormones cycles of MEL and CORT, so that nocturnal levels of MEL and CORT decreased and increased, respectively. In essence, these nocturnal endocrine patterns were often similar to those measured during the day. We interpret that these conspicuous changes in the hormone profiles of nocturnally active turtles are a consequence of heightened activity or arousal. It is possible that activity reduced nocturnal levels of MEL due to elevated metabolism leading to increased hepatic clearance of MEL from the plasma (Owens et al., 1980). While this reasoning is plausible for physically demanding behaviors such as nesting and mating, this study demonstrated that even mild nocturnal activity was associated with reduced plasma MEL in green turtles. Thus, we suspect even simple nocturnal arousal can trigger mechanisms leading to a suppression of MEL during nocturnal activity. In the field, other factors could also possibly account for why nocturnally active turtles possessed low levels of MEL. For instance, prolonged exposure to bright light is known to inhibit nocturnal MEL secretion in captive green turtles (Owens and Gern, 1985). Perhaps, prolonged exposure to even weak ambient light by turtles
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active at night in nature is sufficient to induce this effect. Irrespective of why nocturnally active turtles possessed low levels of plasma MEL, our findings invite speculation about the consequences of this association. One hypothesis is that the reduced levels of MEL in nocturnally active sea turtles provides similar phase properties to the day time profile of MEL when turtles normally conduct most of their activity. In humans, behavioral and physiological disorders that arise with nocturnal shift work, or through jet-lag are due to dissociation between daily activity and the appropriate phases of other physiological, endocrine, and behavioral cycles (Weibel et al., 1996, 1997). Whether, the reduction in plasma MEL is a mechanism that nocturnally active green turtles employ to eliminate similar negative consequences remains to be determined. Another possibility of why nocturnally active green turtles exhibit reduced levels of MEL is to reduce this hormone’s capacity for potentially inhibiting nocturnal activity in otherwise typically diurnal animals. Several studies have reported that exogenous MEL causes sleep-like behavior, drowsiness, and decreased activity in several diurnal vertebrates (Turek, 1989; Chiba et al., 1985; Hyde and Underwood, 2000). In some reptiles, MEL also appears important for initiating the onset of daily inactivity, at least under constant conditions (Underwood, 1990; Hyde and Underwood, 2000). Diurnal birds that use nocturnal activity (Zugunruhe) for vernal migration are reported to reduce nocturnal levels of plasma MEL (Gwinner, 1996). However, if birds receive exogenous MEL during Zugunruhe a suppression in activity can result (Pohl, 2000). Supposing that in green turtles a similar inhibitory effect on nocturnal activity is caused by elevated levels of MEL, then reduced nocturnal levels in this hormone could represent an important mechanism to maintain or promote nocturnal activity in green turtles. Clearly, manipulative experiments using exogenous MEL are needed to test these hypotheses on the role of MEL in influencing the proclivity of nocturnal behavior in green turtles. In conclusion, sea turtles can exhibit temporal shifts in activity to acquire nocturnal resources that presumably influence their survival and reproduction. During nocturnal activity, sea turtles exhibit conspicuous changes in their daily cycles of plasma MEL and CORT. One explanation for these findings could be that compensatory changes in daily hormonal cycles promote and support nocturnal activity. Simply, active behavior and the appropriate phase of the endocrine cycle remained coupled to each other indepen-
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dent of the temporal environment set by physical cues. A cursory glance at the MEL literature reveals that several different daily patterns of MEL secretion can persist in vertebrates, including absence, in addition to the usual nocturnal pattern that typifies most species (Roth, Gern, Roth, Ralph, and Jacobson, 1980; Serino, d’Istria and Montelone, 1993; Taniguchi, Murakami, Nakamura, Nasu, Shinohara, and Etoh, 1993; Mendonca, Tousignant, and Crews, 1995; Van’t Hof and Gwinner, 1998). Again research is needed to determine if these alternative MEL patterns represent facultative or obligatory responses to nondiurnal patterns of activity, as many of these species, including owls, frogs, and crocodilians, exhibit nocturnal or highly variable patterns of daily activity.
ACKNOWLEDGMENTS We thank R. Gnome for help with the husbandry of the captive turtles, and M. Hamann and M. Read for their assistance in blood sampling while on Raine and/or Heron Island. Queensland Parks and Wildlife Service and the Great Barrier Reef Marine Park Authority made important logistical contributions to this study. We thank D. Owens for providing input into an earlier draft of this manuscript. This work was supported by an Australian Postgraduate Award (to T.S.J.) and an Australian Research Council grant (to C.J.L. and J.M.W.). This project was conducted as part of the Queensland Turtle Research Project. This study was approved by the University of Queensland’s Animal Ethics Committee (zoo/ 225/96/urg).
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Pancak, M. K., and Taylor, D. H. (1983). Seasonal and daily plasma corticosterone rhythms in American toads, Bufo americanus. Gen. Comp. Endocrinol. 50, 490 – 497. Pittendrigh, C. S. (1981). Circadian Systems: General perspective. In J. Aschoff (Ed.), Handbook of Behavioral Neurobiology, Vol. 4: Biological Rhythms, pp. 57–77. Plenum Press, New York. Pohl, H. (2000). Circadian control of migratory restlessness and the effects of exogenous melatonin in the brambling, Fringilla montifringilla. Chronobiol. Internat. 17, 471– 488. Reiter, R. (1984). The Pineal Gland. Raven Press. New York. Roth, J. J., Gern, W. A., Roth, E. C., Ralph, C. L., and Jacobson, E. (1980). Nonpineal melatonin in the alligator (Alligator mississippiensis). Science 210, 548 –549. Serino, I., d’Istria, M., and Montelone, P. (1993). A comparative study of melatonin production in the retina, pineal gland and Harderian gland of Bufo viridis and Rana esculenta. Comp. Biochem. Physiol. 106a, 189 –193. Summers, C. H., and Norman, M. F. (1988). Chronic low humiditystress in the lizard Anolis carolinensis: Changes in diurnal corticosterone rhythms. J. Exp. Zool. 247(3), 271–278. Taniguchi, M., Murakami, N., Nakamura, H., Nasu, T., Shinohara, S., and Etoh, T. (1993). Melatonin release from pineal cells of diurnal and nocturnal birds. Brain Res. 620, 297–300. Turek, F. W. (1989). Effects of stimulated physical activity on the
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circadian pacemaker of vertebrates. In S. Daan and E. Gwinner (Eds.), Biological clocks and environmental time, pp. 23–36. Guilford Press, New York. Underwood, H. (1990). The pineal and melatonin: Regulators of circadian function in lower vertebrates. Experientia 45, 914 –922; 46, 120 –128. Van Dam, R. P., and Diez, C. E. (1996). Diving behavior of immature hawksbills (Eretmochelys imbricata) in a Caribbean cliff-wall habitat. Mar. Biol. 127, 171–178. Van-Reeth, O., Sturis, J., Byrne, M. M., Blackman, J. D., L’HermiteBaleriaux, M., Leproult, R., Oliner, C., Refetoff, S., Turek, F. W., and Van-Cauter, E. (1994). Nocturnal exercise phase delays circadian rhythms of melatonin and thyrotropin secretion in normal men. Am. J. Physiol. 266, E96 –E974. Van’t Hof, T. J., and Gwinner, E. (1998). A highly rudimentary circadian melatonin rhythm in a nocturnal bird, the barn owl (Tyto alba). Naturwissenschaften 85, 402– 404. Weibel, L., Spiegel, M., Follenius, M., Erhart, J., and Brandenberger, G. (1996). Internal dissociation of the circadian markers of the cortisol rhythm in night workers. Am. J. Physiol. 270, E608 –E613. Weibel, L., Spiegel, M., Gronfier, C., Follenius, M., and Brandenberger, G. (1997). Twenty-four-hour melatonin and core body temperature rhythms: Their adaptation in night workers. Am. J. Physiol. 41, R945–R954.
Nocturnal Activity in the Green Sea Turtle Alters Daily Profiles of Melatonin and Corticosterone Tim S. Jessop,* ,† ,1 Colin J. Limpus,‡ and Joan M. Whittier† *Department of Zoology and Entomology, University of Queensland, Brisbane Q4072, Australia; †Department of Anatomy, and Developmental Biology, School of Biomedical Sciences, University of Queensland, Brisbane Q4072, Australia; and ‡Department of Environment, P.O. Box 155, Brisbane Q4002, Australia Received June 15, 2001; accepted October 1, 2001
In nature, green turtles (Chelonia mydas) can exhibit nocturnal activity in addition to their typically diurnal activity cycle. We examined whether nocturnal activity in captive and free-living green turtles altered daily plasma profiles of melatonin (MEL) and corticosterone (CORT). In captivity, diurnally active green turtles expressed distinct diel cycles in MEL and CORT; a nocturnal rise was observed in MEL and a diurnal rise was observed in CORT. However, when induced to perform both low- and high-intensity nocturnal activity, captive green turtles exhibited a significant decrease in MEL, compared to inactive controls. In contrast, plasma CORT increased significantly with nocturnal activity, and further, the relative increase in CORT was correlated with the intensity of the nocturnal behavior. In free-living green turtles that performed nocturnal activity including: nesting, mate searching, and feeding/swimming behaviors, plasma profiles in MEL and CORT exhibited relatively little, or no, daily fluctuation. Our findings demonstrate that nocturnal activity in green turtles is often associated with MEL and CORT profiles that resemble those measured during the day. We speculate that these conspicuous changes in MEL and CORT during nocturnal activity could either support or promote behaviors that enable acquisition of transient resources important to the survival and reproductive success of green turtles. © 2002 Elsevier Science (USA) Key Words: daily hormone cycles; melatonin; corticosterone; daily resource availability; nocturnal activity; green turtle; Chelonia mydas.
INTRODUCTION Animals often exhibit regular patterns of daily activity. These patterns are thought to have evolved in 1 To whom correspondence and reprint requests should be addressed. Present address: Center for Reproduction of Endangered Species, Zoological Society of San Diego, California 92112. E-mail: [email protected].
0018-506X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
response to maximizing daily resource acquisition and/or by limiting an individual’s exposure to negative environmental fluctuations (Daan, 1981; Pittendrigh, 1981). However, animals may also dramatically alter their daily or seasonal activity patterns in response to transient ecological factors that could influence their survival or reproductive success (Daan, 1981; Cannicci, Fratini, and Vannini, M. 1999; Cotton and Parker, 2000). While animals may change daily patterns of activity, we could find no published evidence that such temporal shifts in nature are supported by the appropriate phases of endocrine cycles set by endogenous clocks that are entrained to physical cues (Aschoff, 1979). For example, in animals with diurnal activity patterns, daily cycles in corticosterone (CORT) and melatonin (MEL) can exhibit opposing phases entrained by photoperiod and/or temperature (Joseph and Mier, 1973; Reiter, 1981; Underwood, 1990). A peak in basal CORT is often present during the day, coinciding with activity (but see Breuner, Wingfield, and Romero, 1999), and is suspected to support heightened metabolism and energy demands (Pancak and Taylor, 1983; Summers and Norman, 1988; Dallman, Strack, Akana, Bradbury, Hanson, Scribner, and Smith, 1993). In contrast, a peak in MEL is maximal at night during the rest or sleep phase in diurnally active animals (Reiter, 1981; Underwood, 1990). In some diurnally active vertebrates, there is evidence that MEL has an important role in setting the phase of the daily activity cycle as well as mediating inactivity or sleepiness (Hendel and Turek, 1978; Turek, 1989; Underwood, 1990; Hyde and Underwood, 2000). The capacity of animals to exhibit temporal shifts in daily activity patterns raises the obvious question of what might happen to underlying patterns in daily endocrine cycles. For example, do endocrine cycles
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remain coupled to the normal phase, set by environmental zeitgeibers, independent of phase changes in the daily activity cycle? Alternatively, when animals exhibit temporal shifts in activity to what is normally their sleep or inactive phase, are daily cycles in MEL or CORT modulated? Perhaps as an acclimation response to ensure that the appropriate phases of daily endocrine cycles concur with activity? Studies on humans, investigating the effects of night-shift work, nocturnal exercise, or jetlag on the daily MEL and CORT cycles suggest that individuals have a variable capacity to compensate endocrine cycles to temporal shifts in daily activity (Montelone, Maj, Fusco, Orazzo, and Kemali, 1990; Weibel, Spiegel, Follenius, Erhart, and Brandenberger, 1996; Weibel, Spiegel, Gronfier, Follenius, and Brandenberger, 1997). However, to our knowledge, these hypotheses remain to be tested on other species, specifically within a broader ecological framework under natural conditions. This study attempts to address these issues by examining if the green turtle (Chelonia mydas), a marine reptile, modulates endocrine cycles of MEL and CORT during periods of nocturnal activity. Sea turtles typically exhibit diurnal activity patterns (Odgen, Robinson, Whitlock, Daganhardt, and Cebula, 1983; Van Dam and Diez, 1996; Hays, Adams, Broderick, Godley, Lucas, Metcalf, and Prior, 2000), however, they may also exhibit several distinct behaviors that result in intermittent bouts of nocturnal activity. During the nesting season, breeding female green turtles will occupy an internesting habitat and periodically emerge at night from the ocean to nest on a beach. During courtship, male green turtles often rely on scramble competition and will search actively throughout the night to locate receptive females (Jessop, FitzSimmons, Limpus, and Whittier, 1999b). Further, we have observed green turtles feeding or swimming at night within the coral reefs of the Great Barrier Reef. These three examples illustrate the ability of sea turtles to use nocturnal activity in addition to a diurnal activity pattern to acquire transient resources present at night, including a cooler nesting habitat, receptive females, and forage, respectively. In an earlier study on a captive population of green turtles, Owens, Gern, and Ralph (1980) described a typical MEL rhythm with a substantial nocturnal peak. However, when nesting at night, captive female green turtles exhibited low concentrations of plasma MEL similar to those recorded during the day (Owens et al., 1980). These authors attributed this decreased nocturnal MEL to possible stress or heightened metabolism during the physically intense nesting process
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in this species. Although some phases of nesting behaviour are metabolically demanding, it is possible that this result also provides evidence that nocturnal activity, or simply, nocturnal arousal per se, was responsible for depression of the nocturnal MEL peak. In our study, we conducted research under laboratory and field conditions to test interactions between nocturnal activity and daily cycles of MEL and CORT in green turtles. We predicted that green sea turtles showing nocturnal activity could modify daily plasma MEL or CORT cycles by decreasing MEL and increasing CORT levels at night, and thus effectively reduce daily variation in these hormone cycles. For this study, we addressed three aims relating to the possible influence of nocturnal activity upon the daily cycles of MEL and CORT in the green turtle. First, we reconfirmed whether captive-reared green turtles have a typical nocturnal increase in MEL and a diurnal CORT increase. Second, we examined if varying intensities of nocturnal activity in captive turtles were correlated with changes in nocturnal plasma concentrations of MEL and CORT. In this second study, we also sought to identify if there was some threshold of intensity at which activity changed nocturnal concentrations of MEL and CORT. Our third study, undertaken in the field, compared the interactions between three different nocturnal behaviors and endocrine profiles of MEL and CORT in the green turtle.
METHODS Captive Studies Six captive-reared juvenile green turtles, housed at the Department of Zoology and Entomology, at the University of Queensland in Brisbane, Australia were utilized for our two initial studies. Turtles were kept in two 500 l saltwater tanks and fed daily (07:00 –09:00) with a commercial diet of dry trout pellets. Tanks were housed in a semi-enclosed building that provided direct overhead cover but ambient light was introduced through open-sided walls. The water was maintained at a constant temperature of 27 ⫾ 2°C using submersible heaters. In January when these experiments were conducted, turtles were exposed to an ambient summer photoperiod with sunrise and sunset recorded at 6:06 ⫾ 03 and 19:17 ⫾ 03, respectively. To determine the presence of MEL and CORT cycles, turtles were bled twice daily, at two times, selected from 10 time intervals, every three days for a 15-day
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period. Blood samples were collected at 3:00, 6:00, 9:00, 12:00, 15:00, 18:00, 19:30, 21:00, 22:30, and 24:00. We staggered our bleeding protocol to minimize the possible effects of cumulative daily disturbance (through repetitive capture, handling, and blood sampling) on profiles of plasma CORT and MEL. As a general aside, we do not refer to the daily MEL and CORT cycles as circadian rhythms (although they may well be) as we did not establish if these hormone cycles persisted in captive sea turtles under constant conditions.
The Effect of Nocturnal Activity on Hormone Profiles in Captive Green Turtles To determine if nocturnal activity altered daily hormone profiles in diurnally active green turtles, we compared MEL and CORT profiles in response to three different levels of activity: (1) Inactive (Control). Turtles were allowed to rest (akin to sleep) undisturbed during their normal inactive period and blood samples were taken at 22:00 to get reference values for nocturnal levels of plasma MEL and CORT. (2) Low-intensity activity. Turtles were subjected to a passive disturbance administered at night from 21:00 –22:00 by repetitively and gently moving the mesh dividers partitioning turtles in their tanks. This low-intensity activity stimulus was designed to arouse turtles. It induced the turtles to float in the water column or to gently swim, and it prevented inactivity. At 22:00, this low-intensity arousal stimulus was stopped and blood samples were collected. (3) High-intensity activity. Turtles were subjected to a high-intensity activity stimulus by exposing them to a circular water current (⬎1 m/s, created by bilge pumps) in their tanks. The current forced turtles to exhibit bursts of very active swimming. At 22:00, this high-intensity stimulus was ceased and blood samples were collected. Each activity treatment was conducted independently and separated by a three-day interval. Again, we decided against using a simultaneous comparison of treatments (repeated over several nights) as the disturbance of the bilge pumps and water flow was enough to induce nocturnal arousal in turtles in the adjacent tank.
Field Studies A total of 531 green sea turtles were captured and blood sampled in the field. Field studies were conducted in waters adjacent to, or on, the beaches of Heron (23° 26⬘S; 151°57⬘E) and Raine Islands (11° 37⬘S; 144°01⬘E), located within the Great Barrier Reef, off the coast of Queensland, Australia. Our field studies took place during late spring and early summer (mid October to mid December). The mean sunrise and sunset were recorded at 5:15 ⫾ 02 h and 18:05 ⫾ 01 h for Heron Island, and 5:51 ⫾ 01 h and 18:37 ⫾ 02 h for Raine Island. The mean daily air and ocean temperature on Heron and Raine islands during this study were 25.6 ⫾ 0.16°C and 25.25 ⫾ 1.02°C and 32.4 ⫾ 0.12°C and 29.2 ⫾ 0.01°C, respectively. At both islands, blood samples were collected from sea turtles that were hand captured in water using the rodeo method (Limpus and Reed, 1985), or on land during the day and night while displaying one of three behaviors: (1) Female nesting behavior. Individual breeding females were captured and blood sampled throughout the day and night. On Raine Island, females were sampled while exhibiting nesting behavior on the beach throughout the entire day. These blood samples were only analyzed for MEL, as diurnal nesting on this island resulted in heat-stress by mid-afternoon which significantly increased CORT levels (Jessop, Hamann, Read, and Limpus, 2000). To measure the influence of nesting behavior on the daily cycle of plasma CORT, nesting and internesting females (females captured in water during the day when no nesting activity takes place) were sampled from Heron Island. (2) Male courtship behavior. During courtship, male green turtles exhibit various reproductive behaviors (i.e., mate searching and copulation) that persist throughout the night. We compared plasma hormone profiles of MEL and CORT in male turtles captured when exhibiting reproductive behavior during the day (between 9:00 and 15:00) against profiles of males captured at night (21:30 –1:00). (3) General activity behavior. During October fieldwork, swimming or feeding behavior was observed in green turtles around Heron Island throughout the day and night, albeit more commonly during the day. For green turtles in this category, we focused our sampling on juvenile green turtles (less than 65 cm in
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curved carapace length), as overall these turtles were the most similar in size and reproductive status to captive animals. Again, turtles were captured and blood sampled during the day (9:00 –13:00) and night (21:00 –1:00). Blood Sampling Protocols Blood samples (1–2 ml) were taken from the dorsal cervical sinus of sea turtles using a 21gauge needle and 5-ml syringe. All blood samples were transferred to lithium heparin vials and placed in iceboxes or refrigerators for up to 6 h, then centrifuged at 4000 rpm for 5 min. Plasma was decanted into cryotubes and frozen in liquid nitrogen and then transferred to a ⫺ 20°C refrigerator for storage. Extended nocturnal exposure (⬎1–2 h) to bright white light has been demonstrated to suppress nocturnal MEL synthesis in sea turtles, however, shorter periods, up to several minutes, are not thought to suppress plasma MEL levels (Owens et al., 1980; Owens and Gern, 1985; Owens, personal communication). In our captive studies, we took measures to limit any light effects on plasma MEL by capturing and blood sampling turtles under red light, a method widely used to eliminated any inhibitory effects of white light on MEL. In the field, our precautions included limiting the use of small, head-mounted lights to brief periods (⬍2 min for turtle captured in water, ⬍1 min for nesting females) during the final phase of the turtle’s capture. Furthermore, during blood sampling the turtles eyes were covered with our hands to prevent exposure to dull torchlight. Radioimmunoassay Plasma MEL was analyzed by a specific radioimmunoassay (RIA) slightly modified from that used for humans (Fraser, Cowen, Franklin, Francey, and Arendt, 1983). Plasma CORT was analyzed by a direct RIA procedure similar to that previously validated for green turtles (Jessop, Limpus, and Whittier, 1999a). A 100 –300 l aliquot of plasma was extracted twice with 3 ml of diethyl ether, vortexed for 1 min, and centrifuged at 2500 rpm for 5 min. The organic phase was pipetted into a 16 ⫻ 100 mm disposable glass culture tube and placed into a 37°C dry bath, then evaporated to dryness before 0.5 ml Tris buffered saline, pH 7.0, was added. Duplicated samples were incubated overnight at 4°C with tritiated labeled MEL (Amersham, Australia) and MEL specific antibody (Stockgrand Ltd., Surrey, UK), or with tritiated labeled CORT
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(NET-399, Dupont Inc., Sydney, Australia) and CORT specific antibody (Endocrine Sciences, Tarzana, CA). Unbound antigen was separated from free antigen using a charcoal– dextran solution (1.25%/0.125%). The bound fraction was decanted into poly-Q vials (Beckman Scientific) containing 4 ml aqueous based scintillant (BCS, Amersham) and counted on a Beckman LS6000 scintillation counter. Mean extraction efficiencies were 88.6% and 88.5% for MEL and CORT as determined by recovery of tritiated label added to samples prior to adding diethyl ether. The average intraassay coefficient of variation was 8.7 and 12.2%, for MEL and CORT respectively. The interassay coefficient of variation was 7.6 and 11.2%, for MEL and CORT, respectively. Statistical Analysis All hormone data was log transformed (log ⫻ ⫹ 1) prior to analysis to correct for heteroscedasticity. To prevent violation of the assumption of independence, a single blood sample was taken from each individual captured in the field. Repeated measures ANOVA were used to analyze differences in plasma concentrations of MEL and CORT in captive green turtles. A post hoc multiple comparison test was performed using Tukey’s HSD. For data collected from breeding males and juvenile green turtles in the field, a twosample t test or the nonparametric Mann–Whitney test was used to detect differences between day and night levels of MEL and CORT. ANOVA or nonlinear regressions were used to identify significant correlation’s between MEL and CORT and the time of day in nesting green turtles. All differences were considered significant at P ⬍ 0.05.
RESULTS Captive Green Turtles In diurnally active captive green turtles, large daily variation was observed in the daily cycle of plasma MEL (Fig. 1). As expected, MEL increased during the night from 19:30 onward before declining from its peak at 12:00 and reaching basal levels at 9:00 (repeatedmeasures ANOVA, F 9,50 ⫽ 10.62, P ⬍ 0.001). In contrast, CORT displayed a diurnal profile in which plasma levels were significantly elevated during the day from 6:00 to 15:00 before declining to basal levels during the night coinciding with inactivity (repeatedmeasures ANOVA, F 9,50 ⫽ 11.76, P ⬍ 0.001).
Daily Profiles of Melatonin and Corticosterone
FIG. 1. The daily profiles of plasma melatonin (closed circles; mean ⫾ SEM) and corticosterone (open circles; mean ⫾ SEM) in captive juvenile green turtles maintained at 27 ⫾ 5°C under an ambient summer photoperiod. There was significant variation in both hormones with time of day (P ⬍ 0.01). Six turtles were sampled at each time period, the downward pointing arrows represent the onset of sunrise and sunset, respectively, and the horizontal black bars denote the scotophase.
Nocturnal activity in captive green turtles during their inactive rest phase had conspicuous effects on the nocturnal profiles of plasma MEL and CORT. Plasma MEL decreased significantly in response to both low- and high- intensity nocturnal activity compared to levels in inactive turtles sampled at 22:00 (repeated-measures ANOVA, F 2,13 ⫽ 6.19, P ⫽ 0.011; Fig. 2). Post hoc comparisons indicated that MEL levels in the high activity (Tukey HSD, q ⫽ 4.98, P ⬍ 0.05) and low activity treatments (Tukey HSD, q ⫽ 4.26, P ⬍ 0.05) differed significantly from the inactive control group, but not from one another (Tukey HSD, q ⫽ 2.35, P ⬎ 0.05). In contrast, plasma CORT increased significantly and in step with the different intensities of nocturnal activity compared to inactive controls (repeated measures ANOVA F 2,13 ⫽ 23.35, P ⬍ 0.001; Fig. 2). Post hoc comparisons indicated that CORT levels for all treatments were significantly different from each other (Tukey HSD’s all P ⬍ 0.05).
361 F 1,203 ⫽ 7.69, P ⬍ 0.001, Fig. 3a). Despite this general pattern, 40% of nocturnal nesting females had plasma MEL levels that were the equal to or below the mean of 3.69 ⫾ 0.71 pg/ml measured for nesting females sampled throughout the day (6:00 –14:00). Nesting females showed no relationship between plasma levels of CORT and time of day (Polynomial Function: Plasma CORT ⫽ 2.751 ⫹ ⫺ 0.356*Time of Day 2, ⫺ 0.01*Time of Day 2, R 2 ⫽ 0.028, N ⫽ 119; ANOVA: F 1,118 ⫽ 1.33; P ⫽ 0.34, Fig. 3b). CORT levels remained relatively uniform throughout the day in nesting females and overall, levels of plasma CORT were greater in nesting females compared to captive juveniles. Unlike the pronounced daily endocrine cycles of their captive diurnal counterparts, free-living nocturnally active breeding male and juvenile green turtles had either minimal or reversed daily variation in profiles of MEL and CORT. Again, the relative concentrations of plasma MEL and CORT were less and greater, respectively, than that measured for captive turtles. In breeding male green turtles exhibiting nocturnal mate searching we found no difference between day and night levels of plasma MEL (Mann–Whitney Rank Sum Test: T ⫽ 1830, N (Day) ⫽ 24, N (Night) ⫽ 112, P ⫽ 0.29, Fig. 4A) or CORT (T test: t 1,137 ⫽ 1.26, P ⫽ 0.21). Likewise, juvenile green turtles exhibiting swimming or foraging activity had levels of plasma MEL that were similar between day and night sam-
Free-Living Sea Turtles Nesting females showed small daily variation in plasma MEL compared to juveniles in captivity. However, despite this decreased daily variation, plasma MEL was still significantly correlated with time of day, with an overall increase at night (Quadratic Function: Plasma MEL ⫽ 18.08 ⫹ ⫺ 2.23 * Time of Day ⫹ 0.08 * Time of Day 2, R 2 ⫽ 0.071, N ⫽ 204; ANOVA:
FIG. 2. A comparison of plasma melatonin (mean ⫾ SEM) and corticosterone (mean ⫾ SEM) concentrations measured for captive green turtles (at 22:00) which were inactive or that had just performed 1 h of low or high intensity activity prior to blood sampling. Nocturnal activity induced a significant decrease in plasma melatonin (P ⬍ 0.01), but significantly increased plasma corticosterone (P ⱕ 0.01) compared to inactive turtles. Significant post hoc (Tukey’s HSD) differences in plasma hormone levels are denoted by different superscripts.
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free-living sea turtles undertook a range of different nocturnal activities, there was either no or only a small overall increase in nocturnal plasma MEL. Even in nesting turtles where nocturnal levels of MEL were slightly, but significantly, greater in magnitude than daytime values, many females still had plasma MEL levels within the range of those measured during the day. In other diurnal species, including humans and a captive bird, nocturnal exercise or activity may also be associated with decreased levels of plasma MEL (Montelone et al., 1989; Van-Reeth, Sturis, Byrne, Blackman, L’Hermite-Baleriaux, Leproult, Oliner, Refetoff, Turek, and Van-Cauter, 1994; Gwinner, 1996; Buxton, Frank, L’Hermite-Baleriaux, Leproult, Turek, and Van Cauter, 1997). However, in comparison to humans and birds, the intensity of nocturnal activity required by sea turtles to suppress MEL is much less. Relatively mild but sustained nocturnal arousal was
FIG. 3. The daily profiles of (A) plasma melatonin and (B) corticosterone from nesting green turtles sampled at Raine Island (N ⫽ 204) and Heron Island (N ⫽ 119), respectively. Nonlinear regression indicated a significant relationship between time of day and plasma Melatonin. Downward pointing arrows represent the onset of sunrise and sunset, the horizontal black bars denote the scotophase.
pling periods (T test: t 1,35 ⫽ 0.01, P ⫽ 1.00; Fig. 4B). However, juvenile turtles active at night had plasma CORT levels that were significantly greater than those measured in juvenile turtles active during the day (T test: t 1,35 ⫽ 3.07, P ⫽ 0.003; Fig. 4B).
DISCUSSION A typical nocturnal MEL cycle was observed in captive green turtles exhibiting a diurnal activity cycle. This result is consistent with findings in many animals, including a previous study addressing the presence of a daily MEL cycle in green turtles (Owens et al., 1980; Reiter, 1981; Delgado and Vivien-Roels, 1989; Gwinner, 1996). However, when captive and
FIG. 4. Comparison of day (turtles sampled between 9:00 and 15:00) and night (turtles sampled between 21:30 and 1:00) levels of melatonin (mean ⫾ SEM) and corticosterone (mean ⫾ SEM) in breeding male green turtles involved in (A) reproductive behaviors and (B) juvenile green turtles exhibiting feeding/swimming behavior at Heron Island. Nocturnal levels of plasma corticosterone were significantly elevated (P ⬍ 0.05 denoted by asterix) compared to day levels in juvenile turtles captured while swimming or feeding. Sample sizes for each category are numerically represented within the bars.
Daily Profiles of Melatonin and Corticosterone
enough to decrease plasma MEL compared to levels obtained from inactive turtles in captivity. Captive green turtles possessed a broad day time peak in plasma CORT coinciding with daily activity; this pattern was similar to some other diurnal species (Joseph and Mier, 1973; Summers and Norman, 1988). This diurnal peak in the daily CORT cycle was obscured by nocturnal activity that increased plasma CORT concentrations during the night and thus reduced daily variation in this endocrine cycle. Further, it was evident that nocturnal levels of CORT could exceed daytime levels if the intensity of nocturnal activity was great enough, as observed in swimming green turtles. Exercise-induced stress may have also played a factor in increasing plasma CORT in captive turtles exhibiting intense bouts of swimming. This general tendency for increased CORT levels with nocturnal activity is likely to reflect increased metabolism. During greater aerobic demands, catabolic hormones such as CORT serve to catabolize endogenous fuel reserves to maintain the expression of activity (Norris, 1997). Therefore, behavior per se may not actually alter the endogenous basal cycle, instead the presumed metabolic effects of nocturnal behaviors would seem to increase CORT and thus mask any inherent daily variation in this endocrine cycle (Jessop, 2000). Sea turtles exhibiting nocturnal activity typically reduced their daily variation in hormones cycles of MEL and CORT, so that nocturnal levels of MEL and CORT decreased and increased, respectively. In essence, these nocturnal endocrine patterns were often similar to those measured during the day. We interpret that these conspicuous changes in the hormone profiles of nocturnally active turtles are a consequence of heightened activity or arousal. It is possible that activity reduced nocturnal levels of MEL due to elevated metabolism leading to increased hepatic clearance of MEL from the plasma (Owens et al., 1980). While this reasoning is plausible for physically demanding behaviors such as nesting and mating, this study demonstrated that even mild nocturnal activity was associated with reduced plasma MEL in green turtles. Thus, we suspect even simple nocturnal arousal can trigger mechanisms leading to a suppression of MEL during nocturnal activity. In the field, other factors could also possibly account for why nocturnally active turtles possessed low levels of MEL. For instance, prolonged exposure to bright light is known to inhibit nocturnal MEL secretion in captive green turtles (Owens and Gern, 1985). Perhaps, prolonged exposure to even weak ambient light by turtles
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active at night in nature is sufficient to induce this effect. Irrespective of why nocturnally active turtles possessed low levels of plasma MEL, our findings invite speculation about the consequences of this association. One hypothesis is that the reduced levels of MEL in nocturnally active sea turtles provides similar phase properties to the day time profile of MEL when turtles normally conduct most of their activity. In humans, behavioral and physiological disorders that arise with nocturnal shift work, or through jet-lag are due to dissociation between daily activity and the appropriate phases of other physiological, endocrine, and behavioral cycles (Weibel et al., 1996, 1997). Whether, the reduction in plasma MEL is a mechanism that nocturnally active green turtles employ to eliminate similar negative consequences remains to be determined. Another possibility of why nocturnally active green turtles exhibit reduced levels of MEL is to reduce this hormone’s capacity for potentially inhibiting nocturnal activity in otherwise typically diurnal animals. Several studies have reported that exogenous MEL causes sleep-like behavior, drowsiness, and decreased activity in several diurnal vertebrates (Turek, 1989; Chiba et al., 1985; Hyde and Underwood, 2000). In some reptiles, MEL also appears important for initiating the onset of daily inactivity, at least under constant conditions (Underwood, 1990; Hyde and Underwood, 2000). Diurnal birds that use nocturnal activity (Zugunruhe) for vernal migration are reported to reduce nocturnal levels of plasma MEL (Gwinner, 1996). However, if birds receive exogenous MEL during Zugunruhe a suppression in activity can result (Pohl, 2000). Supposing that in green turtles a similar inhibitory effect on nocturnal activity is caused by elevated levels of MEL, then reduced nocturnal levels in this hormone could represent an important mechanism to maintain or promote nocturnal activity in green turtles. Clearly, manipulative experiments using exogenous MEL are needed to test these hypotheses on the role of MEL in influencing the proclivity of nocturnal behavior in green turtles. In conclusion, sea turtles can exhibit temporal shifts in activity to acquire nocturnal resources that presumably influence their survival and reproduction. During nocturnal activity, sea turtles exhibit conspicuous changes in their daily cycles of plasma MEL and CORT. One explanation for these findings could be that compensatory changes in daily hormonal cycles promote and support nocturnal activity. Simply, active behavior and the appropriate phase of the endocrine cycle remained coupled to each other indepen-
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dent of the temporal environment set by physical cues. A cursory glance at the MEL literature reveals that several different daily patterns of MEL secretion can persist in vertebrates, including absence, in addition to the usual nocturnal pattern that typifies most species (Roth, Gern, Roth, Ralph, and Jacobson, 1980; Serino, d’Istria and Montelone, 1993; Taniguchi, Murakami, Nakamura, Nasu, Shinohara, and Etoh, 1993; Mendonca, Tousignant, and Crews, 1995; Van’t Hof and Gwinner, 1998). Again research is needed to determine if these alternative MEL patterns represent facultative or obligatory responses to nondiurnal patterns of activity, as many of these species, including owls, frogs, and crocodilians, exhibit nocturnal or highly variable patterns of daily activity.
ACKNOWLEDGMENTS We thank R. Gnome for help with the husbandry of the captive turtles, and M. Hamann and M. Read for their assistance in blood sampling while on Raine and/or Heron Island. Queensland Parks and Wildlife Service and the Great Barrier Reef Marine Park Authority made important logistical contributions to this study. We thank D. Owens for providing input into an earlier draft of this manuscript. This work was supported by an Australian Postgraduate Award (to T.S.J.) and an Australian Research Council grant (to C.J.L. and J.M.W.). This project was conducted as part of the Queensland Turtle Research Project. This study was approved by the University of Queensland’s Animal Ethics Committee (zoo/ 225/96/urg).
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