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Circadian and circannual rhythms in metabolism and ventilation in red-eared sliders (Pseudemys scripta) Reyes, Catalina 2006

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CIRCADIAN A N D C I R C A N N U A L R H Y T H M S IN M E T A B O L I S M A N D V E N T I L A T I O N IN R E D - E A R E D SLIDERS (PSEUDEMYS SCRIPTA) by C A T A L I N A R E Y E S B . S c , Universidad de los Andes, 2000 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE in T H E FACULTY OF GRADUATE STUDIES (Zoology) T H E UNIVERSITY OF BRITISH COLUMBIA A p r i l , 2006 © Catalina Reyes, 2006 ABSTRACT Circadian and Circannual Rhythms in Metabolism and Ventilation in Red-eared Sliders Pseudemys scripta by Catalina Reyes Temperate reptiles are subjected to drastic daily and seasonal changes in their environment that strongly affect their metabolism. Therefore, reptiles inhabiting in northern latitudes require adaptations that allow them to survive extreme environmental changes. Endogenous biological rhythms such as circadian and circannual cycles are advantageous in allowing organisms to anticipate and prepare for periodic environmental changes. However, to be fully functional these rhythms must be entrained to changing environmental cues. I asked whether red-eared sliders showed circadian and circannual rhythms in metabolism and ventilation, and whether daily oscillations varied across the seasons. Turtles were chronically exposed to either natural seasonal or constant indoor conditions. In addition, turtles were acutely placed under either seasonal or indoor conditions in order to determine the role of temperature and photoperiod in entraining the circadian and circannual rhythms. Daily cycles in metabolism and breathing pattern were measured over one year. I found evidence of endogenous circadian and circannual rhythms in metabolism and ventilation. Both thermocycles and photocycles were important zeitgebers and long-term exposure to these changing environmental cues was required for the rhythms to be expressed. Furthermore, I observed daily and seasonal changes in breathing pattern such that apneas were longer at night and in the winter. These results indicate that endogenous circadian and circannual rhythms entrained by seasonal changes in temperature and photoperiod prompt physiological adjustments in metabolism, ventilation and breathing pattern. These changes may reflect adaptations that prolong dive times and reduce surface intervals at night and in winter. This could serve to reduce the cost of transport, and risk of predation. ii T A B L E OF CONTENTS Topic Page Abstract ii Table of Contents iii List of Tables vi List of Figures xii List of Abbreviations xx Glossary xxi Acknowledgments xxiii C H A P T E R 1: Introduction 1 Circadian rhythms 2 Properties of the circadian rhythm 2 Structures involved in the control of circadian rhythms 3 Circannual rhythms 6 Pseudemys scripta 8 Metabolism 9 Metabolic suppression 10 Ventilation 11 Control of ventilation 11 Observations : 14 Hypothesis 14 C H A P T E R 2: Methods 15 Experimental protocol 15 Experimental set-up 22 Metabolism measurements 23 Data analyses of metabolic variables 24 Temperature quotient (Qio) 25 Ventilation measurements 26 Pneumotachograph calibration 26 Data analyses of ventilatory variables 27 Statistical analyses 28 C H A P T E R 3: Circadian and circannual rhythms in the metabolism of red-eared sliders (Pseudemys scripta) 30 Introduction 30 Results 33 Circadian rhythms of metabolism 33 Chronic acclimatization to seasonal cues 33 Chronic acclimatization to constant conditions (no seasonal cues) 41 Acute exposure to seasonal cues 44 T A B L E O F C O N T E N T S Topic Page Acute exposure to constant conditions (no seasonal cues) 45 Circannual rhythms of metabolism 46 Chronic acclimatization to seasonal cues 46 Chronic acclimatization to constant conditions (no seasonal cues) 51 Acute exposure to seasonal cues 54 Acute exposure to constant conditions (no seasonal cues) 56 Temperature coefficients: seasonal-independent temperature effects on oxygen consumption 58 Discussion 61 Orcadian rhythms 61 Endogenous rhythms 64 Role of external environmental cues in entraining circadian rhythms... 65 Seasonal variation in circadian rhythms 66 Circannual rhythms 67 Seasonal changes in metabolism and role of environmental cues. 68 Endogenous circannual rhythm 70 Circannual and circadian interaction 71 C H A P T E R 4: Circadian and circannual rhythms in ventilation of Red-eared sliders (Pseudemys scriptd) 72 Introduction 72 Results 74 Circadian rhythms in total ventilation 74 Chronic acclimatization to seasonal cues 74 Chronic acclimatization to constant conditions (no seasonal cues) 79 Acute exposure to seasonal cues 79 Acute exposure to constant conditions (no seasonal cues) 80 Circadian rhythms in breariiing frequency and tidal volume 80 Chronic acclimatization to seasonal cues 80 Chronic acclimatization to constant conditions (no seasonal cues) 83 Acute exposure to seasonal cues 86 Acute exposure to constant conditions (no seasonal cues) 86 Circadian rhythms in the components of breathing frequency: breaths per episode, episodes per hour and apnea length 89 Chronic acclimatization to seasonal cues 89 Chronic acclimatization to constant conditions (no seasonal cues) 90 Acute exposure to seasonal cues 95 Acute exposure to constant conditions (no seasonal cues) 96 Circannual rhythms in ventilation 96 Chronic acclimatization to seasonal cues 97 Chronic acclimatization to constant conditions (no seasonal cues) 99 iv T A B L E O F C O N T E N T S Topic Page Acute exposure to seasonal cues 100 Acute exposure to constant conditions (no seasonal cues) 100 Circannual rhythms in breathing frequency and tidal volume 100 Chronic acclimatization to seasonal cues 100 Chronic acclimatization to constant conditions (no seasonal cues) 101 Acute exposure to seasonal cues 104 Acute exposure to constant conditions (no seasonal cues) 104 Circannual rhythms in the frequency components of breathing: breaths per episode, episodes per hour and apnea length 105 Chronic acclimatization to seasonal cues 105 Chronic acclimatization to constant conditions (no seasonal cues) 105 Acute exposure to seasonal cues 110 Acute exposure to constant conditions (no seasonal cues) 110 Discussion 112 Circadian rhythms in total ventilation 112 Role of environmental cues in entraining circadian rhythms 114 Endogenous rhythms 115 Circadian rhythms in the brearliing pattern 116 Breatiiing frequency and its components 116 Tidal volume 117 Role of environmental cues in establishing rhythms in the components of the breathing pattern 118 Respiratory mechanics 118 Circannual rhythms in total ventilation 119 Circannual rhythms in the ventilation components 120 Circannual rhythms in frequency components 120 Conclusions 121 C H A P T E R 5: General discussion 123 Circadian rhythms 123 Circannual rhythms 126 Possible mechanisms underlying circadian and circannual rhythms 130 References 133 Appendix A 147 Appendix B: Metabolism 157 Appendix C: Ventilation 169 Appendix D: Air convection requirement and sensitivity of ventilation 182 L I S T O F T A B L E S Number Caption Page Table 2.1: Temperature and photoperiod used for the three experimental series. Each turtle from each of the two groups underwent 2 experimental runs under each of 2 treatments in each season. Temperature and photoperiod for no cues treatments (daily cues absent) are also shown.... 19 Table 3.1: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles chronically acclimatized to seasonal cues 38 Table 3.2: Mean percentage of time inactive ± SEM in different seasons. Day and night values of inactivity (% time) and P values for turtles chronically acclimatized to seasonal cues. Significant differences between day and night are indicated by (*) 41 Table 3.3: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles chronically acclimatized to constant indoor conditions 43 Table 3.4: Mean percentage of time inactive ± SEM in different seasons. Day and night values of inactivity (% time) and P values for turtles chronically acclimatized to constant indoor conditions. (•) indicates values obtained with the non-parametric Wilcoxon Signed Rank test 43 Table 3.5: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles acutely exposed to seasonal cues 44 Table 3.6: Mean percentage of time inactive ± SEM in different seasons. Day and night values of inactivity (% time) and P values for turtles acutely exposed to seasonal cues 45 Table 3.7: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles acutely exposed to constant indoor conditions 45 Table 3.8: Mean percentage of time inactive ± SEM in different seasons. Day and night values of inactivity (% time) and P values for turtles acutely exposed to constant indoor conditions 46 Table 3.9: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles chronically acclimatized to outdoor seasonal cues. No significant differences in RER were observed between seasons (P=0.212) 47 Table 3.10: Mean percentage of time inactive ± SEM in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles chronically acclimatized to outdoor seasonal cues. Significant differences in inactivity between seasons were observed (P=0.040). Turtles were more active in the fall than in the winter and no cues treatments 47 Table 3.11: Mean temperature coefficients ± SEM of turtles chronically exposed to seasonal cues (outdoor turtles exposed to outdoor conditions). Q 1 0 values for day, night and total (24 hours) oxygen consumption were calculated for three seasonal temperatures (winter, fall and summer), as well as for fall under constant dark (NC) 50 vi LIST OF TABLES (CONTINUED) Number Caption Page Table 3.12: Table 3.13: Table 3.14: Table 3.15: Table 3.16: Table 3.17: Table 3.18: Table 3.19: Table 3.20: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles chronically acclimatized to constant indoor conditions. Significant differences in RER were observed between seasons (P=0.025). Winter RER was significantly higher than other seasons 54 Mean percentage of time inactive ± SEM in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles chronically acclimatized to constant indoor conditions. Significant differences in inactivity between seasons were observed (P=0.020). Turtles were significantly more active in the fall than in the winter and no cues treatments 54 Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles acutely exposed to seasonal cues. No significant differences in RER were observed between seasons (P=0.805) 55 Mean percentage of time inactive ± SEM in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles acutely exposed to seasonal cues. No significant differences in inactivity between seasons were observed (P=0.193) 55 Mean temperature coefficients ± SEM of turtles acutely exposed to seasonal cues (indoor turtles exposed to outdoor conditions). Q 1 0 values for day, night and total (24 hours) oxygen consumption were calculated for three seasonal temperatures (winter, fall and summer), as well as for fall under constant dark (NC) 56 Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles acutely exposed to constant indoor conditions. No significant differences in RER were observed between seasons (P=0.207) 58 Mean percentage of time inactive ± SEM in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles acutely exposed to constant indoor conditions. Significant differences in inactivity between seasons were observed (P=0.026). Turtles were significantly more active in the fall than in the summer and winter 58 Mean Q 1 0 values ± SEM for day, night and total oxygen consumption of turtles chronically acclimatized to seasonal cues (outdoor turtles) at different temperatures (constant and environmental) during fall and winter 59 Mean Q 1 0 values ± SEM for day, night and total oxygen consumption of turtles chronically acclimatized to constant conditions (indoor turtles) at different temperatures (constant and environmental) during fall and winter 60 vii LIST OF TABLES (CONTINUED) Number Caption Page Table A1: Meteorological variables measured daily throughout the experiment. 148 Table B1: Means ± SEM of physiological variables for turtles chronically acclimatized to seasonal cues for each season 157 Table B2: Means ± SEM of physiological variables for turtles chronically acclimatized to constant conditions (seasonal cues absent) for each season 158 Table B3: Means ± SEM of physiological variables for turtles acutely exposed to seasonal cues 159 Table B4: Means ± SEM of physiological variables for turtles acutely exposed to constant conditions (seasonal cues absent) 160 Table B5: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles chronically exposed to seasonal cues. RER, refers to the respiratory exchange ratio 161 Table B6: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles chronically exposed to constant conditions. RER, refers to the respiratory exchange ratio. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 162 Table B7: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles acutely exposed to seasonal cues. RER, refers to the respiratory exchange ratio 163 Table B8: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles acutely exposed to constant conditions. RER refers to the respiratory exchange ratio. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 164 Table B9: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles chronically exposed to seasonal cues. RER, refers to the respiratory exchange ratio 165 Table B10: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles chronically exposed to constant conditions. RER refers to the respiratory exchange ratio 165 Table B11 Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles acutely exposed to seasonal cues. RER, refers to the respiratory exchange ratio 166 viii LIST OF TABLES (CONTINUED) Number Caption Page Table B12: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles acutely exposed to constant conditions. RER, refers to the respiratory exchange ratio 166 Table B13 Statistic values for the One-way repeated measures ANOVA performed to determine the existence of differences in Q 1 0 values of turtles chronically exposed to seasonal cues. Differences between the temperature coefficients calculated for: fall-winter, summer-fall, summer-winter, no cues (fall)-winter and summer-no cues (fall) were determined 167 Table B14 Statistic values for the One-way repeated measures ANOVA performed to determine the existence of differences in Q 1 0 values of indoor turtles acutely exposed to seasonal cues. Differences between the temperature coefficients calculated for: fall-winter, summer-fall, summer-winter, no cues (summer)-fall and no cues (summer)-winter were determined. See table B15 for pairwise multiple comparisons 167 Table B15 Pairwise multiple comparisons (Holm-Sidak) for night temperature coefficients* of indoor turtles acutely exposed to seasonal cues 167 Table B16 Statistic values for the Two-way repeated measures ANOVA performed to determine the existence of differences between Q 1 0 values of turtles chronically exposed to seasonal cues and indoor turtles acutely exposed to seasonal cues. Pair of seasons: fall-winter, summer-fall and summer-winter. 'Significant differences between outdoor and indoor turtles were found for Q10 (night) values calculated between fall-winter (P=0.011) and summer-fall (P=0.019). A Significant differences between outdoor and indoor turtles were only found for Q i 0 (total) values calculated between summer and fall (P=0.024) 168 Table B17 Statistic values for the Two-way repeated measures ANOVA performed to determine the existence of differences between day and night Q i 0 values of turtles chronically exposed to seasonal cues and indoor turtles acutely exposed to seasonal. Only statistics for the difference between outdoor and indoor groups are shown 168 T a b l e d : Means ± SEM of physiological variables for turtles chronically acclimatized to seasonal cues for each season 169 Table .02: Means ± SEM of physiological variables for turtles chronically acclimatized to constant conditions 170 Table C3: Means ± SEM of physiological variables for turtles acutely exposed to seasonal cues 171 Table C4: Means ± SEM of physiological variables for turtles acutely exposed to constant conditions (seasonal cues absent) 172 Table C5: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles chronically exposed to seasonal cues. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 176 ix LIST O F T A B L E S (CONTINUED) Number Caption Page Table C6: Table 3.10: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles chronically exposed to constant conditions. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 177 Table C7: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles acutely exposed to seasonal cues. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 178 Table C8: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles acutely exposed to constant conditions. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 179 Table C9: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to ventilation of turtles chronically exposed to seasonal cues 180 Table C10 Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles chronically exposed to constant conditions 180 Table C11: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles acutely exposed to seasonal cues 181 T a b l e d 2 : Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles acutely exposed to constant conditions 181 Table D1: Means ± SEM of physiological variables for turtles chronically acclimatized to seasonal cues for each season 182 Table .D2: Means ± SEM of physiological variables for turtles chronically acclimatized to constant conditions 182 Table D3: Means ± SEM of physiological variables for turtles acutely exposed to seasonal cues 183 Table D4: Means ± SEM of physiological variables for turtles acutely exposed to constant conditions (seasonal cues absent) 183 Table D5: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles chronically exposed to seasonal cues. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 184 Table D6: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles chronically exposed to constant conditions. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 184 x L I S T O F T A B L E S ( C O N T I N U E D ) Number Caption Page Table D7: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles acutely exposed to seasonal cues. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 185 Table D8: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles acutely exposed to constant conditions. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used 185 Table D9: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to ventilation of turtles chronically exposed to seasonal cues 186 Table D10 Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles chronically exposed to constant conditions 186 Table D11: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles acutely exposed to seasonal cues 186 Table D12: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles acutely exposed to constant conditions 187 xi L I S T O F F I G U R E S Number Caption Page Figure 2.1: Schematic diagram of experimental set-up (adapted from Funk, et al., 1986) 23 Figure 3.1: Mean resting rates of oxygen consumption ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of oxygen consumption for turtles chronically acclimatized to seasonal and indoor conditions. Day and night rates of oxygen consumption are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). Outdoor conditions (seasonal cues): Winter 9°C, 9L:15D; Fall 14.7°C, 10L14D; Summer 20.8°C, 16L8D; No cues: 13.6°C, constant darkness. Indoor conditions (no seasonal cues): Winter 19.65°C; Fall 22.4°; Summer 19.62°C, all seasons under 12L12D; No cues: 19.9°C, constant darkness. Significant differences between day and night values are indicated by (*) (P<0.05) or (e) (P=0.054 and P =0.056, outdoor and indoor turtles respectively) 34 Figure 3.2: Mean hourly oxygen consumption ± SEM over a 24 hour period. Plots on the top show the circadian changes in oxygen consumption for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in oxygen consumption with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 35 Figure 3.3: Difference between photophase and scotophase values of oxygen consumption of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Day and night oxygen consumption (ml 02/min/kg) difference, expressed as the percentage of total oxygen consumption (average over 24 hours) at different seasonal temperatures and under constant darkness. Day and night difference in oxygen consumption at seasonal temperatures are denoted by open bars (winter: 9 °C; fall: 14.71 °C; summer: 20.83 °C). The filled bar indicates day-night difference under constant darkness (13.59 °C) 36 Figure 3.4: Mean daytime and nighttime values of oxygen consumption of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Figure shows nighttime (filled bars) and daytime (open bars) rates of oxygen consumption measured at seasonal temperatures (winter: 9 °C; fall: 14.71 °C and summer: 20.83 °C) and under constant darkness in the fall (13.6 °C) 37 Figure 3.5: Mean resting rates of C 0 2 production ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of C 0 2 production for turtles chronically acclimatized to seasonal and indoor conditions. Day and night rates of C 0 2 production are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) or (e) (P=0.055 and P =0.052, outdoor and indoor turtles respectively) 39 xii LIST OF FIGURES (CONTINUED) Number Caption Page Figure 3.6: Mean hourly C 0 2 production ± SEM over a 24 hour period. Plots on the top show the circadian changes in C 0 2 production for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in C 0 2 production with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 40 Figure 3.7: Proportion of the hour turtles were inactive ± SEM over a 24 hour period. Plots on the top show the circadian changes in the time inactive for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in the time inactive with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 42 Figure 3.8: Seasonal changes in mean resting oxygen consumption ± SEM. Figures show the seasonal values of oxygen consumption for turtles chronically exposed to seasonal and constant conditions. Seasonal rates of oxygen consumption are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Panels E and F show seasonal changes in temperature-corrected fall values of oxygen consumption. Significant differences between seasons (P<0.05) are indicated by different letters 48 Figure 3.9: Seasonal changes in mean resting C 0 2 production ± SEM. Figures show the seasonal values of C 0 2 production for turtles chronically exposed to seasonal and constant conditions. Seasonal rates of C 0 2 production are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters 49 Figure 3.10: Mean oxygen consumption ± SEM of indoor turtles (acclimatized to constant indoor conditions) at different water temperatures. The figure shows the values of oxygen consumption measured at outdoor conditions (seasonal cues, filled symbols) and indoor conditions (no seasonal cues, open symbols). See Table 2.1 for details. Significant differences between seasons at indoor conditions (open symbols) are indicated by (*). Significant differences between seasons at outdoor conditions (filled symbols) are indicated by letters. The line indicates the regression that best fits the data, y = 0.0882 + (0.0004)e(03210> x See figure 3.11 for details of oxygen consumption values at constant conditions 52 xiii LIST OF FIGURES (CONTINUED) Number Caption Page Figure 3.11: Mean oxygen consumption ± SEM measured under constant indoor photoperiod and temperature at different seasons. Rates of oxygen consumption of both outdoor (acclimatized to environmental conditions A) and indoor turtles (acclimatized to indoor conditions «) measured at constant indoor conditions during three seasons (winter: 19.65 °C, fall (corrected) 19.6 °C and summer 19.62 °C). Significant differences between seasons are indicated by (*) for indoor and by (e) for outdoor turtles 53 Figure 3.12: Mean oxygen consumption ± SEM of outdoor turtles (acclimatized to seasonal cues) at different water temperatures. The figure shows the values of oxygen consumption measured at outdoor conditions (seasonal cues, filled symbols) and under indoor conditions (no seasonal cues, open symbols). See Table 2.1 for details. Significant differences between seasons at indoor conditions (open symbols) are indicated by (*). Significant differences between seasons at outdoor conditions (filled symbols) are indicated by different letters. The line indicates the regression that best fits the data, y = 0.1211 + 0.0008 e ( 0 2 8 3 7 ) x . See details of oxygen consumption values at constant conditions in Figure 3.11 57 Figure 3.13: Comparison of metabolism values of Pseudemys scripta with other studies. Panel A compares the rates of oxygen consumption of red-eared sliders (chronically acclimatized to seasonal cues and to constant conditions) averaged over 24 hours with metabolic rates reported for Pseudemys scripta elegans (Jackson, 1971; Jackson et al., 1974) and Chrysemis picta bellii (Glass et al., 1985). Panel B compares the values of oxygen consumption reported in the previously mentioned studies with both day and night metabolic rates of red-eared sliders chronically acclimatized to seasonal cues obtained in the present study. Panel C compares the values of oxygen consumption reported in the previously mentioned studies, with both, day and nighttime metabolic rates of turtles chronically acclimatized to constant conditions found in this study 63 Figure 4.1: Mean resting values of total ventilation ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of ventilation for turtles chronically acclimatized to seasonal and indoor conditions. Day and night rates of ventilation are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) 75 xiv L I S T O F F I G U R E S ( C O N T I N U E D ) Number Caption Page Figure 4.2: Mean values of total ventilation ± SEM over a 24 hour period. Plots on the top show the circadian changes in ventilation for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in ventilation with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 76 Figure 4.3: Mean daytime and nighttime values of total ventilation of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Figure shows nighttime (filled bars) and daytime (open bars) ventilation measured at seasonal temperatures (winter: 9 °C; fall: 14.71 °C and summer: 20.83 °C) and under constant darkness in the fall (13.6 °C) 77 Figure 4.4: Difference between photophase and scotophase values of total ventilation of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Day and night ventilation difference, expressed as the percentage of total (average over 24 hours) ventilation at different seasonal temperatures and under constant darkness. Open bars indicate day-night difference in ventilation at seasonal temperatures (winter: 9 °C; fall: 14.71 °C;. summer: 20.83 °C). Filled bar indicates constant darkness (13.59 °C) 78 Figure 4.5: Mean resting breathing frequency ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of breathing frequency for turtles chronically acclimatized to seasonal and indoor conditions. Day and night breathing frequency are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (PO.05) 81 Figure 4.6: Difference between photophase and scotophase values of breathing frequency of turtles chronically acclimatized to seasonal cues. Day and night breathing frequency difference, expressed as the percentage of total (average over 24 hours) breathing frequency at different seasonal temperatures and under constant darkness. Day and night differences in the frequency of breaths at seasonal temperatures is denoted by open bars (winter: 9 °C; fall: 14.71 °C; summer: 20.83 °C) and under constant darkness (13.59 °C) by the filled bar 82 Figure 4.7: Mean daytime and nighttime values of breathing frequency of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Figures show the daytime difference (open bars) and nighttime (filled bars) values of breathing frequency measured at seasonal temperatures (winter: 9 °C; fall: 14.71 °C and summer: 20.83 °C) and under constant darkness in the fall (13.6 °C) 82 xv LIST OF FIGURES (CONTINUED) Number Caption Page Figure 4.8: Mean resting tidal volume ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of tidal volume for turtles chronically acclimatized to seasonal and indoor conditions. Day and night tidal volumes are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) 84 Figure 4.9: Difference between photophase and scotophase values of tidal volume of turtles chronically acclimatized to seasonal cues. Day and night tidal volume difference, expressed as the percentage of total (average over 24 hours) volumes at different seasonal temperatures and under constant darkness. Day and night differences in the tidal volume at seasonal temperatures is denoted by open bars (winter: 9 °C; fall: 14.71 °C; summer: 20.83 °C) and under constant darkness (13.59 °C) by the filled bar 85 Figure 4.10: Mean breathing frequency ± SEM over a 24 hour period. Plots on the top show the circadian changes in breathing frequency for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in breathing frequency with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 87 Figure 4.11: Mean tidal volume ± SEM over a 24 hour period. Plots on the top show the circadian changes in tidal volume for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in tidal volume with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 88 Figure 4.12: Mean number of episodes per hour ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) number of episodes/hour for turtles chronically acclimatized to seasonal and indoor conditions. Day and night number of episodes/hour are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (PO.05) 91 Figure 4.13: Mean number of breaths per episode ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) number of breaths/episode for turtles chronically acclimatized to seasonal and indoor conditions. Day and night number of breaths/episode are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) 92 xvi LIST O F FIGURES (CONTINUED) Number Caption Page Figure 4.14: Mean apnea length ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) apnea length for turtles chronically acclimatized to seasonal and indoor conditions. Day and night apnea length is also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) or (E) (P=0.053) 93 Figure 4.15: Proportion of time spent in apnea ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) % time in apnea for turtles chronically acclimatized to seasonal and indoor conditions. Day and night % time in apnea is also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) or (e) (P=0.052, 0.051 and 0.051, fall, winter and summer, respectively) 94 Figure 4.16: Seasonal changes in total ventilation ± SEM. Figures show the seasonal values for ventilation for turtles chronically exposed to seasonal and constant conditions. Seasonal values of ventilation are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 98 Figure 4.17: Seasonal changes in breathing frequency ± SEM. Figures show the seasonal values for breathing frequency for turtles chronically exposed to seasonal and constant conditions. Seasonal values of breathing frequency are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 102 Figure 4.18: Seasonal changes in tidal volume ± SEM. Figures show the seasonal values for tidal volume for turtles chronically exposed to seasonal and constant conditions. Seasonal values of tidal volume are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 103 Figure 4.19: Seasonal changes in the mean number of episodes per hour ± SEM. Figures show the seasonal values for episodes/hour for turtles chronically exposed to seasonal and constant conditions. Seasonal values of episodes/hour are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 106 xvii LIST OF FIGURES (CONTINUED) Number Caption Page Figure 4.20: Seasonal changes in the mean number of breaths per episode ± SEM. Figures show the seasonal values for breaths/episode for turtles chronically exposed to seasonal and constant conditions. Seasonal values of breaths/episode are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 107 Figure 4.21: Seasonal changes in apnea length ± SEM. Figures show the seasonal values for apnea length for turtles chronically exposed to seasonal and constant conditions. Seasonal values of apnea length are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 108 Figure 4.22: Seasonal changes in the percent time spent in apnea ± SEM. Figures show the seasonal values for the time spent in apnea for turtles chronically exposed to seasonal and constant conditions. Seasonal values of the percent time spent in apnea are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 109 Figure 5.1: Mean air convection requirement (ACR) ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of ACR for turtles chronically acclimatized to seasonal and indoor conditions. Day and night ACR are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) 124 Figure 5.2: Mean oxygen extraction ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of oxygen extraction for turtles chronically acclimatized to seasonal and indoor conditions. Day and night oxygen extraction are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (PO.05) 125 Figure 5.3: Seasonal changes air convection requirement (ACR) ± SEM. Figures show the seasonal values for ACR for turtles chronically exposed to seasonal and constant conditions. Seasonal values of ACR are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters or (B) (P=0.055 and 0.053, for outdoor and indoor turtles respectively) 127 xviii LIST O F FIGURES (CONTINUED) Number Caption Page Figure 5.4: Seasonal changes in oxygen extraction ± SEM. Figures show the seasonal values for oxygen extraction for turtles chronically exposed to seasonal and constant conditions. Seasonal values of oxygen extraction are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05), are indicated by different letters 128 Figure A1: Ambient air and water temperature. Temperature values were recorded every hour from October 2003 to November 2004 147 F i g u r e d : Number of breaths per episode ± SEM over a 24 hour period. Plots on the top show the circadian changes in breaths/episode for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in breaths/episode with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 173 Figure C2: Number of breathing episodes per hour + SEM over a 24 hour period. Plots on the top show the circadian changes in episodes/hour for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in episodes/hour with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 174 Figure C3: Apnea duration ± SEM over a 24 hour period. Measured as the proportion of time turtles were apneic. Plots on the top show the circadian changes in the proportion of time in apnea for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in the proportion of time turtles spend in apnea with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details 175 xix List of Abbreviations ACR Air Convection Requirement DD Continuous dark fR Breathing frequency F0i Fractional content of oxygen in gas mixture FCOi Fractional content of C0 2 gas mixture LX.-DF Light-dark cycle with X hours of light (L) and Thours of dark (D) per cycle LL Continuous light NVP Non ventilatory period Q i o Temperature quotient. RER Respiratory Exchange Ratio RMR Resting Metabolic Rate sVCOi Mass-specific rate of carbon dioxide production sV0i Mass-specific rate of oxygen consumption VCOi Rate of carbon dioxide production VE Minute ventilation V0i Rate of oxygen consumption VP Ventilatory period Vr Tidal Volume X X Glossary Circadian rhythms: Endogenous rhythm with a period that is close to the period of the solar day (24 hours). Circadian system: The sum of all the circadian oscillators, including the pacemakers and overt circadian rhythms. Circannual rhytiLims: Endogenous rhythm with a period that has a period close to a year. Entrainment: Process by which an endogenous rhythm becomes coupled to an environmental cycle and assumes the period of the external cue. Free-running period: The period of an endogenous rhythm that is not entrained by an external cycle. Period: The time required for a rhythm to complete one full cycle. Photophase: Related to the light phase of a light-dark photocycle. Scotophase: Related to the dark phase of a light-dark photocycle. Spermatogenesis: Formation and development of spermatozoan by meiosis and spermiogenesis. Spermiation: Discharge of spermatozoa from the testis. Spermiogenesis: Transformation of spermatid into spermatozooan. Standard temperature and pressure (dry, STPD): 21 °C, 1 atmosphere (atm). Q10: Compares the rate of a reaction at two different temperatures. xxi V\o-K /MR/ where M R i and M R 2 are the rates of oxygen consumption temperature ti and t2, respectively. Vitellogenesis: Formation of the yolk of an egg. Zeitgeber: Environmental factors that entrain biological rhythms xxii A C K N O W L E D G M E N T S I would like to thank Dr. William K. Milsom for his valuable help and outstanding supervision throughout my master's project. I am very grateful for accepting me in his lab and giving me the opportunity to do my master's project under his supervision and for his helpful guidance and unconditional support. I would like to thank my committee members Dr. Colin Brauner and Dr. Dave Jones for their valuable advice and important feedback in the writing of this thesis and for reading 187 pages in such a short period of time in order to accommodate my defense. I would like to give special thanks to my family for their support, encouragement and advice throughout this process. I would like to dedicate this thesis to them, since their comfort and constant encouragement were the driving force in completing this project. I would like to thank Jonathan Shurin for his incredible patience, care and support throughout my master's. But especially for making life so complete and sharing with me all the laughs and tears that this project brought about. I would like to thank the Milsom lab for their endless support throughout this process. I offer special thanks to Joanna Piercy for her support, company and quality help with formatting and computer problem solving (the master mind behind my power point presentations). Kim Borg who helped me with long and never ending data analyses, as well as animal care, but more so for her unconditional friendship, encouragement and lessons in interpreting dancing. Emily Coolidge who patiently dealt with my prepositions and who appropriately timed the need for a beer or several depending on the stage of thesis writing. xxiii Without her understanding and encouragement I would have missed the deadline. Graham Scott for his support, friendship and "drunk and dial" phone calls which I could barely understand, but were the origin of a good laugh. Charissa for her numerous hugs and support, but especially for the care and interest in my turtles (I think they really appreciated it). Barb who not only offered me her friendship but also made me an awesome "bufanda" that kept me warm and alive!!!! in the winter. My ex-roommate, office and lab mate for her friendship, good times and for waiting home for me with the lights on, so I wouldn't get hunt by vampires. Last, but not least, Jon Chatburn for his fabulous sense of humor and his genius idea of hiding in the paper recycling bin and scaring people as they were walking by. I would like to express my deepest gratitude to Janice Meier and Brian (Bruno) Sardella, for their unconditional friendship and the unmemorable good moments we shared together. I would also like to thank NSERC of Canada, whose operating grant to Dr. William K. Milsom allowed me to do research in this lab. xxiv C H A P T E R 1: I n t r o d u c t i o n Circadian and circannual rhythms are known to exist in a number of behavioural and physiological processes, in a wide variety of organisms ranging from unicells to vertebrates. Circadian and circannual systems confer selective advantages to organisms, by timing biological process so that specific events take place at appropriate times of the day or year. Most efforts towards understanding these time-keeping mechanisms have focused on mammalian biological rhythms. A number of studies however, have focused on reptiles. Even though the mechanisms underlying such rhythms in reptiles are not well understood, studies have found interesting similarities with those underlying mammalian rhythms (Underwood, 1992) . Because of their wide distribution and their vastly different habitats and life strategies, reptiles serve as a useful and interesting model for understanding circadian and circannual systems. Circadian and circannual rhythms in the metabolism of reptiles are of particular interest, since they are highly affected by one of the main external cues entraining biological rhythms, temperature. Furthermore the metabolic demands of organisms for oxygen at any time of the day or year are fulfilled by ventilation, yet not many studies have addressed the role that biological rhythms play in the control of respiration, particularly in reptiles. Therefore, I wished to study the circadian and circannual rhythms in metabolism and respiration of an ectotherm that encounters daily and seasonal environmental challenges due to its distribution, such as Pseudemys scripta. In particular since most reptiles can attain a particular level of ventilation by changing a number of variables that lead to different breathing patterns, I wondered whether certain components of the pattern varied daily and seasonally. My ultimate goal was to determine whether endogenous circadian and circannual rhythms should be considered as another variable in the control of breathing in reptiles. 1 This chapter will examine the mechanisms behind, and the significance of circadian and circannual rhythms. I will focus particularly on the importance of biological rhythms in the metabolism of reptiles. Also, I will briefly describe respiration and control of breathing and address the role that circadian and circannual cycles have on these variables. Finally, I will present the hypothesis of my Masters thesis. Circadian rhythms Organisms have evolved mechanisms that allow them to keep time, such that periodic events related to the 24 hour day-night cycle are expected and prepared for (Underwood, 1992; Tosini et al, 2001; Mortola and Seifert, 2002). These, biological cycles that occur on a 24 hour basis are referred to as circadian rhythms (circadiem, around the day). It is thought that this mechanism evolved in response to light, since DNA replication and biochemical reactions in early organisms would have been negatively affected by solar radiation, particularly by UV light (Menaker et al, 1997; Reddy et al, 2005). A great number of behavioural and physiological processes in vertebrates are temporally organized. For instance, secretion of certain hormones, temperature set-point, metabolism and locomotion amongst others occur during defined phases of the day or night (Tosini et al, 2001; Reddy et al, 2005). Presumably, temporal organization of physiological and behavioural variables is of great importance for organisms that are highly influenced by their environment such as ectotherms, even more so for high latitude species. Properties of the circadian rhythm Circadian rhythms are characterized by three properties: 1) the rhythms persist under constant conditions, such as constant dark or light and constant temperature. Rhythms that oscillate in the absence of external cues are said to be endogenous and they confer an advantage to the organism in that behavioural and physiological processes will not be disrupted with sudden changes in the environment. 2) Circadian rhythms are entrained by periodic external cues or zeitgebers. In order to be fully functional, circadian rhythms need to be synchronized with the 24 hour day. Therefore, environmental cues such as photoperiod and temperature are matched to the period of the clock (Aschoff et al, 1982a; Underwood, 1992). Light is the main zeitgeber in the mammalian circadian rhythm, whereas both photoperiod and ambient temperature play equally important roles in the circadian cycle of reptiles (Firth et al, 1989; Firth et al, 1999; Kortner and Geiser, 2000). 3) Rhythms are temperature compensated; changes in temperature produce small variations in the rhythm, ensuring that sudden temperature fluctuations do not reset the circadian clock (Menaker and Wisner, 1983) Structures involved in the control of circadian rhythms Three structures have been found to be involved in the control of circadian rhythms: the pineal organ, retina and suprachiasmatic nucleus (SCN). Together, these structures constitute the "circadian axis", which is common to all vertebrates. The importance of each structure in regulating circadian rhythms varies between vertebrate classes (Menaker et al., 1997; Tosini, et al., 2001). Pineal organ The pineal organ plays a major role in the organization of circadian rhythms in lower vertebrates and birds, but is not essential for circadian rhythm expression in mammals. The pineal gland occurs as an evagination of the roof of the diencephalon (Aschoff et al, 1982b). Although the pineal organ is found in all vertebrates, its anatomy and physiology vary tremendously among vertebrates, particularly among reptiles. Alligators for instance, posses a very rudimentary organ, while lizards have a well developed pineal organ that in some species is paired with the parietal eye forming a pineal complex. The parietal eye also originates from the roof of the diencephalon and has a lens, cornea and retina. 3 This structure seems to aid in thermoregulation, reproduction and orientation in reptiles (Underwood, 1992; Tosini etal, 2001; Bertolucci and Foa, 2004). Reptilian pineals contain photoreceptive cells, with secretory properties. In snakes, however the pineal gland only contains secretory cells (Underwood, 1992; Tosini and Menaker, 1998). The main product of the pineal gland is the hormone melatonin, which is synthesized and secreted at night in all vertebrates. Melatonin acts as a neuroendocrine transducer of environmental information, since it responds to changes in light and ambient temperature (Lutterschmidt et al, 2003). The pineal may interact with other structures engaged in circadian organization, such as the SCN, which has been found to be the main extra-pineal target of melatonin (Bertolucci and Foa, 1998). Suprachiasmatic nucleus The suprachiasmatic nucleus (SCN) is known to be the "master" circadian clock in mammals. Its importance in reptilian circadian rhythms however has not been fully demonstrated. The SCN is located in the anterior hypothalamus; it lies above the optic chiasm and is subdivided into two structures, the dorsal "shell" and the ventral "core". The core receives most of the input from the eyes through the retinohypothalamic tract, and seems to be important in photic entrainment of the circadian clock (Saper et al, 2005). The shell, which is known as the rhythmic section of the SCN receives input from the core (Antle and Silver, 2005; Piggins and Loudon, 2005). The SCN in mammals interacts with the rest of the circadian system and peripheral tissues by synaptic and hormonal signals, such as the release of glucocorticoids (Herzog and Tosini, 2001). Although peripheral tissues in mammals are known to have circadian pacemakers in gene expression, the SCN essentially drives and entrains such rhythms (Reddy et al, 2005). Furthermore, the SCN indirectly controls circadian rhythms in peripheral tissues by driving the 4 endogenous circadian rhythms of pineal melatonin synthesis and secretion (Arendt, 1998). During the dark cycle, the SCN releases the GABAergic inhibition on the paraventricular nucleus which presumably activates neurons that send signals to the hindbrain and spinal cord, where they contact sympathetic nerve fibers that originate from the superior cervical ganglion, which innervate the pineal (Bowers et al, 1984) and through adrenergic stimulation melatonin is synthesized and secreted to the SCN and peripheral tissues (Herzog and Tosini, 2001; Saper et al, 2005). Although most of the work related to the SCN has been carried out on mammals, it is likely that the suprachiasmatic nuclei in reptiles share the same properties (Underwood, 1992). Tosini and Menaker (1998) suggested that daily activity rhythms in Iguana iguana were driven by two circadian oscillators, the pineal organ and presumably the SCN. Furthermore, both the pineal and the SCN control the circadian rhythm of activity in Podarcis sicula, since changes in the concentration of melatonin receptors in the SCN correlate with enfrainability of locomotor cycles (Bertolucci and Foa, 1998). These studies together provide some evidence of the importance of SCN in reptiles. Retinas and extraretinalphotoreceptors The lateral eyes also form part of the circadian system. Circadian photoreceptors (retinal ganglion cells) in mammals, are only located in the retinas (Menaker, 2003). In reptiles however, the pineal organ and deep brain photoreceptors can also perceive light (Bertolucci and Foa, 2004). Photoreceptors participating in circadian rhythms are independent of the visual system (Menaker, 1982; Underwood, 1992; Menaker, 2003). The eyes of mammals and reptiles not only send photic stimuli to the rest of the circadian system, but also are sites for circadian oscillators. Studies on isolated retinas demonstrated circadian rhythms in melatonin synthesis of Xenopus, Mesocricetus auratus and Iguana iguana (Cahill and Besharse, 1995; Tosini and Menaker, 1996; Tosini and Menaker, 1998). 5 Circannual rhythms Circannual rhythms are endogenous rhythms, with a period of approximately one year and are also entrained by external environmental cues (Underwood, 1992; Kuchling, 1999). Events such as mating, reproduction, growth and overwintering in high latitude species occur at fixed times of the year and conform to an annual cycle. Although, the entire annual cycle does not appear to be driven by endogenous circannual clocks, studies on reptiles and mammals have demonstrated that at least certain stages are under inherent control. For instance, certain phases of the reproductive cycle, as well as the post-reproductive "refractory" period are not disturbed by changes in photic and temperature stimuli and persist in the absence of seasonal cues (Kuchling, 1999). Furthermore, some aspects of hibernation as well as fall fasting prior to the winter season occur at the same time of the year, independent of photoperiod and temperature conditions (Cagle, 1950; Mayhew, 1965; Bennett and Dawson, 1976). These endogenous circannual rhythms will ensure that crucial stages of the life cycle of animals, particularly those with high latitude distributions, take place at appropriate times of the year (Underwood, 1992). Like circadian rhythms, circannual cycles need to be synchronized with periodic environmental cues to be fully functional for the organism. Both temperature and photoperiod entrain circannual rhythms, although their contribution varies between the different phases of the annual cycle as well as between species. For instance, Mayhew (1965) suggested independent control of dormancy and metabolic suppression in the lizard Phrynosoma m'calli, so that dormancy was initiated by reduced photoperiod, while low temperatures initiated metabolic suppression. It is worth mentioning that the effects of these environmental cues on the metabolic state only occurred during the appropriate season, proving to be permitted by endogenous rhythms. For some hibernators, such as ground squirrels photoperiod acts as a weak zeitgeber (Kortner and Geiser, 2000), 6 whereas in reptiles, external cues, particularly photoperiod in the initial stages of overwintering, play an important role in entraining the rhythms (Rismiller and Helmaier, 1982; 1991). Interestingly, the brain structures involved in the control of circadian rhythms are also engaged in producing endogenous seasonal rhythms. In mammals, the SCN, which drives circadian cycles, only modulates circannual rhythms (Kortner and Geiser, 2000). The role that the SCN plays in reptilian seasonal rhythms is not well known. Seasonal changes in the concentration of melatonin receptors in the SCN of lizards has been suggested, which would cause seasonal changes in the responsiveness of circadian rhythms and affect behavioural and physiological processes (Bertolucci and Foa, 1998). The pineal gland, on the other hand controls seasonal rhythms in mammalian and reptilian species. Pinealectomy has been shown to have long-term effects on the synchronization of hibernation and reproductive events (Arendt, 1998; Kortner and Geiser, 2000). As mentioned previously, the pineal gland acts as a transducer of environmental information into endocrine signals, by means of its hormone melatonin. Therefore, seasonal changes in daylength and thermoperiod will change the duration and amplitude of the melatonin rhythm, which ultimately regulates circannual rhythms in a number of physiological processes in vertebrates (Arendt, 1998; Tosini et al, 2001). Although, the roles of the SCN and pineal gland differ between circadian and circannual rhythms, the interaction of the different structures of the "circadian axis" seems to be necessary to produce both rhythms. Evidence for this arises from studies showing that mammalian SCN activity was not reduced and that circadian rhythms persisted during hibernation, although with very small amplitudes, despite the fact that the SCN only serves a modulating function in the circannual rhythms of hibernating species (Kortner and Geiser, 2000). 7 Biological rhythms in behaviour and to some extent physiology of ectotherms have been thoroughly studied; with emphasis on the circadian rhythms of lizards. Turtles, however have not received as much attention, and very few studies have focused on the circadian and circannual endogenous cycles in metabolism. This is somewhat surprising since the high diversity of chelonians and the variety of environments they inhabit will convey important information in the field of biological rhythms. Pseudemys scripta in particular is an excellent model to study circadian and seasonal rhythms in a number of physiological variables. Due to their distribution red-eared sliders encounter both daily and drastic annual changes in their environment, thus endogenous circadian and circannual rhythms could be advantageous in that Pseudemys scripta could anticipate and prepare for periodic environmental changes. Pseudemys scripta Pseudemys scripta (red-eared sliders) inhabits ponds over most of the Eastern United States, as well as Central America, the Greater Antilles and the northern South American countries (Harles and Morlock, 1978); their distribution ranges up to a latitude of 50 0 N (Ultsch, 1989). Red-eared sliders are primarily diurnal (Harles and Morlock, 1978). Their daily activities consist of feeding, and basking presumably for thermoregulation (Ernst and Barbour, 1989). Sliders feed mainly in the morning and late afternoon and basking begins at eight in the morning and peaks between ten and eleven, in the months of August and September. This behaviour shifts later into the day during October and November (Ernst and Barbour, 1989; Boyer, 1965). Throughout the night P. scripta sleep by resting on the bottom or floating at the surface (Ernst and Barbour, 1989). Some temperate populations of red-eared sliders undergo hibernation in October, when water temperature falls below 10 °C, until March or April (Ernst and Barbour, 1989). Pseudemys scripta commonly hibernates submerged in the body 8 of water or burrowed in the mud (Cagle, 1950). In this way they avoid freezing and dehydration, but hypoxia or even anoxia might be experienced in these environments (Ultsch, 1989). Their reproductive cycle is widely determined by seasonality. Mating occurs in late spring and occasionally in the fall, and eggs are laid in June and July (Cagle, 1950; Ernst and Barbour, 1989). Hatchlings might emerge from their nests in the late summer or early fall (Costanzo et al, 2000). Metabolism As ectotherms, the metabolism of Pseudemys scripta is highly influenced by environmental temperature. Reptiles are known to control body temperature to some extent, mainly by means of behavioural thermoregulation, such as basking, postural adjustments and selection of appropriate microclimates (Bennett and Dawson, 1976; Bartholomew, 1982). Reptiles however, also demonstrate some physiological temperature control, mostly by changing the rates of temperature gain or loss (Bartholomew, 1982; Rismiller and Heldmaier, 1985; Lutterschmidt et al, 2003). Despite the limited thermoregulatory capacities of reptiles, they seem to maintain their body temperature within a narrow range that facilitates and ensures proper physiological function (Bennett and Dawson, 1976; Bennett, 1988). Temperature control might be difficult for aquatic ectotherms, such as Pseudemys scripta, since the water environment tends to be homogenous, complicating behavioural thermoregulation (Seebacher and Franklin, 2005). Therefore, P. scripta take advantage of the heterogeneous terrestrial environment by basking on land. It has been suggested that this behaviour aids in digestion, ovule maturation and removal of parasites and fungi (Avery, 1982). The small surface to volume ratios of fresh water turtles minimizes their heat loss to the environment, which could be advantageous when exposed to sudden cold conditions once entering the water after basking (Avery, 1982; Bartholomew, 1982). Furthermore, turtles are capable of controlling warming and cooling rates, so that aquatic turtles tend to 9 heat faster than they cool, while the opposite is true for terrestrial turtles, to avoid overheating (Bartholomew, 1982). Physiological control of rates of cooling and heating are mainly achieved by adjustments in the cardiovascular system, particularly to blood flow (Bartholomew, 1982; Seebacher and Franklin, 2005). Behavioural and physiological thermoregulation of Pseudemys scripta however, is not sufficient to eliminate daily and seasonal temperature fluctuations, particularly those associated with the drastic winter environmental conditions (see below). Therefore, changes in the metabolic rate of red-eared sliders occur on a daily and annual basis. Such adjustments in the metabolic rate of reptiles are known to be influenced by both photoperiod and temperature, which could also be driven by circadian rhythms and to some extent by circannual endogenous cycles (Mayhew, 1965; Mautz, 1979; Rismiller and Heldmaier, 1985; Rismiller and Heldmaier, 1991; Underwood, 1991). Metabolic suppression A common environmental stress experienced by various vertebrates inhabiting the northern hemisphere is the low temperature experienced throughout the winter season. Vertebrates implement different overwintering strategies. Some remain active or migrate to warmer regions during the cold seasons (Ultsch, 1989), while other vertebrates enter a state of sequestration, called hibernation, which involves a decrease in the basal metabolic rate (Boutilier, et al, 1997; Hochachka and Guppy, 1987). Turtles that inhabit such environments have adapted to the low ambient temperatures experienced during the winter by entering such a hypometabolic state that ensures survival until favorable conditions return (Boutilier et al, 1997). Moreover, there is evidence of metabolic inhibition beyond the effects of temperature in hibernating ectotherms (Boutilier, et al, 1997; Ultsch, 1989; 10 Penney, 1987; Herbert and Jackson, 1985; Seymour, 1973). The metabolic rate was significantly reduced in dormant toads compared to awake individuals at constant temperatures. Furthermore, a high Qi0 (8.5) was also observed in aerobic turtles, which experienced a change in ambient temperature from 10 to 3°C (Boutilier et al, 1997; Ultsch, 1989; Penney. 1987; Herbert and Jackson, 1985). Although, turtles can undergo seasonal metabolic suppression (temperature independent reduction in the metabolic rate), there is no evidence that they can actively suppress metabolism on a daily basis (torpor). Ventilation One key role of the cardiorespiratory system is to supply oxygen to the tissues to satisfy their metabolic requirements. In order to fulfill this role, ventilation and perfusion must be tightly correlated to metabolic rate (Shelton et al, 1986). For instance, lung ventilation in Pseudemys scripta occurs in episodes (VP), consisting of a variable number of breaths clustered together. The ventilatory period is followed by a breath-holding period (NVP) of variable length. (Milsom, 1988; Shelton et al, 1986; Burggren et al, 1977). Apnea occurs at the end of inspiration, which normally corresponds to the diving interval in aquatic species (Milsom, 1989; Shelton et al, 1986; Shelton and Boutilier, 1982). Changes in central vascular blood flow are associated with the arrhythmic breathing pattern. Pulmonary blood flow and heart rate increase during the ventilatory period and a right to left shunt might develop during the breath-holding period, due to increasing pulmonary resistance (Wang and Hicks, 1996; Burggren, 1988). Thus, ventilation and perfusion are regulated to match metabolic demand in turtles. Control of ventilation The respiratory system not only supplies oxygen to the organism to meet the metabolic requirements, it also maintains the arterial blood gases and the acid-11 base balance within determined ranges, in order to preserve the homeostasis of the system (Milsom, 1990; Shelton, et al, 1986). Inputs from different receptor groups allow the central pattern generator to modify the breathing pattern in order to fulfill that role, as well as to co-ordinate respiratory movements with other behavioral and physiological activities and to optimize the respiratory pattern (Milsom, 1990). Very little is known about the mechanisms that generate the arrhythmic breathing pattern of reptiles (Shelton et al, 1986). The existence of a common central rhythm generator responsible for both the rhythmic pattern seen in birds, fish and most mammals and the arrhythmic pattern of marine mammals, amphibians and reptiles, is still speculative. Evidence, such as the periodic breathing experienced by mammals during hibernation and the fact that changes in the respiratory drive of arrhythmic breathers primarily affects the length of the apnea, whereas the time of expiration and inspiration remain relatively constant; supports the hypothesis of a common central rhythm generator (Milsom, 1990). This pattern generator appears to rely more on peripheral inputs than higher centers in periodic breathers (Shelton, era/., 1986). The peripheral inputs come from different groups of receptors, such as defense receptors, pulmonary stretch receptors and chemoreceptors. The defense receptors are located in the posterior nares, larynx and glottis; they are innervated by the trigeminal nerve and are responsible for the apnea that occurs upon submergence (Jones and Milsom, 1979a; Shelton et al, 1986). Pulmonary stretch receptors are generally located in the subepithelial connective tissues in the walls of the lung. This group consists of three different types of receptors; 1) tonic, slowly adapting stretch receptors (SAR) provide volume information, and the increase in the discharge of the receptor adapts slowly when the airway wall tension is maintained constant (Schelegle and Green, 2001); 2) rapidly adapting stretch receptors (RAR) sense information on the rate of change, producing a burst when the lung volume changes rapidly, but adapting when the volume is constant; and; 3 ) an intermediate class of receptors that responds to both tonic and phasic volume change. These various groups of receptors are innervated by the vagus 12 nerve. In addition, pulmonary stretch receptors show chemosensitivity to C 0 2 (Milsom, 1990; Milsom and Jones, 1980; Jones and Milsom, 1979a; Jones and Milsom, 1979b). The chemoreceptors consist of an association of three types of cells; the glomus cells, sustentacular cells and nerve terminals. The glomus cells make synaptic contact with the nerve terminals, as well as with arterial capillaries; the sustentacular cells on the other hand are believed to confer nourishment to both types of cells. This association of cells is believed to have a role in intravascular chemoreception (Milsom, 1990; Shelton et al, 1986). Both peripheral and central chemoreceptor responses have been demonstrated in turtles. Tissue homologous to the aortic bodies are located in the truncal region in turtles, however, tissue homologous to the carotid bodies has yet to be accurately located in turtles (Milsom, 1990). Reflex responses to stimulation of the various groups of receptors have been widely studied, the response to hypoxia is an increase in minute ventilation, although the magnitude of this response varies among species; indeed some turtles tolerate high levels of hypoxia (Boyer, 1966; Shelton et al, 1986). The increase in minute ventilation is due mainly to an increase in the breathing frequency, as a result of a reduction in the non-ventilatory period, maintaining the frequency of breaths within each ventilatory period constant, but changing the number of breaths per ventilatory period. Tidal volume and breath duration, on the other hand, remain relatively unchanged (Milsom, 1990; Milsom, 1988; Shelton et al, 1986). The response to hypercapnia includes an increase in the tidal volume (Milsom, 1988; Milsom and Jones, 1980; Shelton et al, 1986), due to an increase in force and rate of expiration and inspiration; although, the increase in minute ventilation is primarily due to changes in the respiratory frequency (Milsom and Jones, 1980). 13 Observations Very few studies to date have examined "true" circadian and seasonal rhythms (rhythms persisting under constant darkness) in the metabolism and respiration of turtles. Several questions arise. First do these variables change with circadian and circannual rhythms. If so, are these changes partially or completely independent of the daily and seasonal changes seen in temperature? Finally, if daily and seasonal changes in metabolism and ventilation occur, are they accompanied by changes in the breathing pattern? If so, what are the implications for diving patterns? Hypothesis I hypothesize that metabolism changes with both endogenous circadian and circannual rhythms. In order to fulfill the metabolic requirements of the organism both ventilation and perfusion should remain linked to metabolism in both cases. Thus, ventilation should oscillate with the same daily and seasonal rhythms as metabolism. Circadian and circannual changes in metabolism and ventilation may be accompanied by changes in breathing and diving patterns, producing longer non-ventilatory periods at night and in the winter. During the ventilatory periods, breathing will still re-establish gas homeostasis, and net ventilation relative to metabolism will remain the same, regardless of the changes in the breathing pattern and diving profiles. Thus, whereas ventilation will be influenced by metabolic rate, the breathing pattern will be affected such that dive times will be enhanced and surface intervals reduced even more than would be predicted by changes in metabolic rate alone. 14 C H A P T E R 2: M e t h o d s Adult red-eared sliders (Pseudemys scripta) of either sex, and weighing between 0.52 - 2.92 kg (average mass = 1.09 kg; n = 16) were obtained from two commercial suppliers, Lemberger Company (Oshkosh, WI) and Sullivan Company Inc. (Nashville, TN). Eight animals were housed outdoors in a semi-natural pond, experiencing the environmental temperatures and natural photoperiod throughout the year (seasonal cues present). The remaining eight turtles were kept in four indoor tanks containing 89.92 gallons of water each at room temperature (2i°C), with a flow through system. Turtles were provided with basking platforms and full spectrum lights that were set by a timer on a 12 hour light and 12 hour dark photoperiod (i2L:i2D) (seasonal cues absent). All the turtles were fed a mixture of trout chow (Aquamax 400, 5D04), vegetables, and fruits, as well as vitamin D and calcium supplements three times a week. During the winter, turtles ate less, so food was provided only once a week. Turtles were fasted seven days prior to the trials to avoid confounding effects of digestion on the metabolic rate and respiratory exchange ratio measurements. The animals were weighed every month and prior to each experimental treatment. Experimental Protocol Experiments were carried out from the 13th of October 2003 until the 26 t h of November 2004. The two groups of turtles were each acclimatized to three different conditions to produce six treatment groups: 1. Chronic acclimatization to seasonal cues (throughout the year). Outdoor turtles exposed to environmental conditions. 15 2. Chronic acclimatization to constant conditions (seasonal cues absent throughout the year). Indoor turtles exposed to indoor conditions. 3 . Acute exposure to seasonal cues (duration of the experiment, 3 - 4 days). Indoor turtles acutely exposed to the prevailing outdoor conditions. 4 . Acute exposure to constant conditions (seasonal cues absent for the duration of the experiment, 3 - 4 days). Outdoor turtles acutely exposed to indoor conditions. 5 / 6 . Acute exposure to constant dark (daily and seasonal cues absent for the duration of the experiment, 3 - 4 days). Both groups of turtles exposed to constant darkness. 1. Chronic acclimatization to seasonal cues To determine if the physiological variables of interest exhibited circadian or circannual rhythms I acclimatized this group of turtles to environmental conditions throughout the year (treatment #1). Turtles were undisturbed at all times and were only removed from the semi-natural pond to run the experimental series during each season. Two data loggers recorded temperature every hour (DS1921 Thermocron iButton, Dallas Semiconductor Corporation). One data logger was submerged in the pond to measure water temperature and the second was placed on the basking area to record ambient temperature. Daily photoperiod, rainfall (Meteorological service of Canada website) and atmospheric pressure (barometer measurement was temperature corrected) were documented1. 1 Figure A1 shows hourly recordings of air and water temperature from October 2003 to November 2004 (Appendix A). Daily photoperiod, rainfall and atmospheric pressure recordings from October 2003 to November 2004 are shown in Table A1 (Appendix A). 16 2. Chronic acclimatization to constant conditions (seasonal cues absent) To understand the role of seasonal cues, such as temperature and photoperiod, in entraining circadian or circannual rhythms, I acclimatized a second group of turtles, using indoor tanks, to constant conditions (treatment #2) throughout the year (i2L:i2D photoperiod and 21 °C). The objective was to determine whether biological rhythms remained in the presence of daily cues but in the absence of seasonal cues. Data loggers were submerged in two of the four tanks to obtain the water temperature every hour. A third temperature logger placed in the environmental chamber, recorded air temperatures. 3. Acute exposure to seasonal cues I also wished to determine whether acute exposure to seasonal changes in temperature and photoperiod could entrain the rhythms. Turtles maintained under constant conditions (treatment #2) throughout the year were exposed to the prevailing seasonal cues during each season for 3 to 4 days. 4. Acute exposure to constant conditions (seasonal cues absent) To determine if acute removal of environmental cues would affect the circadian and circannual rhythms, turtles acclimatized to daily and seasonal changes in temperature and photoperiod (treatment #1) were exposed to constant conditions (i2L:i2D photoperiod and 2i°C water temperature; seasonal cues removed) for 3 to 4 days. All of the treatments were repeated in each of three seasons; summer, fall and winter. The temperature and photoperiod for each treatment are shown in Table 2.1. Water temperatures of experiments carried out at constant indoor conditions (seasonal cues absent) in the fall were slightly warmer than in winter and summer, due to the difficulty of obtaining a precise water temperature in the experimental tank with the temperature control system I was using. 17 5 and 6. Acute exposure to constant dark (daily cues absent) To remove the effects of photoperiod in establishing circadian rhythms I also ran experiments on both groups of turtles under constant darkness and at the temperature to which they were acclimatized. This constitutes the fifth and sixth treatments which were only run once for each group. Outdoor turtles chronically acclimatized to seasonal cues (treatment #1) were exposed to constant darkness in the fall of 2004; therefore, the water temperature chosen for this experiment (no cues) was 13.6 °C. Indoor turtles chronically acclimatized to constant conditions (treatment #2) were exposed to constant darkness in the summer at 19.89 °C. 18 Table 2.1: Temperature and photoperiod used for the three experimental series. Each turtle from each of the two groups underwent 2 experimental runs under each of 2 treatments in each season. Temperature and photoperiod for no cues treatments (daily cues absent) are also shown. Season Group Treatment Experimental run Photoperiod Temperature FALL Outdoor Outdoor conditions (seasonal cues) ioL:i4D 14.7 °C Air Experimental series #1 FALL Outdoor Outdoor conditions (seasonal cues) Hypoxia-Hypercapnia Experimental series #1 FALL Outdoor Indoor conditions (no seasonal cues) 12L:12D 22.4 °C Air Experimental series #1 FALL Outdoor Indoor conditions (no seasonal cues) Hypoxia-Hypercapnia Experimental series #1 FALL Outdoor No cues oL:24D 13.6 °C Air Experimental series #1 FALL Outdoor No cues Hypoxia-Hypercapnia Experimental series #1 FALL Indoor Outdoor conditions (seasonal cues) ioL:i4D 14.7 °c Air Experimental series #1 FALL Indoor Outdoor conditions (seasonal cues) Hypoxia-Hypercapnia Experimental series #1 FALL Indoor Indoor conditions (no seasonal cues) 12L:12D 22.4 °c Air Experimental series #1 FALL Indoor Indoor conditions (no seasonal cues) Hypoxia-Hypercapnia Experimental series #1 19 Season Group Treatment Experimental run Photoperiod Temperature WINTER Outdoor Outdoor conditions (seasonal cues) 9L:i5D 9 ° C Air Experimental series #2 WINTER Outdoor Outdoor conditions (seasonal cues) Hypoxia-Hypercapnia Experimental series #2 WINTER Outdoor Indoor conditions (no seasonal cues) 12L:12D 19-65 °c Air Experimental series #2 WINTER Outdoor Indoor conditions (no seasonal cues) Hypoxia-Hypercapnia Experimental series #2 WINTER Indoor Outdoor conditions (seasonal cues) 9L:i5D 9 ° C Air Experimental series #2 WINTER Indoor Outdoor conditions (seasonal cues) Hypoxia-Hypercapnia Experimental series #2 WINTER Indoor Indoor conditions (no seasonal cues) 12L:12D 19.65 °c Air Experimental series #2 WINTER Indoor Indoor conditions (no seasonal cues) Hypoxia-Hypercapnia Experimental series #2 20 Season Group Treatment Experimental run Photoperiod Temperature SUMMER Outdoor Outdoor conditions (seasonal cues) i6L:8D 20.8 °C Air Experimental series #3 SUMMER Outdoor Outdoor conditions (seasonal cues) Hypoxia-Hypercapnia Experimental series #3 SUMMER Outdoor Indoor conditions (no seasonal cues) 12L:12D 19.62 °C Air Experimental series #3 SUMMER Outdoor Indoor conditions (no seasonal cues) Hypoxia-Hypercapnia Experimental series #3 SUMMER Indoor Outdoor conditions (seasonal cues) i6L:8D 20.8 °C Air Experimental series #3 SUMMER Indoor Outdoor conditions (seasonal cues) Hypoxia-Hypercapnia Experimental series #3 SUMMER Indoor Indoor conditions (no seasonal cues) 12L:12D 19.62 °C Air Experimental series #3 SUMMER Indoor Indoor conditions (no seasonal cues) Hypoxia-Hypercapnia Experimental series #3 SUMMER Indoor No cues oL:24D 19.89 °c Air Experimental series #3 SUMMER Indoor No cues Hypoxia-Hypercapnia Experimental series #3 21 Within each season, two experimental runs were carried out for each treatment in each group. Turtles were acclimated to the experimental set-up for one or two days. Each experimental run was then carried out for 24 hours. On the first day post-acclimation, the turtle was given air to breathe, and on the second day the turtle was given a hypoxic-hypercapnic gas mixture (8% 02 - 3% CO2) to breathe (details in Experimental set-up section below). Experimental set-up The experimental set up consisted of a tank filled with water to a depth of 62 cm (Figure 2.1). The water entering the tank was filtered and the temperature was controlled by a solenoid system, while a flow-through system ensured that the water temperature remained constant within treatments. Thus, day and night water temperatures were equivalent. A data logger (DS1921 Thermocron iButton, Dallas Semiconductor Corporation) placed in the tank monitored the water temperature every hour for the total length (24 hours) of the experimental run, after which the temperature data was downloaded to a computer. The tank was placed inside a wooden box to insulate the turtle from external stimuli (i.e.: disturbance, noise, and light). Full spectrum lights were attached to the lid of the box. The lights were controlled by a timer to simulate a light-dark cycle. For the initial period of experimental series 1, two infrared cameras were placed inside the box, in order to monitor and record the turtle's behaviour for 24 hours. 22 Ventilation chamber The tank was divided in two, allowing us to run experiments on one individual while another turtle was being acclimated to the experimental tank for one or two days. Turtles were trained to respire through a funnel (11 cm in diameter), which was flushed continuously with humidified air on the first day following acclimation and the hypoxic-hypercapnic gas mixture2 on the second day, as mentioned above. The remaining surface of the tank was covered with mesh to prevent turtles from breathing outside the funnel, while allowing volume changes to be dispersed over the surface of the tank when the turtle raised its head to breathe. Each experiment was conducted on unrestrained turtles for one full circadian cycle (24 hours). Metabolism Measurements The rate of oxygen consumption (VoJ was measured using an open-flow respirometry system. A port in the side of the breathing funnel delivered either air 2 Data obtained from turtles breathing the hypoxic-hypercapnic gas mixture is not shown in this document. 23 or the hypoxic-hypercapnic gas mixture at a rate of 350 ml/min. The air leaving the funnel was passed through a drying column containing Calcium Sulfate (drierite) to remove water vapor, and then sampled by an 0 2 and C 0 2 gas analyzer (model 222A version 1.02, Raytech Instruments). The gas analyzer was calibrated prior to each trial by using 100% nitrogen and a mixture of 21% oxygen, 5% C 0 2 and 74% nitrogen. Since the error involved in calculating V0l without removing C 0 2 from the air sample is nominal (3%) (Withers, 1977), I did not use any C 0 2 absorbent. The fractional compositions of 0 2 and C 0 2 (in percent) in the exhalent gas were recorded onto a computer using WINDAQ acquisition software (version 2.19). Data analyses of metabolic variables The data channels where the fractional compositions of 0 2 (FQi) and C 0 2 (FC O i) were recorded were subsequently subtracted from the levels being administered to the funnel, and the area under the 0 2 and C 0 2 curves was integrated to obtain the oxygen consumed and C 0 2 produced. (The difference in F0i and FCOi entering and leaving the funnel was multiplied by the flow rate to obtain the amount of 0 2 and C 0 2 removed from or added to the gas leaving the funnel). All Vo2 and Vco, values were converted to STPD and normalized to the weight of the animal (kg). Because water vapor had been removed from the air/gas sample, the rate of oxygen consumption was further corrected as per Withers (1977): eq. 2.1 Vo; Vo2-Vco^FIOi 2 corr 1 r-. Where F10i was 0.2093 ml 0 2 on air and 0.08 ml 0 2 on the hypoxic-hypercapnic gas mixture. 24 The respiratory exchange ratio (RER) was calculated for each hour from the oxygen consumption and C 0 2 production values. eq. 2.2 RER = '-^ In order to filter out the confounding effects of activity on the metabolic rate, the percentage of time the turtle remained inactive was determined for each hour by calculating the duration of segments of the trace where activity-induced noise was observed. Activity-induced noise on the breathing traces was determined by comparing the signal and the video recording of the turtles during the initial experiments. Segments of metabolic rate traces were taken out from the analyses at times when turtles performed exploratory swimming, ensuring that values of oxygen consumption used for the analyses were in fact resting metabolic rates. Oxygen consumption values were used to calculate the percentage of oxygen extracted (%£„2) from each breath and the air convection requirement (ACR, ml air/ml 0 2 ) for each hour, using the following equations: eq.2.3 o/oE^=_Z^_ VEXFIOI eq.2.4 ACR = -^— Vo2cm Temperature quotient (Qw) Oxygen consumption data were averaged over both the daytime and nighttime measurements, and over the full 24 hours (total Vo2) for each turtle in each 25 season. To determine the effects of temperature on the metabolism of red-eared sliders I calculated the Qio's of oxygen consumption using the following relation: where MRi and M R 2 are the rates of oxygen consumption at temperature ti and t2, respectively. I determined the temperature sensitivity (Qio) of day, night and total V0l for three seasons: winter (9 °C), fall (14.7 °C) and summer (20.8 °C), as well as for the experiment with no cues (constant darkness) for turtles maintained under seasonal and under constant conditions. To determine the seasonally-independent effects of temperature on the metabolism of turtles, I determined the Qio's for Vo, at the environmental and constant (« 2i°C) temperature within each season for both constant and seasonal acclimatized groups of turtles. Ventilation Measurements A pneumotachograph was attached to the outflow of the breathing funnel. The pressure changes across the pneumotachograph were measured using a Validyne DP 103-18 differential pressure transducer. A constant flow of 350 ml/min was maintained through the funnel and was regulated by a gas mixing flow meter (Cameron Instrument Company). Pneumotachograph calibration The pneumotachograph was calibrated for each turtle. The funnel contained a calibration port through which known volumes (1-5 ml) of air could be pumped at known rates (10-60/min, in increments of 10) using a syringe-pump (Harvard apparatus model 683). In this calibration, withdrawal of air from the funnel 26 simulated inspiration by the turtle and injection of air simulated expiration. The differential pressure signal was recorded to a computer with WINDAQ data acquisition software (version 2.19). Both flow rate and volume were linear over this range. The voltage produced by the turtle's inspiratory and expiratory efforts was converted to volume using the polynomial equation obtained with the calibration (equation 2.7). The pressure deflections resulting from the volumes generated by the syringe-pump were integrated to obtain tidal volumes and the number of peaks counted to obtain the pumping rate (Funk er al, 1986). Data analyses of ventilatory variables Data traces were analyzed in one hour-long segments. All of the fall and winter data under outdoor conditions (seasonal cues present) were analyzed every hour for the total length of the experiment (24 hours). All other runs were analyzed every other hour (even hours were analyzed for a total of 12 hours). The breathing trace was analyzed for total frequency (/k, breaths/min) and average tidal volume ( V T , ml). These values were used to calculate total ventilation (VE, ml/min/kg). eq.2.6 VF.=fRxVT To determine total breathing frequency (fR, breaths/minute) the number of breaths per hour was calculated. The instantaneous frequency (frequency between individual breaths in an episode) was obtained by counting the number of breaths in each episode and dividing it by the total length of the breathing episode. Tidal volume was obtained by integrating the area under the differential pressure transducer trace. Values of voltage were converted to volumes using the regression equation obtained from calibration as described in the previous section. eq.2.7 Volume = [ v ° l t a g e " Z° " (~b) * ^ u e n ^ a 27 Where a, b and z0 are parameters given by the regression equation using values of volume, flow rate and voltage obtained during the calibration. The tidal volumes obtained each hour were averaged and standardized for body weight (ml/kg). Since turtles can increase ventilation through changes in apnea length (end-inspiratory pause), number of episodes and/or the number of breaths per episode; I analyzed a number of respiratory variables that have an effect on the breathing pattern. The duration (minutes) and the number of episodes were calculated for each data segment (one-hour) of the breathing trace, as were the number of breaths in each episode. Apnea was defined as a respiratory pause that exceeded the duration of two missed breaths. The length of the apneic periods (minutes) was determined for each hour. The percent times spent breathing (eupnoea) and not breathing (apnea) were also calculated as a percentage of each analyzed hour. Statistical analyses Statistical analyses were performed using Sigma Stat (version 3.11). Data for all the physiological variables were averaged over the daytime, nighttime and total time of a run (24 hours) for each turtle at each season. The Kolmogorov-Smirnov test was used to test normality and Levene's test was used to determine homogeneity of variance between samples. Data that did not meet the assumptions of normal distribution or equal variances were either natural log (Ln) transformed or were analyzed with a non-parametric equivalent. Circadian rhythms To determine whether circadian rhythms were present in the measured physiological variables a paired t-test was carried out comparing the average day and night values of the physiological variables within each season. 28 Circannual differences in circadian rhythms A two-way repeated measures ANOVA was used to determine if the season had an effect on the differences seen between day and night values. At times when significant differences were observed between the treatments a Pairwise multiple comparisons test (Holm-Sidak method) was used to find which groups were responsible for the differences. Circannual rhythms Values of the physiological variables were averaged over 24 hours for each turtle. The values for each season were compared using one-way repeated measures ANOVA. If significant differences between the seasons were observed a post-hoc test (Holm-Sidak test) was used to determine which particular seasons were different. 29 C H A P T E R 3: C i r c a d i a n a n d C i r c a n n u a l R h y t h m s i n the M e t a b o l i s m o f R e d -e a r e d S l i d e r s (Pseudemys scripta) INTRODUCTION The metabolism of reptiles is highly influenced by both environmental factors and internal biological rhythms (Aschoff and Pohl, 1970; Bennett, 1988). As ectotherms, reptiles depend largely on the environment to supply the heat necessary for thermoregulation. Although reptiles are capable of some metabolic heat production, it is often negligible and can not be sustained for thermoregulation for long periods of time (Bartholomew, 1982; Bennett, 1988; Seebacher and Franklin, 2005). Because of the limited ability of reptiles to thermoregulate, they often depart from their preferred temperature, as environmental temperature fluctuates widely with daily and seasonal cycles. Therefore, reptiles, particularly the ones living in northern latitudes, such as red-eared sliders require adaptations that allow them to survive extreme changes in their environment (Ultsch, 1989). It has been proposed that mechanisms that keep time such as circadian and circannual rhythms are advantageous in that organisms can anticipate and prepare for periodic environmental changes (Underwood, 1992; Mortola and Seifert, 2002; Tosini et al., 2001). Circadian rhythms have been demonstrated in a number of physiological variables, including the metabolism of reptiles (Mautz, 1979; Feder and Feder, 1981; Rismiller and Heldmaier, 1991). Daily rhythms in the metabolism of reptiles are often associated with daily changes in activity and ambient temperature (Bennett and Dawson, 1976; Mautz, 1979). Although it is somewhat difficult to determine independent daily changes in these variables, evidence from other studies demonstrated day-night differences in the metabolic 30 rate of lizards at rest and at a uniform temperature (Rismiller and Heldmaier, 1991). It has been suggested that photoperiod acts as a "noise free" signal that consistently indicates the past or upcoming season, while ambient temperature is more variable, and could change unexpectedly within an individual day or during an entire year (Underwood, 1992; Rismiller and Heldmaier, 1991). Thus, photoperiod will aid in anticipating drastic seasonal changes primarily in ambient temperature, which have substantial effects on the metabolic rate of ectotherms. During the winter months, for instance, most temperate reptiles enter a state of dormancy, which involves a decrease in the basal metabolic rate (Ultsch, 1989, Boutilier et al, 1997). Metabolic adjustments commence in advance of the winter season in preparation for the upcoming cooler months (Bennett and Dawson, 1976), suggesting the prevalence of an endogenous circannual rhythm. Together, circadian and circannual rhythms could prompt daily and seasonal adjustments in the metabolism of Pseudemys scripta that enhance their survival. Very few studies to date have focused on the effects that changing seasonal cues have on the circadian rhythms of metabolism in reptiles. Most research has concentrated on one or the other, ignoring the close link between circadian and circannual cycles. Thus, in seasonal studies oxygen consumption is often measured only at particular times of the day and conversely, daily studies are often only conducted in one season. This will obscure interesting information on seasonal changes in daily cycles of physiological variables. Therefore, the purpose of this chapter was to address this issue, by experimentally evaluating daily and seasonal changes in the metabolic rate of Pseudemys scripta held under various conditions. I was interested in determining whether circadian rhythms in metabolism prevailed under constant darkness (i.e., endogenous rhythms), and if they were independent of daily activity. I also wondered whether 31 the changes that occur seasonally, in ambient temperature and photoperiod significantly affected circadian rhythms. I asked whether acute changes in day length and temperature could also alter the circadian rhythms of metabolism. Another objective was to establish the role that environmental cues play in determining circannual cycles in the metabolism of red-eared sliders. 32 R E S U L T S 3 Circadian rhythms of metabolism Mean values of oxygen consumption and C0 2 production normalized to body weight were combined for animals in each group (n=8 for winter and fall, n=6 for summer and no cues experiments) to establish trends in metabolic rate over the course of a day. Values for winter (seasonal cues absent), summer and no circadian cues are shown every other hour. Values for winter (seasonal cues present) and fall are shown every hour. Chronic acclimatization to seasonal cues Pseudemys scripta maintained outdoors under natural conditions showed a daily rhythm in their metabolic rate when examined over 24 hours indoors at a "constant" (steady day and night) seasonally specific temperature, under natural photoperiod. Rates of oxygen consumption during the day were significantly higher than at night for the fall and winter seasons (P <0.05) (Figure 3.1). Despite the difference in photoperiod between fall and winter, oxygen consumption increases started at around six in the morning and decreases occurred between five and six in the afternoon for both seasons. Furthermore, peak V0l occurred at noon for all seasons (0.4569, 0.1081 and 0.5185 ml 02/min-kg, fall, winter and summer respectively) (Figure 3.2). Even though a peak in Vo2 was evident during the day in the summer, the circadian pattern was largely lost, primarily due to elevated V0l during the night. In fact, day and night rates of oxygen consumption were not significantly different (P >0.05) (Figure 3.1). 3 Mean daytime and nighttime measurements for all the physiological variables referred to in this chapter are shown in Tables B1-B4. P values obtained with statistical tests performed on the physiological variables referred to in this chapter are shown in Tables B5-B12. P values obtained with statistical tests performed on temperature coefficients (Q 1 0) are shown in Tables B13-B17 in Appendix B. 33 1.0 0.8 cn £ 0.6 E o •> Circadian Changes in Oxygen consumpt ion (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues B I Fall Winter Summer No cues (Fall) 1.0 Fall Winter Summer Acute Exposure to Constant Conditions Acclimatization to Constant Conditions 0.8 1 0.6 £ 0.4 CM o > 0.2 0.0 C r UN Fall Winter Summer Season Fall Winter Summer No cues _ (Summer) Season Figure 3.1: Mean resting rates of oxygen consumption i SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of oxygen consumption for turtles chronically acclimatized to seasonal and indoor conditions. Day and night rates of oxygen consumption are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). Outdoor conditions (seasonal cues): Winter 9°C, 9L15D; Fall 14/TC, 10L14D; Summer 20.8°C, 16L8D; No cues: 13.6°C, constant darkness. Indoor conditions (no seasonal cues): Winter 19.65°C; Fall 22.4°; Summer 19.62°C, all seasons under 12L12D; No cues: 19.9°C, constant darkness. Significant differences between day and night values are indicated by (*) (P<0.05) or (e) (P=0.054 and P =0.056, outdoor and indoor turtles respectively). 34 W inter —A— Fall • Sum mer No cues 14 1.2 1.0 1 1 0.8 CM o £ 0.6 CM 0 0.4 0.2 0.0 1.4 1.2 1.0 J ; o.8 CM o 1 0.6 CM . § 0.4 0.2 0.0 Seasonal Cues Outdoor An ima l s No Seasonal Cues Oxygen C o n s u m p t i o n (Air) Seasonal Cues 10 15 Hour 20 25 10 15 20 Hour 25 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Indoor An ima ls No Seasonal Cues 10 15 20 25 Hour 10 15 20 25 Hour Figure 3.2: Mean hourly oxygen consumption ± S E M over a 24 hour period. Plots on the top show the circadian changes in oxygen consumption for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in oxygen consumption with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 35 On further analyses it can be seen that the difference between day and night values of oxygen consumption increased at colder water temperatures (Figure 3.3). Only with the warmer water temperatures during the summer season were differences in day and night values of oxygen consumption not significant. 100 1 80 A 4 6 8 10 12 14 16 18 20 22 24 26 Tempera tu re (°C) Figure 3.3: Difference between photophase and scotophase values of oxygen consumption of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Day and night oxygen consumption (ml 02/min/kg) difference, expressed as the percentage of total oxygen consumption (average over 24 hours) at different seasonal temperatures and under constant darkness. Day and night difference in oxygen consumption at seasonal temperatures are denoted by open bars (winter: 9 °C; fall: 14.71 °C; summer: 20.83 °C). The filled bar indicates day-night difference under constant darkness (13.59 °C). Although both day and night metabolic rates decreased with cooler temperatures, the decline in night oxygen consumption at low water temperatures was larger (Figure 3.4). 36 0.5 0.4 E CM o i 0.3 H CM o > 0.2 0.1 H 0.0 6 1 i 1 r 8 10 12 14 16 18 20 22 24 26 winter no cues fall summer Temperature °C Figure 3.4: Mean daytime and nighttime values of oxygen consumption of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Figure shows nighttime (filled bars) and daytime (open bars) rates of oxygen consumption measured at seasonal temperatures (winter: 9 °C; fall: 14.71 °C and summer: 20.83 °C) and under constant darkness in the fall (13.6 °C). In order to verify the occurrence of true circadian rhythms I removed all external cues that could function as an indicator of time of the day. Therefore, experiments were carried out under constant darkness and at the temperature turtles were acclimatized to in the fall. Since this experiment (no cues) occurred in the fall season the temperature chosen was 13.6 °C. The "daytime" and "nighttime" averaged hours corresponded to the fall photoperiod as well. Turtles showed circadian rhythms after being placed in the experimental tank under constant darkness for two days (Figure 3.1). The pattern, however, was shifted; peak V0l (0.2734 ml 02/min-kg) occurred at eight in the morning and the day-night difference was reduced compared to the corresponding fall season (cues present) 37 (Figures 3.2 and 3.3). "Day" levels of oxygen consumption measured under constant darkness differed substantially from day-fall values, while "night" Vo2 values were only slightly lower than night-fall values (Figure 3.4). As a consequence, only a trend in the day-night difference was observed ( P =0.054). Carbon dioxide production behaved in the same fashion as oxygen consumption; daytime values were significantly higher than nighttime values for fall and winter ( P <o.05), but differences were not significant for the summer ( P > o . o s ) . Under constant darkness (no cues), daytime rates of C0 2 production were higher than nighttime Vco,, although not significant a trend in day-night difference was observed ( P =0.055) (Figure 3.5 and 3.6). As a consequence of the parallel in oxygen consumption and C0 2 production, the respiratory exchange ratio remained relatively constant throughout the 24 hours for all seasons ( P > o . o s ) (Table 3.1). Table 3.1: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles chronically acclimatized to seasonal cues. SEASONS DAY NIGHT P Fall 1.02 ± 0.008 1.015 ± 0.0083 0.43 Winter 1.02 ± 0.005 1.025 ± 0.012 0.64 Summer 1.02 ± 0.006 1.019 ± O.0066 0.41 No cues 1.04 ± 0.015 1.065 ±0.026 0.36 38 1.0 0.8 c E 0.6 Circadian Changes in C 0 2 production (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues B I Fall Winter Summer NC (Fall) 1.0 0.8 I 0.6 0.4 0.2 0.0 Fall Winter Summer Acute Exposure to Constant Conditions Acclimatization to Constant Conditions nu Fall Winter Summer Season Fall Winter Summer NC (Summer) Season Figure 3.5: Mean resting rates of C 0 2 production ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of C 0 2 production for turtles chronically acclimatized to seasonal and indoor conditions. Day and night rates of C 0 2 production are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) or (e) (P=0.055 and P =0.052, outdoor and indoor turtles respectively). 39 W i n t e r A F a l l —m- S u m m e r N o c u e s 1.4 1.2 10 0.8 0.6 0.4 0.2 0.0 1.4 1.2 1.0 0-8 O Seasonal Cues C 0 2 Production (Air) Outdoor Animals No Seasonal Cues 06 0.4 0.2 00 10 15 Hour 20 25 10 15 Hour 20 25 1.4 1.2 1.0 0 8 0.6 0.4 0 2 0.0 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Seasonal Cues Indoor Animals No Seasonal Cues 10 15 Hour 20 25 10 15 Hour 20 25 Figure 3.6: Mean hourly C 0 2 production ± S E M over a 24 hour period. Plots on the top show the circadian changes in C 0 2 production for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in C 0 2 production with no external with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 40 Day and night activity levels did not differ in the fall and summer seasons (P >o.o5). In winter and when no cues were present, daytime activities were significantly higher than nighttime activities (P < o . o s ) . Overall, however, the level of activity was minor, consisting of movements around the breathing funnel rather than exploratory swimming, as noted with the video monitoring system. Episodes of activity for all seasons never exceeded 20% of the total time (24 hours), ruling out the confounding effects of activity on the circadian changes in oxygen consumption and C 0 2 production (Figure 3.7 and Table 3.2). Table 3.2: Mean percentage of time inactive ± S E M in different seasons. Day and night values of inactivity (% time) and P values for turtles chronically acclimatized to seasonal cues. Significant differences between day and nig ht are indicated by (*). SEASONS DAY NIGHT P Fall 89.48 ±2.59 94.41 ± 2.10 0.110 Winter 94. 9 ± 2.25 98.59 ± 0.56 0.042* Summer 94.07 ± O.99 96.54 ± 1.32 0.296 No cues 94.86 ± 1.11 98. 7 ± 1.11 0.006* Chronic acclimatization to constant conditions (no seasonal cues) The circadian effects on the rate of oxygen consumption and C 0 2 production seen in outdoor turtles were lost in turtles chronically acclimatized to constant conditions (21 °C, 12L:12D) for all seasons (P > o . o s ) (Figure 3.1 and 3.5). Similar trends were present but the differences were not significant (Figures 3.2 and 3.6). These results suggest that chronic exposure to daily and seasonal variations in temperature and photoperiod are important for entraining circadian rhythms in the metabolic rate of turtles. Under constant darkness (no cues), both rates of Vo2 and Vco2 were higher during the "day" than at "night"; however only trends were observed (Po 2 : P =0.056; VCOl: P =0.052). It is rather interesting that, the 41 110 100 I 90 TJ 03 c CD 80 E 70 60 50 110 100 4) > 90 CD c 0) 80 E 70 60 50 W inter —A— Fall • Sum mer No cues % T ime Inactive (Air) Seasonal Cues Outdoor A n i m a l s No Seasonal Cues 10 15 Hour 20 25 10 15 Hour 20 25 110 Seasonal Cues Indoor An ima ls No Seasonal Cues 10 15 Hour 20 25 Figure 3.7: Proportion of the hour turtles were inactive ± S E M over a 24 hour period. Plots on the top show the circadian changes in the time inactive for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in the time inactive with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 42 circadian effect on metabolism appeared to be stronger in constant darkness than under a 12L:12D photocycle. Given that oxygen consumption and C0 2 production changed in parallel, mean respiratory exchange ratios were approximately l for all seasons and did not vary with time of the day (P > 0.05) (Table 3.3). Table 3.3: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles chronically acclimatized to constant indoor conditions. SEASONS DAY NIGHT P Fall 1.01 ± 0.002 1.02 ± 0.006 0.28 Winter 1.03 ± 0.009 1.04 ± 0.008 0.64 Summer 1.02 ± 0.004 1.01 ± 0.003 O.067 No cues 1.02 ± 0.003 1.02 ± 0.0024 0-75 Turtles held under constant conditions remained relatively inactive throughout the experiment, and day and night activity did not differ significantly (P >0.05) (Table 34J-Table 3.4: Mean percentage of time inactive ± SEM in different seasons. Day and night values of inactivity (% time) and P values for turtles chronically acclimatized constant indoor conditions. (•) indicates values obtained with the non-parametric Wilcoxon Signed Rank test. SEASONS DAY NIGHT P Fall 89.43 ± 2.5 88.41 ± 3.84 0.84 Winter 98.24 ± 0.59 95.48 ± 3.14 !• Summer 97.41 ± 0.41 90.36 ± 5.88 0.27 No cues 96.58 ± 1.54 96.62 ± 2.64 043 43 Acute exposure to seasonal cues Acute exposure to seasonal cues did not entrain circadian rhythms in the rates of oxygen consumption or C 0 2 production in any of the seasons (P >o.05) (Figure 3.1 and 3.5). This suggests that long term exposure to varying environmental cues, such as temperature and photoperiod is required to entrain such biological cycles. Even though differences between daytime and nighttime metabolism did not become apparent when turtles were acutely exposed to environmental cues, it is worth mentioning that the trend in the circadian pattern was enhanced in the summer season (Figures 3.2 and 3.6). Since V0l and Vco, were tightly correlated, the RER did not vary between day and night (P >o.05) (Table 3.5). Table 3.5: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles acutely exposed to seasonal cues. SEASONS DAY NIGHT P Fall 1.02 ± 0.006 1.03 ± 0.006 0-35 Winter 1.02 ± 0.01 1.03 ± 0.01 0.62 Summer 1.02 ± 0.004 l.Ol ± 0.003 O.13 Turtles with acute exposure to seasonal cues remained inactive the majority of the experiment, day and night percent time inactive did not vary significantly (P >o.05) (Table 3.6). 44 Table 3.6: Mean percentage of time inactive ± SEM in different seasons. Day and night values of inactivity (% time) and P values for turtles acutely exposed to seasonal cues. SEASONS DAY NIGHT P Fall 88.31 ± 3.07 92.41 ± 3.59 0.13 Winter 97.72 ± 0.92 94.14 ± 1.82 0.16 Summer 92.65 ± 2.02 95.65 ± 1.88 0.22 Acute exposure to constant conditions (no seasonal cues) When turtles acclimatized to outdoor conditions (seasonal cues) were acutely exposed to constant indoor conditions (21 °C and 12L:12D photoperiod), differences between daytime and nighttime values of oxygen consumption were lost in all seasons C P > o . o s ) (Figure 3.1). Although no significant differences between day and night rates of oxygen consumption were evident, a clear circadian trend in V0l was apparent in the fall and summer seasons (Figure 3.2). As well, C 0 2 production did not vary significantly between day and night in any season ( P >0-05) (Figure 3.5), and the RER remained relatively constant throughout the experiment. No significant differences between day and night RER values were observed ( P > o . o s ) (Table 3.7). Turtles also remained inactive for most of the experiment ( P > o . o s ) (Table 3.8). Table 3.7: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Day and night mean RER values and P values for turtles acutely exposed to constant indoor conditions. SEASONS DAY NIGHT P Fall 1.02 ± 0.007 1.019 ± 0.002 0.84 Winter 1.07 ± 0.03 1.07 ± O.03 0-75 Summer 1.07 ± 0.03 1.06 ± 0.03 0.84 45 Table 3.8: Mean percentage of time inactive ± SEM in different seasons. Day and night values of inactivity (% time) and P va lues for turtles acutely e x p o s e d to constant indoor conditions. SEASONS DAY NIGHT P Fall 82.9647 ± 5-79 90.96 ± 2.29 0.64 Winter 93.203 ± 3.86 98.54 ± 0.81 0.15 Summer 95.412 ± 1.534 96.5 ± 1.32 0.67 Circannual rhythms of metabolism To examine the effects of the circannual cycle on the metabolism of Pseudemys scripta, all physiological variables referred to in this chapter were averaged over the entire experimental run (24 hours) for each season. Chronic acclimatization to seasonal cues Pseudemys scripta showed a circannual rhythm in the rate of oxygen consumption under seasonal temperature and natural photoperiod. Fo2was higher in the summer and gradually decreased in subsequent colder seasons ( P < o . o s ) (Figure 3.8). Similar seasonal trends were observed in C0 2 production, Vco, varied significantly between seasons, with lower values in the cooler seasons ( P < o . o s ) (Figure 3.9). Fall values of oxygen consumption and C0 2 production did not vary significantly when turtles were measured under constant darkness (no cues) in the corresponding season, suggesting that photoperiod on its own does not induce seasonal changes in metabolism. As a consequence of parallel changes in the seasonal cycles of oxygen consumption and C0 2 production the respiratory exchange ratio remained relatively constant throughout the seasons ( P > o . o s ) (Table 3.9). 46 Table 3.9: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles chronically acclimatized to outdoor seasonal cues. No significant differences in R E R were observed between seasons (P=0.212) SEASON TOTAL (24 hours) Fall 1.02 ± 0.007 Winter 1.02 ± 0.009 Summer 1.02 ± 0.006 No cues 1.05 ± 0.014 Turtles were slightly more active in the fall compared to winter. Turtles with seasonal cues were also more active than those exposed to constant darkness (no cues) (P <o.040) (Table 3.10). Increased activity did not seem to be caused by higher temperatures, since periods of movement in the summer were not different from other seasons. Overall, turtles remained inactive for the majority of the experimental treatment. Table 3.10: Mean percentage of time inactive ± S E M in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles chronically acclimatized to outdoor seasonal cues. Significant differences in inactivity between seasons were observed (P=0.040). Turtles were more active in the fall than in the winter and no cues treatments. SEASON TOTAL (24 hours) Fall 92.38 ± 1.9 Winter 97.20 ± 1.05 Summer 94.89 ± 0.49 No cues 96.14 ± 1.04 47 Circannual Changes in Oxygen Consumpt ion (Air) 0.8 - i °> 0.6 H E C ^ 0 . 4 i CM O 0.2 -I Acclimatized to Seasonal Cues 0.0 Acute Exposure to Seasonal Cues ..1 20.8C •f 14.7C C 8.8'C • • b.13.6 0.8 -0.6 -E O 0.4 -I CM O 0.2 -> 0.0 -Summer Fall Winter No cues (Fall) Acute Exposure to Constant Conditions a 22.4C J-19.6C 0.8 9> 0.6 O 0.4 0.2 0.0 Summer Fall Winter Acute Exposure to Constant Conditions E a 19.6C T Corrected I • T 1 9 . 6 C Summer Fall Winter Seasons B a T 20.8 C -14.7C 8.8C Summer Fall Winter Acclimatized to Constant Conditions D r ' -22.4C ab 19.6C 6 C 19.8C Summer Fall Winter No cues (Summer) Acclimatized to Constant Conditions 19.8C Summer Fall Winter No cues (Summer) Seasons Figure 3.8: Seasonal changes in mean resting oxygen consumption ± S E M . Figures show the seasonal values of oxygen consumption for turtles chronically exposed to seasonal and constant conditions. Seasonal rates of oxygen consumption are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Panels E and F show seasonal changes in temperature-corrected fall values of oxygen consumption. Significant differences between seasons (P<0.05) are indicated by different letters. 48 C i r c a n n u a l C h a n g e s in C O 2 P r o d u c t i o n (Air) 0.8 TI "55 0.6 C £ 8 0.4 CM O . § 0.2 0.0 Acc l imat ized to Seasona l C u e s Acu te Exposure to Seasona l C u e s B a T2O.8C "14.7C b 13.6C c 8.8 C a T 2 0 . 8 C b 8 .8C Summer Fall Winter No cues (Fall) Summer Fall Winter Acute Exposure to Constant Cond i t i ons Acc l ima t i zed to Cons tan t Cond i t i ons 0.8 at 0.6 c E ~c\i 8 0.4 CM O S 0.2 I 0.0 a "[19.6C b J 19.8C Summer Fall Winter Seasons Summer Fall Winter No cues (Summer) Seasons Figure 3.9: Seasonal changes in mean resting C 0 2 production ± S E M . Figures show the seasonal values of C 0 2 production for turtles chronically exposed to seasonal and constant conditions. Seasonal rates of C 0 2 production are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 49 Temperature coefficient Temperature coefficients (Qio) are often used to express metabolic sensitivity to temperature changes. Theoretically the oxygen consumption of reptiles increases at higher temperatures according to van't HofFs rule. To determine the effects that changes in ambient temperature have on the metabolism of Pseudemys scripta, I calculated the Qio's of day, night and total (24 hour) oxygen consumption for three seasons (winter 9 °C, fall 14.71 °C and summer 20.8 °C) and for experiments under constant darkness (13.6 °C) (Table 3.11). Table 3.11: Mean temperature coefficients ± SEM of turtles chronically exposed to seasonal cues (outdoor turtles exposed to outdoor conditions). Q 1 0 values for day, night and total (24 hours) oxygen consumption were calculated for three seasonal temperatures (winter, fall and summer), as well Qu> Fall-Winter (14.7-8.8 °c) Sum.-Fall (20 .8-14 .7 °c) Sum.-Wint ( 20 . 8 - 8 . 8 °c) NC (fall)-Wint (13.6-8.8 °c) Sum-NC (fall) (20 .8 -13 .6 °c) Day 4.02±i.3i 2.i4±o.59 2.56±0.43 3.20±i.48 3.o6±o.74 Night 5.89±i.30 3.33±o.9i 3.89±o.35 4.84±1.39 5.27±2.22 Total ( 2 4 hours) 4.70±i.io 2.83±o.74 3.30±o.38 4.i9±o.97 3.io±o.55 As mentioned previously, turtles chronically exposed to environmental cues throughout the year showed seasonal changes in their metabolic rate (Figure 3.8). The drop in the metabolic rate of red-eared sliders from summer to fall appeared to result from the changes in water temperature between these two seasons, in other words temperature effects can fully explain the changes seen in metabolism, as indicated by the Qi0 values calculated (Table 3.11). On the other hand Qi0 values suggest that turtles actively suppressed their metabolism in winter. Intriguingly, metabolism was more temperature sensitive over the ranges from fall to winter than over the entire range from summer to winter, as revealed by higher fall-winter Q10 values (Table 3.11), although no significant differences were found 50 (P>0.05). Furthermore, active metabolic suppression in Pseudemys scripta seemed to be greater at night than in the day (^ =0.025). The temperature coefficient calculated between summer (natural photoperiod) and fall values under conditions of constant darkness, suggest there was active suppression of the metabolism as well. It is rather interesting that constant darkness in the fall elicits metabolic suppression, while changes between summer and fall under natural photoperiod may be exclusively due to temperature effects. I should point out that indications of metabolic suppression were only evident at night and over the whole 24 hours, but daytime changes in oxygen consumption did not suggest active suppression when measured under constant darkness. Chronic acclimatization to constant conditions (no seasonal cues) Turtles acclimatized to constant conditions (21 ° C and 12L:12D) did not show the same circannual patterns in oxygen consumption and C 0 2 production as turtles chronically exposed to seasonal cues. Nevertheless, fall metabolic rate was higher compared to that of other seasons (Figure 3.8). It is possible though, that the higher temperature used during the fall trial (22.4 ° C ) compared to other seasons (» 19.6 ° C ) was partially responsible for such results. To determine if fall rates of oxygen consumption were indeed higher than those of other seasons I used the following relationship to correct for the temperature difference between trials: where M R 2 was the variable to be determined (corrected fall Vo2 at t2 = 19.6 ° C ) . M R i was the fall rate of oxygen consumption measured at 14.71 ° C (ti) of turtles chronically exposed to constant conditions. The temperature coefficient ( Q i 0 ) used was 6.56 (Q10 obtained for V0l values measured at 14.71 ° C and 22.4 ° C in the fall, see Table 3.20). The Q J 0 was calculated by inserting the values of oxygen 51 consumption measured in the fall at 22.4 °C ( M R 2 ) and at 14.71 °C (MRi) in to eg. 3.1. After calculating the value of fall oxygen consumption at 19.6 °C, fall V a. was significantly higher than winter values (P =0.004) (Figure 3.8). Rates of oxygen consumption and C 0 2 production in the summer versus winter did not differ significantly. However, corresponding summer values under constant darkness were higher than Vo2 and Vco2 winter values (P <0.05) (Figure 3.10). Oxygen consumption trends of turtles under constant darkness (summer) were exceptionally similar to trends measured in the corresponding season under natural photoperiod. Figure 3.10: Mean oxygen consumption ± SEM of indoor turtles (acclimatized to constant indoor conditions) at different water temperatures. The figure shows the values of oxygen consumption measured at outdoor conditions (seasonal cues, filled symbols) and indoor conditions (no seasonal cues, open symbols). See Table 2.1 .for details. Significant differences between seasons at indoor conditions (open symbols) are indicated by (*). Significant differences between seasons at outdoor conditions (filled symbols) are indicated by letters. The line indicates the regression that best fits the data, y = 0.0882 + (0.0004)e<03210>x See figure 3.11 for details of oxygen consumption values at constant conditions. 52 Regardless of the almost identical experimental temperatures and photoperiods used at each season, winter rates of oxygen consumption and C0 2 production were lower (see turtles acclimatized to constant conditions curve, Figure 3.11). Oxygen consumption declines in a progressive fashion from fall to summer to winter despite of the slight increase in water temperature. These temperature-independent changes in V0l and VCo2 suggest evidence of an endogenous circannual rhythm in the metabolic rate of Pseudemys scripta. Figure 3.11: Mean oxygen consumption ± S E M measured under constant indoor photoperiod and temperature at different seasons. Rates of oxygen consumption of both outdoor (acclimatized to environmental conditions • ) and indoor turtles (acclimatized to indoor conditions •) measured at constant indoor conditions during three seasons (winter: 19.65 °C, fall (corrected) 19.6 °C and summer 19.62 °C). Significant differences between seasons are indicated by (*) for indoor turtles and by (e) for outdoor turtles. Interestingly, the respiratory exchange ratio was higher in winter compared to other seasons. Turtles had higher rates of C0 2 production relative to their oxygen consumption (P <o.os) (Table 3 .12). Turtles remained more active in the fall compared to winter and under constant darkness (P <o.os) (Table 3 .13). Since fall 53 and summer activity did not differ, it is unlikely that the high fall metabolic rate is caused by periods of activity. Table 3.12: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles chronically acclimatized to constant indoor conditions. Significant differences in R E R were observed between seasons (P=0.025). Winter RER was significantly SEASON TOTAL (24 hours) Fall 1.08 ±0.003 Winter 1.036 ± 0.008 Summer 1.016 ± 0.003 No cues 1.018 ± 0.002 Table 3.13: Mean percentage of time inactive ± SEM in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles chronically acclimatized to constant indoor conditions. Significant differences in inactivity between seasons were observed (P=0.020). Turtles were SEASON TOTAL (24 hours) Fall 89.00 ± 2.16 Winter 96.85 ± 1.55 Summer 93.89 ± 2.99 No cues 96.60 ± 1.94 Acute exposure to seasonal cues When turtles maintained under constant indoor conditions were acutely exposed to seasonal cues (natural temperature and photoperiod at each season) a circannual cycle became apparent. Higher values of oxygen consumption were observed in the summer, and Vo2 was significantly reduced in the cooler seasons (P <o.05) (Figure 3.8). Even though seasonal trends in C0 2 production were similar 54 to those of oxygen consumption, fall and winter Vco2 were not different, although both were significantly lower than summer Vco2 values (P <o.os) (Figure 3.9). Respiratory exchange ratios did not change throughout the seasons, indicating that oxygen consumption and C 0 2 production varied correspondingly throughout the year (P >o.os) (Table 3.14). Differences in metabolic rates in various seasons were not caused by activity (P >o.os) (Table 3.15), since turtles remained inactive for the majority of the experimental trials. Table 3.14: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles acutely exposed to seasonal cues. No significant differences in RER were observed between seasons (P=0.805). SEASON TOTAL (24 hours) Fall 1.02 ± 0.005 Winter 1.02 ± 0.009 Summer 1.09 ± 0.004 Table 3.15: Mean percentage of time inactive ± SEM in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles acutely exposed to seasonal cues. No significant differences in inactivity between seasons were observed (P=0.193). SEASON TOTAL (24 hours) Fall 90.74 ± 3.18 Winter 95.48 ± 1.12 Summer 93.65 ± 1.69 Temperature coefficient The seasonal differences found in the metabolism of this group of turtles can not be fully explained by temperature effects, as indicated by high Q10 values calculated between seasons (Table 3.16). Pseudemys scripta acutely exposed to 55 environmental conditions appear to actively suppress their metabolic rates with changing seasons. Acute and chronic exposure to environmental conditions shows interesting differences. For instance, temperature coefficients were overall higher in turtles acutely exposed to seasonal cues than those calculated for turtles chronically acclimatized to seasonal cues (Table 3.16). These results suggest that continuous exposure to seasonal conditions allow for more subtle changes in the rates of oxygen consumption between seasons. Furthermore, metabolic suppression was higher in the day than at night (although not significant, P=o.245), except for temperature coefficients calculated between summer and fall, and no cues and fall. Table 3.16: Mean temperature coefficients ± SEM of turtles acutely exposed to seasonal cues (indoor turtles exposed to outdoor conditions). Q 1 0 values for day, night and total (24 hours) oxygen consumption were calculated for three seasonal temperatures (winter, fall and summer), as well as for fall Q10 Fall-Winter (14.7-8.8 °C) Summer-Fall (20 .8 -14 .7 °C) Sum.-Winter ( 2 0 8 - 8 . 8 °c) NC (sum)-Fall (19.8-14.71 °c) NC (sum)-Wint (19 .8 -8 .8 °c) Day 8.oi±2.i8 5.27±1.54 5-37±0.53 7.i6±2.6 6.66±i.28 Night 2.26±0.6l 6.64±i.24 3.8o±o.8i 7.68±i.64 3.75±o.9i Total ( 2 4 hours) 3.i9±o.58 6.i6±i.57 4-39±o.95 6.78±i.48 4.29±0.55 Acute exposure to constant conditions (no seasonal cues) Acute exposure of turtles to constant conditions revealed interesting seasonal rhythms in metabolism independent of the effects of temperature (Figure 3.8). Fall and summer rates of oxygen consumption and C 0 2 production were significantly higher than winter values, regardless of the similar experimental temperatures used ( P < o . o s ) . Equation 3.1 was used to correct for the higher fall 56 temperature as well, and the Q i 0 used was 3.47 (calculated by inserting the Vo2 measured in the fall at 22.4 °C and at 14.71 °C in to eq. 3.1). In fact, when comparing winter (19.65 °C), fall (corrected, 19.6 °C) and summer (19.62 °C) values of oxygen consumption in Figure 3.12 the winter metabolic suppression becomes apparent. There is an accelerating decrease in the metabolic rate levels from fall (19.6 °C) to winter (19.65 °C) (Figure 3.11). Furthermore, the steep decline in winter oxygen consumption suggests active suppression of the metabolism of overwintering turtles. 0.8 -I 0.7 -0.6 -0.5 -I 0.4 -O i CM 0.3 -O > 0.2 -0.1 -0.0 -F: 14.71°C. NC: 13.6°C rW: 8.8 °C F: 22.4°C F: 19.6°C(corr) T S: 19.62°C S: 20.84°C 1 W : 19.65°c"^" 10 12 14 16 18 Temperature (°C) 20 22 24 Figure 3.12: Mean oxygen consumption ± S E M of outdoor turtles (acclimatized to seasonal cues) at different water temperatures. The figure shows the values of oxygen consumption measured at outdoor conditions (seasonal cues, filled symbols) and under indoor conditions (no seasonal cues, open symbols). See Table 2.1 for details. Significant differences between seasons at indoor conditions (open symbols) are indicated by (*). Significant differences between seasons at outdoor conditions (filled symbols) are indicated by different letters. The line indicates the regression that best fits the data, y = 0.1211 + 0.0008 E<0 2837>\ See details of oxygen consumption values at constant conditions in Figure 3.11. Although it is difficult to tease out the effects of temperature on the metabolism of reptiles, these results together with observations of lower winter rates of oxygen 57 consumption in turtles chronically acclimatized to constant conditions, suggest that there is in fact an endogenous circannual rhythm in the metabolic rate of Pseudemys scripta (Figure 3.11). The respiratory exchange ratio remained relatively constant throughout the year (P >0-05) (Table 3.17). Longer activity periods were measured in the fall (P <o.05) (Figure 3.18). On the whole, mean activity periods did not exceed more than 13% of the total time (24 hours) in this season. Table 3.17: Mean respiratory exchange ratio (RER) ± SEM in different seasons. Mean RER values calculated over 24 hours and P values for turtles acutely exposed to constant indoor conditions. No significant differences in RER were observed between seasons (P=0.207). SEASON TOTAL (24 hours) Fall 1.02 ± 0.004 Winter 1.07 ± 0.03 Summer 1.07 ± 003 Table 3.18: Mean percentage of time inactive ± SEM in different seasons. Mean values of inactivity (%time) calculated over 24 hours and P values for turtles acutely exposed to constant indoor conditions. Significant differences in inactivity between seasons were observed (P=0.026). Turtles were significantly more active in the fall than in the summer and winter. SEASON TOTAL (24 hours) Fall 86.880 ± 3.23 Winter 95.87 ± 2.30 Summer 96.04 ± 0.73 Temperature coefficients: seasonal-independent temperature effects on oxygen consumption Temperature coefficients (Qi0) of day, night and total (24 hours) rates of oxygen consumption, within the fall and winter seasons were calculated. My objective was to determine temperature effects on the metabolism of turtles independent of 58 seasonal effects. To calculate the temperature coefficients I used the metabolic rates measured for each group of turtles (chronically acclimatized to seasonal cues and to constant conditions) at the seasonal (fall: 14.71 °C and winter 8.8 °C) and constant water temperatures within each season. Chronic acclimatization to seasonal cues Changes in temperature within the fall season appeared to evoke active suppression of the metabolic rate of Pseudemys scripta, particularly at night, as indicated by high Qi0 values (Table 3.19), while in winter changes in the rates of oxygen consumption could be explained by changes in temperature alone. Table 3.19: Mean Q 1 0 values ± S E M for day, night and total oxygen consumption of turtles chronically acclimatized to seasonal cues (outdoor turtles) at different temperatures (constant and environmental) during fall and winter. S E A S O N F A L L W I N T E R Condition Constant cond.-Seasonal cues (22.4-14.7 °C) Constant cond.-Seasonal cues (19.6-8 .8 °C) Day 2.85±o.4i i .63±o.4i Night 3.93±0.37 2.7i±0.45 Total ( 2 4 hours) 3-47±0.4 2.n±0.44 Chronic acclimatization to constant conditions (no seasonal cues) Turtles underwent a strong metabolic suppression when the water temperature was reduced to environmental levels in the fall. Although Qi0 values obtained for changes in temperature within the winter season were not as high, there is still evidence of an active suppression of the metabolism of turtles in the daytime (Table 3.20). 59 Table 3.20: Mean Q 1 0 values ± S E M for day, night and total oxygen consumption of turtles chronically acclimatized to constant conditions (indoor turtles) at different temperatures (constant and environmental) during fall and winter. SEASON FALL WINTER Condition Constant-Seasonal cues (22.4-14.7 °C) Constant-Seasonal cues (19.6-8 .8 °C) Day 6.96±1.55 3.3i±0.43 Night 6.i4±o.73 2.57±o.63 Total ( 2 4 hours) 6.56±o.93 2.53±o.34 60 DISCUSSION CIRCADIAN RHYTHMS I found evidence of circadian rhythms with an endogenous component in the metabolism of Pseudemys scripta chronically exposed to seasonal cues. The strength of the circadian rhythm varied seasonally, with larger amplitudes in colder seasons. Despite the seasonal differences in these rhythms, metabolism was always higher during the photophase. Higher daytime rates of oxygen consumption are expected in diurnal species, such as red-eared sliders (Cagle, 1950; Boyer, 1965). The circadian rhythm in oxygen consumption prevailed under constant darkness, demonstrating the endogenous nature of the circadian clock. I found that interactions between temperature and photoperiod affected the phase and amplitude of the circadian rhythms observed in the metabolism of Pseudemys scripta, however and thus the amplitude of the circadian rhythm was greater, at the same temperature when photoperiod was provided. Even more importantly, chronic exposure to seasonal changes in temperature and day length were required for this rhythm to be expressed, since turtles chronically acclimatized to constant temperature and photoperiod did not show circadian rhythms in their metabolism. While my results are consistent with results from other reptiles indicating that metabolism has a significant circadian component (Glass et al, 1979; Mautz, 1979; Rismiller and Heldmaier, 1991) such patterns are not well described in turtles. The incidence of circadian rhythms in metabolism and other physiological variables has often been overlooked, and might account for discrepancies among studies in the literature. The values of total oxygen consumption obtained in the present study (average over 24 hours) are somewhat lower than values previously obtained for Pseudemys scripta elegans (Jackson, 1971; Jackson et al, 1974) and Chrysemys picta bellii (Glass er al, 1985) (Figure 3.13, panel A). My daytime values of metabolism (Figure 3.13, panel A and B), for turtles acclimatized to 61 seasonal cues (Figure 3.13, panel A) best approximate values reported in other studies. Given the significant circadian component in the metabolism of these animals, my data indicate that comparing metabolic rates among studies requires that we account for the period of the daily cycle in which measurements were taken. Diurnal species like Pseudemys scripta often have higher resting metabolic rates during the photophase. In fact diurnal species in general have higher daytime levels of a number of physiological variables, such as body temperature, locomotion and ventilation; whereas the opposite is true for nocturnal species (Bennett and Dawson, 1976; Rismiller and Heldmaier, 1982; Rismiller and Heldmaier, 1985; Rismiller and Heldmaier, 1991; Menaker et al., 1997; Mortola and Seifert, 2002; Lutterschmidt et al, 2003). Underground dwelling mammals (fossorial) and lizards, on the other hand, show uniformity of day and night resting metabolic rates (Mautz, 1979; Kenagy and Vleck, 1982;). These findings together suggest that endogenous rhythms in behavioural and physiological variables prompt the animal for activity patterns that are independent of environmental photo and thermocycles. Red-eared sliders carry out activities such as basking and feeding during the day (Cagle, 1950; Boyer, 1965). Higher daytime ambient temperatures will facilitate behavioural thermoregulation, ensuring that physiological processes, for instance digestion, take place more efficiently (Bartholomew, 1982). The tight correlation between metabolism, temperature and activity poses difficulties for the study of circadian rhythms in the metabolism of reptiles. In fact, many studies to date lack appropriate controls for these interactions (Bennett and Dawson, 1976; Mautz, 1979). In my study, the circadian rhythm in the metabolism of Pseudemys scripta was not caused by activity. Furthermore, daily changes in ambient temperature were not responsible for 62 1.4 i 1-2 4 1.0 i 0.8 i 0.6 H 0.4 j • © A 6 • Outdoor turtles A Indoor turtles • Jackson, 1971 T Jackson et al., 1974 • C. picta bellii o T • Outdoor turtles-day Outdoor turtles-night Indoor turtles-day Indoor turtles-night Jackson, 1971 Jackson et al., 1974 C. picta bellii T f I • ft 10 15 20 25 o Temperature ( C) 35 5 ~t— 10 15 20 25 30 35 Temperature ( C) Figure 3.13: Comparison of metabolism values of Pseudemys scripta with other studies. Panel A compares the rates of oxygen consumption of red-eared sliders (chronically acclimatized to seasonal cues and to constant conditions) averaged over 24 hours with metabolic rates reported for Pseudemys scripta elegans (Jackson, 1971; Jackson et al., 1974) and Chrysemis picta bellii (Glass et al., 1985). Panel B compares the values of oxygen consumption reported in the previously mentioned studies with both day and night metabolic rates of red-eared sliders chronically acclimatized to seasonal cues obtained in the present study. Panel C compares the values of oxygen consumption reported in the previously mentioned studies, with both, day and nighttime metabolic rates of turtles chronically acclimatized to constant conditions found in this study. 63 differences in daytime and nighttime rates of oxygen consumption, since the water temperature was maintained at a constant environmental level throughout each treatment (3-4 days). Endogenous rhythms Three properties characterize circadian rhythms: 1) they are entrained by light-dark and temperature cycles; 2) they are temperature compensated, so that changes in ambient temperature cause small variations in the period of the rhythm; and 3) they oscillate under constant conditions, such as oL:24D or 24L:oD photoperiods (an endogenous or "free-run" rhythm) (Menaker and Wisner, 1983). The importance of the two latter properties resides in maintaining a time-keeping mechanism that is not immediately disturbed by acute changes in temperature or photoperiod. A number of studies have shown endogenous circadian rhythms in both behavioural and physiological variables, such as melatonin synthesis (Menaker and Wisner, 1983; Firth et al, 1999); body temperature, locomotion (Tosini and Menaker, 1998); and oxygen consumption (Rismiller and Heldmaier, 1991). In my study, both turtles chronically acclimatized to seasonal cues (outdoor turtles under natural conditions) and indoor turtles exposed to an constant indoor photoperiod (i2L:i2D) and temperature throughout the year showed endogenous circadian rhythms in metabolism, in other words the rhythm persisted after 2 days of exposure to constant darkness. The amplitude of the rhythm, however, decreased in both groups in constant dark and shifted to slightly earlier in the day compared to the corresponding season when temperature and photoperiod cues were present. "Free-running" rhythms are known to have periods that slightly deviate from 24 hours (Underwood, 1992; Tosini et al, 2001; Mortola and Seifert, 2002), and the amplitude of circadian rhythms have been shown to be greater under light-dark cycles than under constant darkness (Kenagy and Vleck, 1982). In fact, usually prolonged removal of zeitgebers will cause further reduction in the amplitude of the rhythm, until the rhythm is lost altogether. 64 Role of external environmental cues in entraining circadian rhythms Although the advantage of an internal time-keeping mechanism is evident, circadian rhythms must be synchronized with the 24 hour day to be functional and confer a selective advantage to the organism. Consequently, these rhythms are entrained by periodic environmental cues or zeitgebers, such as temperature and photoperiod. This ensures that events take place at the appropriate time of the day (Aschoff et al, 1982a; Underwood, 1992). The circadian rhythm in the metabolic rate of Pseudemys scripta is evidently entrained by photoperiod, since increases and declines in their metabolism occurred in anticipation of sunrise Qights on) and sunset Qights off) and under constant daytime and nighttime environmental temperatures (but see below). Similar results have been reported for metabolism and body temperature selection in Lacerta viridis (Rismiller and Heldmaier, 1991), as well as melatonin synthesis in Anolis (Menaker and Wisner, 1983). These studies together provide evidence for the important role that photoperiod plays in the circadian system of reptiles. Daily changes in ambient temperature are also known to be an important zeitgeber for reptiles. Pseudemys scripta maintained at a constant temperature throughout the year (turtles chronically acclimatized to constant conditions) did not show daily rhythms in their metabolism, despite the daily changes in photoperiod. Furthermore, Pseudemys scripta acutely exposed to constant conditions lost the circadian rhythms in oxygen consumption, presumably due to the lack of a thermocycle or, as discussed below, exposure to high constant water temperatures. The importance of thermocycles as an entraining cue of circadian rhythms has also been shown in the daily rhythms of melatonin. Firth and Kennaway (1980) demonstrated higher amplitudes in melatonin synthesis of Trachydosaurus rugosus under a 24 hour light-dark and temperature cycle than after exposure to a 24 hour light-dark cycle and constant temperature. It appears, however, that there is no need for large amplitude in temperature cycles, since differences as low as 2 °C between daytime and nighttime temperatures were sufficient to entrain daily 65 pineal melatonin rhythm in Anolis carolinensis (Underwood, 1992). Interestingly, however, Hochscheid et al. (2004) did not observe circadian rhythms in the rates of oxygen consumption in Caretta caretta under constant photoperiod (i3L:iiD) but with seasonal changes in water temperatures throughout the year. My results suggest that circadian rhythms in Pseudemys scripta are greatest when they receive both daily cycles in photoperiod and temperature cues. Rismiller and Heldmaier (1991) also found that photoperiod and temperature act synergistically in influencing the metabolic rate of Lacerta viridis. Moreover, in the present study acute exposure of Pseudemys scripta to seasonal cues did not elicit circadian rhythms in oxygen consumption. This suggests that long term exposure to seasonally changing environmental cues is necessary to entrain the rhythms. Clearly, more than three daily cycles need to elapse in order for the rhythm to be entrained. This may ensure that the rhythm is entrained by the environmental cue and is not a masking effect (Bertolucci and Foa, 1998). Seasonal variation in circadian rhythms Circadian rhythms in the metabolism of Pseudemys scripta varied seasonally. Larger changes between day and night were observed in cooler seasons, suggesting that either cold water temperature, short photoperiods or an interaction of both cues increased the circadian rhythms in winter and fall (Figure 3.3). Day-night oscillations of oxygen consumption in many species of reptiles are known to be large. This characteristic of the reptilian circadian rhythm is believed to assist in energy conservation during the inactive phase of the day (Bennett and Dawson, 1976). In this respect it is not surprising that Pseudemys scripta in this study had very low night metabolic rates in the winter, since this season is known to be highly challenging for animals inhabiting high latitudes. Survival during the winter is enhanced by reducing the overall metabolism compared to other seasons. It is not clear why daytime metabolic rates did not fall as much but there is no doubt that energy conservation will be maximized if metabolism is further reduced 66 during the night. Thus, higher Qi0 values were obtained at night in turtles chronically exposed to seasonal cues compared to daytime temperature coefficients (Table 3.11). Preparation for the winter season begins during the fall. Photoperiod is thought to act as a "noise free" signal that conveys information about the upcoming season, since acute changes in ambient temperature can occur at any time (Rismiller and Heldmaier, 1991). Therefore, increasingly shorter photoperiods in the fall could have triggered the large reduction in night metabolic rates seen in Pseudemys scripta, which would explain the large day-night amplitude in fall rates of oxygen consumption. Further reductions in metabolism will result from low water temperatures, particularly in the winter. Photoperiod is also known to act on body temperature selection (Rismiller and Heldmaier, 1982,1987) and an interaction of both cues is responsible for seasonal adjustments in the metabolism of Lacerta viridis (Rismiller and Heldmaier, 1991). While photoperiod seems to be important in daily and seasonal adjustments of the metabolism of reptiles, water temperature also plays an important role in allowing photoperiod effects to be expressed on the circadian rhythm of Pseudemys scripta. High temperatures in the summer (seasonal cues present) and at constant indoor conditions appear to have suppressed the circadian rhythms in both groups of red-eared sliders. In conclusion my results suggest that an interaction between photoperiod and temperature determine the expression of circadian rhythms in the metabolism of turtles. Circannual Rhythms I found evidence of seasonal rhythms under natural photoperiod and temperature in turtles chronically exposed to environmental conditions. The circannual rhythm, however, was largely the result of the changes in temperature between seasons, as turtles acutely exposed to seasonal cues showed the same annual cycles 67 as those chronically acclimatized to environmental conditions (Figure 3.8). Despite the large effect of temperature on the seasonal rhythms of Pseudemys scripta, however, my results also demonstrate the existence of an endogenous seasonal cycle, since turtles acclimatized to indoor and outdoor conditions showed metabolic suppression during the winter even at high constant temperatures (Figure 3.11). Seasonal rhythms in the metabolism of Pseudemys scripta were similar to those found in other temperate reptiles and mammals, with higher oxygen consumption values in the summer and decreases throughout the fall and winter (Hochscheid et al, 2004; Kortner and Geiser, 2000; Rismiller and Heldmaier, 1991; Armitage and Shulenberger, 1972; Mayhew, 1965). High levels of oxygen consumption in the summer and reduced winter metabolism are characteristic of most temperate species. Different seasonal metabolic adjustments are adopted by species which occupy habitats that pose other challenges to them. For instance, the warm climate lizard Sceloporus cyanogenys, has higher metabolic rates when acclimated to short photoperiods (simulating fall or winter) than long photoperiods (summer), independent of acclimation temperature, presumably to conserve water and energy during the hot season (Mayhew, 1965; Songdahl and Hutchison, 1972; Hailey and Loveridge, 1997). In any case, the life cycle of Pseudemys scripta is highly seasonal. Timing events such as reproduction with favorable times of the year and undergoing dormancy at times when activity can not be sustained due to extreme weather confers an advantage to any high latitude species. Seasonal changes in metabolism and role of environmental cues High summer and fall metabolic rates of turtles chronically acclimatized to seasonal cues correspond to the reproductive cycle of Pseudemys scripta. Many turtles, red-eared sliders included, mate in both spring and fall (Cagle, 1950; Moore and Lindzey, 1992). Furthermore, male temperate chelonians undergo spermatogenesis in spring and spermiogenesis and spermiation peak in the 68 summer or early fall, after which a refractory period occurs from late fall to early spring. Temperate female turtles, on the other hand, show a period of ovarian quiescence during the summer when nesting occurs (June and July) (Cagle, 1950; Ernst and Barbour, 1989). Vitellogenesis starts in the late summer or fall and progresses until spring, with a break during hibernation (Kuchling, 1999; Duvall et al., 1982). High metabolic rates during mating and oogenesis have also been reported for a number of reptiles (Rismiller and Heldmaier, 1991). The reproductive pattern described above may explain the inherent higher fall metabolic rates observed in turtles chronically and acutely exposed to constant conditions (Figure 3.11). Differences in the metabolic rate of P. scripta chronically acclimatized to seasonal cues between summer and fall are the result of changes in water temperature between seasons (20.8 °C in the summer and 14.71 °C in the fall), as indicated by the calculated temperature coefficients (Table 3.11). During winter, however, turtles appear to actively suppress their metabolism. Inverse acclimation or metabolic suppression has been widely reported in a number of temperate and subtropical reptiles: Phrynosoma m'calli, Ptyodactylus hasselquistii, Caretta caretta and should aid in energy conservation during winter inactivity (Mayhew, 1965; Bennett and Dawson, 1976; Gregory, 1982; Ultsch, 1989; Zari, 1999; Hochscheid et al, 2004). The reduction in metabolism commences well in advance of the winter season. P. scripta in my study, for instance, underwent fall anorexia in late autumn. It is believed that the progressively shorter photoperiod in the fall triggers behaviours such as fasting (Cagle, 1950; Gregory, 1982) and selection of colder body temperatures (Rismiller and Heldmaier, 1982) that will gradually initiate a decline in metabolism (Bennett and Dawson, 1976). However, photoperiod on its own does not seem to be enough to cause a significant reduction in V0l, since the metabolism of Pseudemys scripta (chronically acclimatized to seasonal cues) under constant darkness did not differ 69 significantly from the corresponding fall (natural photoperiod) season (Figure 3.8). Therefore, interaction between both photoperiod and temperature might be necessary for winter inactivity. In fact, Mayhew (1965) proposed that metabolic suppression was the result of low temperatures, while dormancy was cued by changes in photoperiod. In support of this, Rismiller and Heldmaier (1982) demonstrated that exposure of Lacerta viridis to short photoperiod and a constant temperature of 23 °C induced brumation. Further evidence for the role of photoperiod as an indication of changes in season comes from P. scripta acutely exposed to seasonal cues. The larger summer to fall metabolic suppression compared to that occurring between fall and winter suggests that changes in photoperiod with changing seasons could have induced such abrupt results. Turtles chronically exposed to seasonal cues underwent more subtle changes in their metabolism. In fact, Mayhew (1965) and Kortner and Geiser (2000) proposed that the decline in day length from late summer to mid-autumn acts as an important stimulus for winter dormancy. Temperature on the other hand seems to be a stronger zeitgeber during winter (Kortner and Geiser, 2000). Both photoperiod and temperature cues are important in entraining circannual rhythms, but unlike circadian rhythms it seems that each environmental cue plays more central roles at different times of the year. Endogenous circannual rhythm My data demonstrate the existence of endogenous circannual rhythms in the metabolism of P. scripta. Chronic and acute exposure to constant conditions revealed both an inherent high fall metabolic rate, as well as winter metabolic suppression (Figure 3.8 and 3.11). As mentioned above the high rates of oxygen consumption during the fall might correspond to the reproductive cycle. Very few studies have shown endogenous control of the reproductive cycle in reptiles. Kuchling (1999) showed intrinsic spermatogenesis in male Testudo hermanni under hibernation conditions. A complete circannual cycle however, has not yet 70 been demonstrated in any chelonian. On the other hand, evidence for endogenous control of metabolic suppression and dormancy is stronger. For instance, photoperiod and temperature cues only prompted changes in physiological variables in Phrynosoma m'calli if they occurred at the appropriate season (Mayhew, 1965; Bennett and Dawson, 1976; Underwood, 1992). Furthermore, no differences in oxygen consumption were found when Chelonia mydas was exposed to simulated winter and summer conditions; it is likely that an endogenous seasonal rhythm overrode their results, since exposure to the simulated conditions did not correspond with the season (Southwood et al, 2003). An endogenous circannual rhythm in the metabolism of Pseudemys scripta will be advantageous if the environmental cues can not be sensed during winter dormancy. Red-eared sliders often overwinter buried in the mud in the bottom of ponds, where seasonal changes may be less perceptible therefore a mechanism that measures time and allows the animal to prepare for arousal in the absence of environmental cues would be beneficial. Circannual and circadian interaction Results from my study strongly suggest an interaction between circadian and seasonal rhythms in the metabolism of Pseudemys scripta. Four of my findings give evidence for this interaction. First, the circadian rhythms in the metabolism of Pseudemys scripta varied seasonally. Second, daily and seasonal changes in photoperiod and temperature were necessary to entrain the circadian rhythms. Third, even though Pseudemys scripta chronically exposed to constant conditions had circadian cues, but not seasonal cues, the circadian rhythm in metabolism was suppressed. Presumably, if we remove circadian cues from Pseudemys scripta chronically acclimatized to environmental conditions, the seasonal effects would disappear. Fourth, although both circadian and circannual rhythms proved to be endogenous, the interaction of photoperiod and temperature influenced and entrained both rhythms. The interaction between day-night and annual cycles is not surprising, since both appear to be controlled by the same mechanisms. 71 C H A P T E R 4: C i r c a d i a n a n d C i r c a n n u a l R h y t h m s i n V e n t i l a t i o n o f R e d - e a r e d S l i d e r s (Pseudemys scripta) INTRODUCTION Respiration in reptiles is arrhythmic with different species displaying one of two characteristic breathing patterns, single breaths interspaced by relatively short non-ventilatory periods or a variable number of breaths clustered together in an episode or ventilatory period (VP) followed by a breath hold or apnea of variable length (Milsom and Jones, 1980; Glass and Wood, 1983; Milsom, 1988; Milsom, 1990). The later breathing pattern is typical of aquatic reptiles such as Pseudemys scripta. In aquatic species the non-ventilatory period often corresponds to a dive (Wood and Lenfant, 1976; Burggren and Shelton, 1979; Shelton et al., 1986). Although one of the main roles of the cardiorespiratory system is to supply oxygen for metabolism, the respiratory system plays a crucial role in a number of other physiological and behavioural functions, such as pH balance, thermoregulation, buoyancy control, and in some species, defense and mating behaviours (Jackson, 1971; Wood and Lenfant, 1976; Glass and Wood, 1983; Glass et al., 1985; Shelton et al, 1986). Thus, although total ventilation must be tightly correlated to metabolism, reptiles may vary the breathing pattern, so that the respiratory system can subserve these other functions as well (Glass and Wood, 1983). The metabolism of reptiles is highly affected by daily and seasonal changes in the environment, particularly temperature (Bennett and Dawson, 1976). Additionally, there is strong evidence for endogenous circadian and seasonal rhythms in both metabolic rate and body temperature selection in reptiles (Mayhew, 1965; Mautz, 1979; Rismiller and Heldmaier, 1982, 1987, 1991; Underwood, 1992, Chapter 3: Circadian and circannual rhythms in the metabolism of red-eared sliders). Since 72 ventilation is very sensitive to both of these variables it is expected to oscillate with the same daily and seasonal rhythms and several studies have demonstrated the existence of circadian (Glass et al, 1979; Hicks and Riedesel, 1983; Stephenson, et al, 2000, Seifert et al, 2000; Seifert and Mortola, 2002a, 2002b; Mortola and Seifert, 2002; Mortola 2004) and circannual rhythms in ventilation (McArthur and Milsom, 1991; Andrade and Abe, 1999). It is worth mentioning that most of these studies have focused on the mammalian circadian rhythm of breathing. Only two studies to date have studied the circadian cycle of breathing in reptiles. Although this is not surprising given that breathing in reptiles is constrained by functions other than gas exchange, this makes studies of such rhythms in reptiles all the more interesting. Since I found evidence for circadian and circannual rhythms in the metabolism of Pseudemys scripta and, as previously mentioned, ventilation is tightly correlated to metabolism, I wished to investigate whether total ventilation varied with the same circadian and seasonal rhythms as metabolism in this species. I also wanted to determine the effects of environmental cues on the biological rhythms of ventilation and whether these rhythms prevailed under constant conditions. A third objective was to determine if the circadian and circannual rhythms in ventilation were accompanied by daily and seasonal changes in the breathing pattern, independent of changes in total ventilation. 73 RESULTS4 Circadian rhythms in total ventilation Mean values of total ventilation for individual animals normalized to body weight were combined (n=8 for winter and fall, n=6 for summer and no cues experiments). Chronic acclimatization to seasonal cues Pseudemys scripta maintained outdoors under natural conditions showed a daily rhythm in their ventilation when examined over 24 hours indoors at a "constant" (steady day and night) seasonally specific temperature, and under natural photoperiod. Daytime total ventilation (VE) was significantly higher than nighttime f^in the fall and winter seasons (P <o.05) (Figure 4.1). Despite the difference in photoperiod between fall and winter, ventilation began increasing at approximately seven in the morning and decreases occurred at six in the afternoon for both seasons. In summer ventilation increased when the lights were turned on and decreased at around 10 at night. Peak ventilation occurred at 10,11 and 12 in the morning for winter, fall and summer respectively (17.41, 30.29 and 23.97 ml/min/kg, winter, fall and summer respectively) (Figure 4.2). Even though a peak in ventilation was evident in the summer, day and night rates of ventilation did not vary significantly (P>o.os) (Figure 4.1). Interestingly, daytime values of VE were higher during fall compared to summer, regardless of the 6 °C water temperature increase during summer (Figure 4.3). Nighttime VE, however changed with seasonal temperature variation. 4 For methods see Chapter 2. Mean daytime and nighttime measurements for all the physiological variables referred to in this chapter are shown in Tables CT-C4. Trends in the physiological variables over a 24 hour period are shown in Figures C1-C3. P values obtained with statistical tests (paired t-test and One way repeated measures ANOVA, for circadian and circannual rhythms respectively) performed on the physiological variables referred to in this chapter are shown in Tables C5-C12 in Appendix C. 74 40 30 ] cn J 20 E IB > 10 Circadian C h a n g e s in Total Ventilation (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues B Fall Winter Summer No cues (Fall) Fall Winter Summer Acute Exposure to Constant Conditions Acclimatization to Constant Conditions 40 i C D £ UJ > Fall Winter Summer Season Fall Winter Summer No cues _ (Summer) Season Figure 4.1: Mean resting values of total ventilation ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of ventilation for turtles chronically acclimatized to seasonal and indoor conditions. Day and night rates of ventilation are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05). 75 Winter —A— Fall —•— Summer - 0 - No cues Total Vent i lat ion (Air) Seasonal Cues Outdoor An ima l s No Seasonal Cues Seasonal Cues Indoor An ima ls No Seasonal Cues 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 Hour Hour Hour Hour Figure 4.2: Mean values of total ventilation ± S E M over a 24 hour period. Plots on the top show the circadian changes in ventilation for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in ventilation with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 76 25 20 4 £ 15 c I 1 UJ 10 5 ^ 8 10 12 14 16 18 20 22 24 26 Tempera ture °C Figure 4.3: Mean daytime and nighttime values of total ventilation of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Figure shows nighttime (filled bars) and daytime (open bars) ventilation measured at seasonal temperatures (winter: 9 ° C ; fall: 14.71 ° C and summer: 20.83 ° C ) and under constant darkness in the fall (13.6 °C) . The difference between day and night values of ventilation increased proportionately with colder water temperatures (Figure 4.4). A s a result of the high nighttime ventilation at warmer water temperatures during the summer season, the difference between day and night values of VE in summer was not significant (Figure 4.1). 77 140 120 J 100 © 80 H cn | 60 H o as 40 H 20 0 8 10 12 14 16 18 20 22 24 26 Temperature (°C) Figure 4.4: Difference between photophase and scotophase values of total ventilation of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Day and night ventilation difference, expressed as the percentage of total (average over 24 hours) ventilation at different seasonal temperatures and under constant darkness. Open bars indicate day-night difference in ventilation at seasonal temperatures (winter: 9 °C; fall: 14.71 °C; summer: 20.83 °C). Filled bar indicates constant darkness (13.59 °C). Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to outdoor conditions in the fall were also held in constant darkness at their acclimatization temperature (13.6 °C) for 2-3 days. This was done to determine i f circadian rhythms in ventilation were endogenous. Even though ventilation during the day was higher, the difference between day and night values was not significant (P>o.05) (Figure 4.1). However, the peak ventilation (17.61 ml /min/kg) occurred earlier in the day at eight in the morning. This phase shift of the circadian rhythm in VE under constant darkness masked the day-night difference in this variable as I averaged daytime and nighttime values based on the natural day (7:00 to 17:00) under natural photoperiod (Figures 4.1 and 4.2). Significant differences between day and night were found when the 4 hour shift was taken into account (P=0.007). The circadian 7X change when ho cues were present was lower compared to the corresponding fall season when daily cues were present (Figure 4.4). "Day" levels of ventilation measured under constant darkness differed substantially from fall daytime values, while "night" Rvalues under constant darkness were only slightly lower than fall nighttime values under natural photoperiod (Figure 4.3). Chronic acclimatization to constant conditions (no seasonal cues) Circadian effects on ventilation appeared to be lost with chronic acclimatization to constant indoor conditions during all seasons (P >o.05) (Figure 4.1). Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to constant indoor conditions showed significantly higher "daytime" rates of ventilation than "nighttime" values, when kept under constant darkness and at their acclimatization temperature (19.8 °C) for 2-3 days (Figure 4.1). It is rather interesting that under constant darkness circadian effects on ventilation appeared to be stronger than under a chronic constant light-dark photoperiod. This reflects the higher daytime ventilation rates under constant darkness than in the corresponding summer season under natural photoperiod. Nighttime values under constant darkness were only slightly higher than summer values. Acute exposure to seasonal cues Acute exposure to seasonal cues (indoor turtles at outdoor conditions) did not entrain circadian rhythms in ventilation in any of the seasons (P XJ.05) (Figure 4.1). This further confirms that long term exposure to varying environmental cues, such as temperature and photoperiod is required to entrain such biological cycles. 79 Acute exposure to constant conditions (no seasonal cues) P. scripta chronically acclimatized to outdoor conditions (seasonal cues) were exposed to constant indoor conditions (no seasonal cues). Unexpectedly, daytime ventilation was significantly higher than nighttime ventilation in the summer (P<0.05) while daily rhythms in ventilation during the fall and winter became non-significant (P > o . o s ) (Figure 4.1). Circadian rhythms in breathing frequency and tidal volume To determine whether changes in breathing frequency or tidal volume produced the daily changes in ventilation, I measured both variables under the five treatments described previously. Mean values of tidal volume for individual animals normalized to body weight and breathing frequency were combined (n=8 for winter and fall, n=6 for summer and no cues experiments) to establish trends in these variables over the course of one day. Chronic acclimatization to seasonal cues P. scripta maintained outdoors under natural conditions showed daily rhythms in breathing frequency when measured indoors at a "constant" seasonally specific temperature but under natural photoperiod. Breathing frequency during the day was significantly higher than at night in all seasons (P<0.05) (Figure 4.5). Larger differences between day and night values of breathing frequency occurred at colder temperatures (Figure 4.6). Reduction of breathing frequency at colder seasonal temperatures was more prominent during the night than in the day (Figure 4.7), resulting in the larger day-night difference observed in the winter. 80 Circadian C h a n g e s in Breathing Frequency (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues m 3 CD 8 2 CD 0) 0 UilJ .—I—. DL Fall Winter Summer No cues (Fall) Fall Winter Summer Acute Exposure to Constant Conditions Acclimatization to Constant Conditions *= 3 1 CD J 1 £ 2 i CD cr CD i— L L 1 J ^ * Fall Winter Summer Season Fall Winter Summer No cues „ (Summer) Season Figure 4.5: Mean resting breathing frequency ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of breathing frequency for turtles chronically acclimatized to seasonal and indoor conditions. Day and night breathing frequency are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05). XI >> u 140 -i c CD ZS CT 120 -CD LL 100 -CD _C 'sz 80 -ro 0 60 -CO c CD 40 -CO d CO 20 -u 0 -T r -i 1 r -i 1 1 4 6 8 10 12 14 16 18 20 22 24 26 Temperature (°C) Figure 4.6: Difference between photophase and scotophase values of breathing frequency of turtles chronically acclimatized to seasonal cues. Day and night breathing frequency difference, expressed as the percentage of total (average over 24 hours) breathing frequency at different seasonal temperatures and under constant darkness. Day and night differences in the frequency of breaths at seasonal temperatures is denoted by open bars (winter: 9 °C; fall: 14.71 °C; summer: 20.83 °C) and under constant darkness (13.59 °C) by the filled bar. Figure 4.7: Mean daytime and nighttime values of breathing frequency of turtles chronically acclimatized to seasonal cues (outdoor turtles exposed to outdoor conditions). Figures show the daytime difference (open bars) and nighttime (filled bars) values of breathing frequency measured at seasonal temperatures (winter: 9 °C; fall: 14.71 °C and summer: 20.83 °C) and under constant darkness in the fall (13.6 °C). 8 2 Pseudemys scripta chronically exposed to outdoor conditions (seasonal cues) did not show daily rhythms in tidal volume in any of the seasons (P>0.05) (Figure 4.8). Values of daytime tidal volume were somewhat higher in the winter and fall, while summer VT was slightly higher at night (Figure 4.9). The changes in breathing frequency and tidal volume taken together indicate that daily rhythms in ventilation are attained mainly by daily changes in breathing frequency, while changes in tidal volume play only a minor role in producing the circadian rhythm in V E. The lack of a significant daily rhythm in ventilation in the summer was the result of opposite daily changes in summer breathing frequency and tidal volume. Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to outdoor conditions did not show circadian rhythms in either breathing frequency or tidal volume when measured under constant darkness (P>0.05) (Figure 4.5 to 4.9). Chronic acclimatization to constant conditions (no seasonal cues) Circadian rhythms in breathing frequency in turtles chronically exposed to constant indoor conditions were observed in all seasons except winter (P<o.05) (Figure 4.5). Breathing frequency was higher during the day than at night. Daily changes in tidal volume were only significant in the summer (Figure 4.8), although there was a trend for nighttime tidal volumes to be higher than daytime values in all seasons. Opposite changes in breathing frequency and tidal volume during the photophase and scotophase could explain, to some extent, why daily changes in ventilation were not observed under constant indoor conditions. 83 C i r cad ian Change s in T ida l V o l ume (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues Fall Winter Summer No cues Winter Summer Acute Exposure to Constant Condition Acclimatization to Constant Conditions Fall Winter Season Summer Fall Winter Summer No cues „ (Summer) Season Figure 4.8: Mean resting tidal volume ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of tidal volume for turtles chronically acclimatized to seasonal and indoor conditions. Day and night tidal volumes are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05). 8 4 50 -1 CD E 40 -o > 30 -" r o T3 20 -H c CD 10 -cn c ro .c 0 -o vP 0 s -10 -1 1 1 1 1 1 1 1 1 1 1 4 6 8 10 12 14 16 18 20 22 24 26 Temperature (°C) Figure 4.9: Difference between photophase and scotophase values of tidal volume of turtles chronically acclimatized to seasonal cues. Day and night tidal volume difference, expressed as the percentage of total (average over 24 hours) volumes at different seasonal temperatures and under constant darkness. Day and night differences in the tidal volume at seasonal temperatures is denoted by open bars (winter: 9 °C; fall: 14.71 °C; summer: 20.83 °C) and under constant darkness (13.59 °C) by the filled bar. Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to constant indoor conditions only showed daily differences in breathing frequency (P<o.05) (Figure 4.5), but not in tidal volume (P>o.05) (Figure 4.8) when measured under constant darkness. Breathing frequency was higher during the day, while daytime and nighttime tidal volumes were remarkably similar. T h e resting level of breathing frequency was higher and the daily change in breathing frequency was larger under conditions of constant darkness than under natural photoperiod in the corresponding summer season (Figure 4.5). T h e large increase in daytime fR under constant darkness caused the larger day-night difference in VE seen in the red-eared sliders. It is worth mentioning that the daily 85 cycle in breathing frequency measured under constant darkness shifted to later in the day; peak breathing frequency occurred at four in the afternoon (4.10 breaths/min). Acute exposure to seasonal cues When indoor turtles were acutely exposed to seasonal cues (outdoor conditions) the day-night differences in breathing frequency were lost in the summer season (P>0.05) (Figure 4.5). Both day and night fR during fall and winter declined when indoor turtles were exposed to the colder seasonal temperatures, but the decrease in daytime values was more pronounced. It is noteworthy that acute exposure to seasonal conditions suppressed the rhythms in the summer, despite the similar water temperatures (20.8 °C and 19.6 °C, seasonal and constant indoor conditions respectively). Thus, longer seasonal photoperiods could have suppressed day-night differences in breathing frequency (i6L:8D and i2L:i2D, seasonal and constant indoor conditions respectively). Pseudemys scripta acutely exposed to outdoor seasonal conditions did not show significant daily differences in tidal volume (P>0.05) (Figure4.8), although VT tended to be higher at night in all seasons. The daily difference seen in summer under constant indoor conditions was lost due to increases in daytime values, while nighttime tidal volumes remained approximately the same (Figure 4.8). Acute exposure to constant conditions (no seasonal cues) When P. scripta chronically acclimatized to seasonal cues were exposed to constant indoor conditions, breathing frequency during the day remained significantly higher than at night in all seasons (P<0.05) (Figure 4.5). Day- night differences in breathing frequency during fall and winter however were not as large when turtles were acutely exposed to indoor conditions. The summer differences between outdoor seasonal and constant indoor conditions were similar, 86 I S 2 c r Winter —A— F a l l -m-Summer -0- No cues Breathing Frequency (Air) Seasonal Cues Outdoor Animals No Seasonal Cues Seasonal Cues Indoor Animals No Seasonal Cues Hour 0 S 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 Hour Hour Hour Figure 4.10: Mean breathing frequency ± SEM over a 24 hour period. Plots on the top show the circadian changes in breathing frequency for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in breathing frequency with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 87 14 12 10 g me 8 o > 6 P 4 2 0 14 12 cn 10 me 8 o > 6 i- 4 W inter —A— Fall • Summer No cues Tidal Volume (Air) Seasonal Cues Outdoor Animals No Seasonal Cues 10 15 Hour 10 15 20 Hour Seasonal Cues Indoor Animals No Seasonal Cues 10 15 20 Hour 10 16 20 Hour Figure 4.11: Mean tidal volume ± SEM over a 24 hour period. Plots on the top show the circadian changes in tidal volume for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in tidal volume with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 88 however, suggesting that temperature, not photoperiod, was more important in controlling the daily cycle infR. Although tidal volume was slightly higher at night, no significant differences were observed in any of the seasons (P>0.05) (Figure 4.8). Interestingly, acute exposure to constant indoor conditions reversed the trend seen in day and night tidal volumes during the fall and winter (Figures 4.8 and 4.11). Higher tidal volumes were now seen at night just as they were in the summer in both trials. The opposite changes in fR and VT during day and night seen in winter and fall resulted in there being no daily changes in ventilation. Circadian rhythms in the components of breathing frequency: breaths per episode, episodes per hour and apnea length Since breathing frequency was the major variable demonstrating circadian rhythms. I wanted to determine whether the number of breaths in each episode, the number of breathing episodes or the length of the apnea (or non-ventilatory periods) were responsible for the daily changes in breathing frequency. These variables were calculated from the breathing traces and mean values for individual animals were combined (n=8 for winter and fall, n=6 for summer and no cues experiments) to establish trends in these variables over the course of one day. Chronic acclimatization to seasonal cues P. scripta maintained outdoors under natural conditions showed daily rhythms in the number of episodes per hour when measured indoors at a "constant" (steady day and night) seasonally specific temperature and under natural photoperiod. The number of episodes per hour during the day was higher than at night in all seasons (P<o.os) (Figure 4.12). As a consequence, the non-ventilatory periods, or apneas were longer at night in all seasons (P<0.05) (Figures 4.14 and4.i5). Daily 89 changes in the number of breaths per episode were observed only in the winter (P<0.05) (Figure 4.13). Daytime values in breaths per episode during the fall where highly variable between individuals. Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to outdoor conditions did not show circadian cycles in the number of episodes/hour, breaths per episode, apnea length or proportion of time spent apneic (P>o.05) (Figures 4.12, 4.13, 4.14 and 4.15) when exposed to constant darkness. Although no significant differences between day and night were found, trends of daily cycles in the number of episodes/hour were evident. The cycle shifted to earlier in the day with a peak number of episodes at eight in the morning (19.67 episodes/hour). Breaths per episode and apnea length, on the other hand, did not show any daily cycles, remaining constant throughout the day. Chronic acclimatization to constant conditions (no seasonal cues) Pseudemys scripta chronically exposed to constant indoor conditions showed daily rhythms in the number of episodes per hour in fall and winter (P<0.05), with higher values during the day (Figure 4.12). The number of episodes during the summer remained relatively constant over 24 hours, with only a slight increase during the day (P>o.05). Interestingly the length of the average apnea and the % time spent apneic only increased at night during the summer (Figures 4.14 and 4.15). Breaths per episode did not vary daily in any of the seasons (Pxj.05) (Figure 4.13). Thus, changes in breathing frequency are likely to be caused by daily cycles in the number of episodes and not the number of breaths within each episode. In the summer, however, daily increases in breathing frequency seem to be caused by combined increases in both variables, with breaths per episode having a more profound effect on fR during this season. 90 Circadian C h a n g e s in E p i s o d e s per Hour (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues 30 25 20 | 15 o </> CL U 10 iiU l i Fall Winter Summer No cues (Fall) Winter Summer Acute Exposure to Constant Conditions Acclimatization to Constant Conditions Fall Winter Summer Season Fall Winter Summer No cues Season ( S u m m e r ) Figure 4.12: Mean number of episodes per hour ± S E M during different seasons. Figures show the day (open bars) and night (filled bars) number of episodes/hour for turtles chronically acclimatized to seasonal and indoor conditions. Day and night number of episodes/hour are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05). 9! Ci r cad ian C h a n g e s in Brea ths per E p i s o d e (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues B Hi Hi Fall Winter Summer No cues (Fall) Fall Winter Summer Acute Exposure to Constant Conditions Acclimatization to Constant Conditions 40 © 30 T3 O co CL *D £ 20 CD CD CQ 10 I ni ni ni Fall Winter Summer Season i • III Fall Winter Summer No cues _ (Summer) Season Figure 4.13: Mean number of breaths per episode ± S E M during different seasons. Figures show the day (open bars) and night (filled bars) number of breaths/episode for turtles chronically acclimatized to seasonal and indoor conditions. Day and night number of breaths/episode are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (PO.05). 92 Circadian C h a n g e s in Apnea Length (Air) Acclimatization to Seasonal Cues A Acute Exposure to Seasonal Cues Fall Winter Summer No cues (Fall) Fall Winter Summer Acute Exposure to Constant Conditions Acclimatization to Constant Conditions Winter Season Summer Fall Winter Season Figures show the day (open bars) Summer No cues (Summer) Figure 4.14: Mean apnea length ± SEM during different seasons and night (filled bars) apnea length for turtles chronically acclimatized to seasonal and indoor conditions^ Day and night apnea length is also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) or (E) (P=0.053). 93 Circadian C h a n g e s in the Proport ion of T ime in A p n e a (Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues CD 120 100 80 60 40 20 I 0 120 Fall Winter Summer No cues Fall Winter Summer (Fall) Acute Exposure to Constant Conditions Acclimatization to Constant Conditions CD E Winter Summer Season Winter Summer No cues _ (Summer) Season Figure 4.15: Proportion of time spent in apnea ± S E M during different seasons. Figures show the day (open bars) and night (filled bars) % time in apnea for turtles chronically acclimatized to seasonal and indoor conditions. Day and night % time in apnea is also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05) or (e) (P=0.052, 0.051 and 0.051, fall, winter and summer, respectively). 94 Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to constant indoor conditions showed daily differences only in the number of breaths per episode (P<0.05) (Figure 4.13), but not in the number of episodes per hour (Pxj.05) (Figure 4.12) and the % time spent apneic (Figure 4.15) when measured under constant darkness. The number of breaths per episode was higher during the day than at night. Furthermore, both the difference between day and night and the number of breaths in each episode were higher under conditions of constant darkness than in the corresponding summer season under natural photoperiod (Figure 4.13). Thus, the daily changes in breathing frequency of Pseudemys scripta exposed to constant darkness were attained by changes in the number of breaths within each episode and not the number of episodes per se. Interestingly, changes in breaths per episode play a similar role in the changes seen in fR during the summer season under natural photoperiod. It appears that at higher summer water temperatures turtles increase breathing frequency by varying the number of individual breaths rather than changing the number of episodes and this is accentuated under conditions of constant darkness. Acute exposure to seasonal cues Indoor turtles acutely exposed to outdoor seasonal cues displayed daily rhythms only in the number of episodes per hour, the length of apneas and the % time spent apneic during the fall (P<o.05), when they showed higher daytime values (Figures 4.12, 4.14 and 4.15). Trends in the winter daily differences were largely lost (P>0.05), mainly by a reduction in the number of episodes during the day. Interestingly, during this season turtles exhibited a higher number of episodes per hour at night. Although summer differences were not significant (P>o.os), the difference between day and night was larger when turtles were exposed to seasonal conditions compared to constant indoor conditions. 95 Turtles acutely exposed to seasonal cues did not show daily rhythms in breaths per episode in any of the seasons (P>0.05) (Figure 4.13). In fact, no trends were observed; the number of breaths in each episode remained relatively constant throughout the day. Changes in fall breathing frequency were the result of daily rhythms in the number of episodes, while the number of breaths in each episode did not play a major role in any of the seasons. Acute exposure to constant conditions (no seasonal cues) P. scripta maintained outdoors and exposed to constant indoor conditions showed higher number of episodes per hour during the day in all the seasons (P<o.05). The length of the. apneas, however, was only greater at night in the winter (P=0.c>53) (Figure 4.14). The number of breaths per episode, on the other hand, did not differ between day and night (P>o.05) (Figure 4.13). Interestingly, fall daytime values decreased greatly and winter nighttime values increased with exposure to constant indoor conditions. Since summer day and night breaths per episode did not vary appreciably from exposure to outdoor seasonal and constant indoor conditions (similar temperature, but different photoperiod) one could argue that temperature, not photoperiod, played a larger role in regulating this variable. Furthermore, this suggests that the change in fall and winter breaths per episode were the result of the higher water temperatures to which turtles were exposed during this treatment, and not due to photoperiod. Circannual rhythms in ventilation To determine whether there were circannual cycles in the respiratory variables of Pseudemys scripta, all physiological variables referred to in this chapter were averaged over the entire experimental run (24 hours) for each season. 96 Chronic acclimatization to seasonal cues Pseudemys scripta showed a circannual rhythm in total ventilation under seasonal temperature and natural photoperiod. Overall ventilation was higher in the summer and gradually decreased in the fall. From fall to winter however, ventilation changed rapidly (Figure 4.16). Thus, significant differences were only observed in the winter (P<o.05). It is rather interesting that despite the 6 °C difference between summer and fall, ventilation differed only slightly between these two seasons. As mentioned previously, the high fall VE was mainly due to high daytime, rather than nighttime values (Figure 4.3). Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to outdoor seasonal conditions showed lower levels of ventilation when measured under constant darkness than in the corresponding fall season under natural photoperiod but these differences were not significant (Figure 4.16). This suggests that although photoperiod may influence ventilation to some extent, the effect is not sufficient to have a significant effect. 97 Circannual Changes in Total Ventilation (Air) Acclimatization to Seasonal Cues 30 at 1 20 LU •> 10 T 2 0 . 8 C 14.7C be 5-8.8C 13.6C Summer Fall Winter No cues (Fall) Acute Exposure to Constant Conditions 19.6C Summer Fall Winter Acute Exposure to Constant Conditions 30 -\ •S 20 10 ab -p I M . O ^ ' | Corrected u Summer Fall Winter Season Acute Exposure to Seasonal Cues B a T 2 0 . 8 C a b jr 14.7C Summer Fall Winter Acclimatization to Constant Conditions D 22.4C r19.6C a 19.8C 1 9 . 6 C ^ H ~ Summer Fall Winter No cues (Summer) Acclimatization to Constant Conditions £. 19.6C | Corrected ab ^^ 9.8 0 T19 6CI T ^ . e c ^ B I I II Summer Fall Winter No cues (Summer) S e a s o n Figure 4.16: Seasonal changes in total ventilation ± SEM. Figures show the seasonal values for ventilation for turtles chronically exposed to seasonal and constant conditions. Seasonal values of ventilation are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 98 Chronic acclimatization to constant conditions (no seasonal cues) Turtles acclimatized to constant indoor conditions did not show the same circannual patterns in total ventilation as turtles chronically exposed to seasonal cues. Nevertheless, VE in the fall was higher than that in other seasons (Figure 4.16). To determine whether the fall values of total ventilation were higher than those of other seasons due to the higher temperature used during the fall trial (22.4 ° C ) compared to other seasons (* 19.6 ° C ) I used the following relationship to correct for the temperature difference between treatments: where VE2 was the fall ventilation (VE) measured at 22.4 ° C (t2), and VEi was the fall ventilation measured at 14.71 ° C (ti) taken from turtles chronically exposed to constant conditions under outdoor seasonal conditions. This yielded a temperature coefficient ( Q i 0 ) of 2.87 which was then used to correct VE1 to 19.6 ° C . This corrected fall total ventilation was significantly higher only compared to summer values (P =0.035). Ventilation in the winter versus summer did not differ significantly (P>o.os) (Figure 4.16). Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to constant indoor conditions did not show differences in the ventilatory rates measured under constant darkness (P>0.05) (Figure 4.16). eq. 4.1 99 Acute exposure to seasonal cues When turtles maintained under constant indoor conditions were acutely exposed to seasonal cues, a similar circannual cycle to the one observed in turtles acclimatized to outdoor seasonal conditions was apparent. Higher and lower levels of ventilation occurred in the summer and winter, respectively. However, ventilation declined gradually from summer to fall, while winter VE declined more abruptly. Differences were significant only between summer and winter (P<o.05) (Figure 4.16). Acute exposure to constant conditions (no seasonal cues)s Circannual rhythms in total ventilation occurred when outdoor turtles were acutely exposed to constant indoor conditions. Despite the almost identical water temperatures used in this treatment for all seasons, winter ventilation rates were extremely reduced (Figure 4.16). Significant differences however, were only found between summer and winter (P<o.05). These results, together with observations of higher fall ventilation values in turtles chronically acclimatized to constant indoor conditions, suggest the existence of an endogenous circannual rhythm, with inherent high fall and suppressed winter ventilatory rates in Pseudemys scripta (Figure 4.16). Circannual rhythms in breathing frequency and tidal volume Chronic acclimatization to seasonal cues Pseudemys scripta chronically acclimatized to seasonal cues showed circannual rhythms in breathing frequency. fR was higher in the summer and gradually decreased in the following colder seasons (P<0.05) (Figure 4.17). Despite similar 5 The temperature coefficient (Q10) used to correct for the temperature difference between treatments was 2.16. 100 water temperature changes («6 °C), from fall to summer and fall to winter, breathing frequency was further reduced during the winter season. Tidal volume, on the other hand, did not differ significantly between seasons (P>0.05) (Figure 4-1.8). Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically acclimatized to outdoor seasonal conditions had significantly lower breathing frequencies when measured under constant darkness than in the fall season under natural photoperiod CP<o.c>5) (Figure 4.17). Values of tidal volume of P. scripta exposed to constant darkness were not different from those measured in other seasons (P>0.05) (Figure 4.18). Chronic acclimatization to constant conditions (no seasonal cues)6 Turtles acclimatized to constant indoor conditions did not show the same circannual differences in breathing frequency or tidal volume (P>o.05) (Figures 4.17 and 4.18). Acute exposure to constant dark (daily cues absent) Pseudemys scripta chronically exposed to constant indoor conditions did not show differences in breathing frequency or tidal volume when measured under constant darkness (P>o.os) (Figures 4.17 and 4.18). Both respiratory variables were slightly higher in the absence of daily cues than in the corresponding summer season under natural photoperiod. 6 Temperature coefficients (Q 1 0) used to correct for the temperature difference between treatments was 2.3 and 1.16, for breathing frequency and tidal volume respectively. 101 Circannual Changes in Breathing Frequency (Air) Acclimatization to Seasonal Cues ro 3 CD i l 1 A 20.8C •14.7-C £.13.6C .8C Summer Fall Winter No cues (Fall) 5 Acute Exposure to Constant Conditions 22.4 C 19.6C S u m m e r Fall Winter 5 Acute Exposure to Constant Conditions £ 4 " | f l 9 6 C a b 1 9 6 C g 3 " ^ T O r r e c t e d £ 2 - T-19.6C l l Summer Fall Season Winter Acute Exposure to Seasonal Cues B a J 20 8C -b T 14.7-C j- 8.8 C Summer Fall Winter Acclimatization to Constant Conditions D 22.4 C -19.6C T 1 9 . 8 C 19.6 C l — — I Summer Fall Winter No cues (Summer) Acclimatization to Constant Conditions 19.8C Summer Fall Season Winter No cues (Summer) Figure 4.17: Seasonal changes in breathing frequency ± SEM. Figures show the seasonal values for breathing frequency for turtles chronically exposed to seasonal and constant conditions. Seasonal values of breathing frequency are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 102 Circannual Changes in Tidal Volume (Air) 10 Acclimatization to Seasonal Cues £ 6 e e I 4 ro •o F Acute Exposure to Seasonal Cues 13.6-C T 2 0 . 8 ' C _ a 4 7 C 1 ^ 1 ' Summer Fall Winter No cues (Fall) 1 0 Acute Exposure to Constant Conditions £ 6 4 H -19.6C Summer Fall Winter Acute Exposure to Constant Conditions 10 i b 19.6C T19.6-C _ ^ ° r r e c t e d a €. 6 > 4 Summer Fall Winter Season B I 20.8C Summer Fall Winter Acclimatization to Constant Conditions 19.8C Summer Fall Winter No cues (Summer) Acclimatization to Constant Conditions 19.8C Summer Fall Winter No cues (Summer) Season Figure 4.18: Seasonal changes in tidal volume ± SEM. Figures show the seasonal values for tidal volume for turtles chronically exposed to seasonal and constant conditions. Seasonal values of tidal volume are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8) See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 103 Acute exposure to seasonal cues Circannual changes in breathing frequency became apparent when indoor turtles were acutely exposed to outdoor seasonal conditions. Breathing frequency was higher in the summer and gradually decreased in colder seasons (P<0.05) (Figure 4.17). Although winter /flwas lower than fall fR, this decline was not significant, unlike the changes observed in outdoor turtles chronically exposed to seasonal cues, where changes from fall to winter were larger. If one assumes that the seasonal changes in the breathing frequency of indoor turtles were only the result of changes in temperature, then in outdoor turtles, perhaps both temperature and endogenous rhythms control this variable. Tidal volume of Pseudemys scripta, conversely, did not vary between seasons despite the difference in temperature and photoperiod (P>o.os) (Figure 4.18). Acute exposure to constant conditions (no seasonal cues) Fall breathing frequency and tidal volume were corrected for the difference in temperature between treatments as described earlier. The temperature coefficients (Qi0) used were 1.69 and 1.3283 for breathing frequency and tidal volume, respectively (Q10 obtained for fR and VT values measured at 14.71 °C and 22.4 °C in the fall). Despite the same water temperatures used in each season, fR during the summer was significantly higher than in the winter (P<o.os) (Figure 4.18). Tidal volume, on the other hand, was higher in the fall (P<o.os). Results from the four treatments (chronic and acute exposure of outdoor and indoor turtles to seasonal and constant indoor conditions) suggest that changes in breathing frequency play the major role in producing the circannual changes observed in the ventilation of red-eared sliders. Changes in tidal volume make a 104 lesser contribution. Furthermore, breathing frequency seems to be actively suppressed in winter, beyond the effects of temperature, as shown by the reduced winter breathing frequencies of outdoor turtles chronically acclimatized to seasonal cues and acutely exposed to constant indoor conditions. Circannual rhythms in the frequency components of breathing: breaths per episode, episodes per hour and apnea length Chronic acclimatization to seasonal cues Outdoor red-eared sliders chronically exposed to seasonal conditions breathed with a significantly higher number of episodes per hour during the summer season (P<o.05) (Figure 4.19). This was accompanied by a shorter apnea length (Figure 4.21). The number of breaths in each episode however, was significantly reduced in the winter (P<o.05) (Figure 4.20). Acute exposure to constant dark (daily cues absent) The number of episodes per hour and breaths per episode of outdoor red-eared sliders measured under constant darkness were not significantly different than values measured in the corresponding fall season under natural photoperiod (P>0.05) (Figure 4.19). Chronic acclimatization to constant conditions (no seasonal cues) Pseudemys scripta chronically exposed to constant indoor conditions did not show differences in the number of episodes per hour, the length of each apnea, or the number of breaths in each episode between seasons (P>0.05) (Figures 4.19-4.22), after correcting fall values for the difference in temperature between treatments (Qio's used were 2.03, 0.96 and 1.15 for episodes per hour, apnea length and breaths per episode respectively). These results are consistent with the lack of 105 Circannual Changes in Episodes per Hour (Air) 40 30 ® 20 H o 'o. 10 H Acclimatization to Seasonal Cues A a J20 .8C Acute Exposure to Seasonal Cues 13.6C B 20.8c Summer Fall Winter 40 i 30 «? 20 i 10 -\ Summer Fall Winter No cues (Fall) Acute Exposure to Constant Conditions Acclimatization to Constant Conditions 19.6'' 19.8C Summer Fal l Winter Summer Fall Winter No cues (Summer) 4 0 Acute Exposure to Constant Conditions Acclimatization to Constant Conditions 30 H © 20 10 19.6C 19.6C T Corrected 19.8-C Summer Fal l Season Winter Summer Fall Winter No cues (Summer) S e a s o n Figure 4.19: Seasonal changes in the mean number of episodes per hour ± SEM. Figures show the seasonal values for episodes/hour for turtles chronically exposed to seasonal and constant conditions. Seasonal values of episodes/hour are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 106 Circannual Changes in Breaths per Ep i sode (Air) Acclimatization to Seasonal Cues 13.6C Summer Fall Winter No cues Acute Exposure to Constant Conditions C 19.6C "22.4C b T 19.6C Summer Fall Winter Acute Exposure to Constant Conditions 19.6C 19.6C Corrected Summer Fall Season Winter Acute Exposure to Seasonal Cues B 20.8C j 14.7 C T 8 . 8 C Summer Fall Winter Acclimatization to Constant Conditions D 19.8C [19.6C S u m m e r Fall Winter No cues (Summer) Acclimatization to Constant Conditions 19.6C _ Corrected 119.6 JMM 6 C J 19.8C Summer Fall Winter No cues (Summer) Season Figure 4.20: Seasonal changes in the mean number of breaths per episode ± SEM. Figures show the seasonal values for breaths/episode for turtles chronically exposed to seasonal and constant conditions. Seasonal values of breaths/episode are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 107 Circannual Changes in Apnea Length (Air) Acclimatization to Seasonal Cues b 8.8C b 13.6C Summer Fall 6 0 Winter No cues (Fall) Acute Exposure to Constant Conditions :es) 58 -— c I 56 -leng' 54 -Apnea 52 -5 0 19.6C 60 n Summer Fall Winter Acute Exposure to Constant Conditions co 58 -B c § 56 -O) c iS 54 -03 CD c Q . < 52 -50 -19.6C Acute Exposure to Seasonal cues B a -J 20.8 C 14.7C Summer Fall Winter Acclimatization to Constant Conditions 19.8C Summer Fail Winter No cues (Summer) Acclimatization to Constant Conditions 19.8C Summer Summer Fall Winter No cues (Summer) Season Figure 4.21: Seasonal changes in apnea length ± SEM. Figures show the seasonal values for apnea length for turtles chronically exposed to seasonal and constant conditions. Seasonal values of apnea length are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 108 Circannual Changes in the Proportion of Time in Apnea (Air) 120 n 100 S 80 Acclimatization to Seasonal Cues a20 .8C D14.7C D8.8C D13 .6C Acute Exposure to Seasonal Cues B a 20.8C a b 14.7C | 8.8C 120 Summer Fall Winter No cues (Fall) Acute Exposure to Constant Conditions Summer Fall Winter 100 1 o 80 Acclimatization to Constant Conditions D 19.6C 19.8C CL < 60 -\ 40 H 20 -\ Summer Fall Winter Summer Fall Winter No cues (Summer) 120 Acute Exposure to Constant Conditions 100 -\ 80 H 60 40 4 20 a 19.6C 19.6C a b Corrected D 1 9 . 6 C Summer Acclimatization to Constant Conditions 19.6C 19.6 C Corrected 19.6C 19.8C Summer Fall Winter No cues (Summer) Season Figure 4.22: Seasonal changes in the percent time spent in apnea ± S E M . Figures show the seasonal values for the time spent in apnea for turtles chronically exposed to seasonal and constant conditions. Seasonal values of the percent time spent in apnea are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters. 109 seasonal differences in breathing frequency of indoor turtles exposed to constant indoor conditions. Acute exposure to constant dark (daily cues absent) The number of episodes per hour, length of apnea or breaths per episode of indoor red-eared sliders exposed to constant darkness did not differ significantly from values obtained in the same season in animals experiencing a constant photoperiod (P>0.05) (Figures 4.19,4.20 and 4.21). Acute exposure to seasonal cues Pseudemys scripta kept under constant indoor conditions showed remarkably similar seasonal changes in the number of episodes per hour as turtles chronically maintained outdoors, when both groups were exposed to outdoor seasonal cues. The number of episodes per hour was higher in the summer and decreased correspondingly with the colder seasons. The change from summer to fall was quite large, while a subtle decrease in the number of episodes occurred from fall to winter. Nevertheless, values differed significantly in all seasons (P<o.os) (Figure 4.19). Similar changes were seen in the length of apnea and the % time spent apneic (Figures 4.21 and 4.22). On the other hand, the number of breaths in each episode did not differ between seasons when turtles were acutely exposed to seasonal cues (P>o.05) (Figure 4.20). Acute exposure to constant conditions (no seasonal cues) The number of episodes, length of apneas and breaths within each episode in the fall season were corrected for the difference in temperature between treatments (temperature coefficients (Q10) used were 1.92, 0.984 and 0.41 for episodes per hour, apnea length and breaths per episode respectively). 110 Turtles acutely exposed to constant indoor conditions did not show seasonal differences in any of the variables (P>o.05) (Figures 4.19 and 4.22). In conclusion, seasonal changes in breathing frequency seem to be the result of seasonal differences in the number of episodes per hour rather than the number of breaths per episode. I observed significant differences in the number of breaths per episode only in turtles chronically exposed to outdoor seasonal conditions during the fall. On the other hand, seasonal changes in the number of episodes per hour were observed in both groups of turtles under outdoor seasonal conditions. Changes in the number of episodes seems to be caused by changes in temperature, since neither group of turtles showed seasonal differences under constant indoor conditions. However, when both groups of turtles where exposed to outdoor seasonal cues, the number of episodes per hour was elevated during the summer, whereas changes from fall to winter were more subtle. I l l D I S C U S S I O N Circadian rhythms in total ventilation I found a daily rhythm in total ventilation in Pseudemys scripta in response to seasonal changes in environmental cues. The magnitude of this rhythm varied with season with larger day-night differences occurring in colder seasons. Ventilation was higher during the photophase in all seasons. My results are consistent with those from other studies on reptiles which show circadian rhythms in total ventilation (e.g., Varanus exanthematicus, Terrapene ornata and Thamnophis elegans, Wood et ah, 1977; Glass et al, 1979; Hicks and Riedesel, 1983). Because these species are diurnal like Pseudemys scripta, higher ventilatory rates occurred during the day. Circadian rhythms in breathing have also been reported in mammals. Higher daytime ventilation has been reported in humans, while nocturnal rats showed higher values during the night (Seifert et al, 2000; Seifert and Mortola, 2002a, 2002b). These results indicate that circadian rhythms are common across diverse taxa and follow daily activity patterns. Ventilation is largely determined by metabolic rate, which is strongly affected by temperature and activity in reptiles (Bennett and Dawson, 1976). Metabolism, temperature and activity display circadian cycles in a number of reptiles (Rismiller and Heldmaier, 1982; Rismiller and Heldmaier, 1984; Rismiller and Heldmaier, 1991; Underwood, 1992; Mortola and Seifert, 2002). Thus, circadian oscillations in ventilation were expected. However, studies have found that circadian patterns in body temperature, metabolism and activity occur independent from each other (Mortola, 2004). Daily changes in ventilation of Pseudemys scripta were not prompted by activity since turtles generally remained inactive throughout the experiment and ventilation values during activity periods were removed from the analyses. Studies on resting garter snakes, humans under "routine conditions" and rats have also shown circadian patterns in breathing independent of activity 112 (Hicks and Riedesel, 1983; Stephenson et al, 2000; Mortola and Seifert, 2002). The breathing cycles in my study were also independent of daily temperature changes. Water temperature was constant throughout the treatment, therefore the day-night differences in ventilation of Pseudemys scripta I observed were internally driven. These findings also agree with results in other reptiles (Glass et al, 1979; Hicks and Riedesel, 1983). Although the present study showed evidence of circadian rhythms in ventilatory and metabolic rates independent from activity and temperature, it is difficult to determine whether the biological rhythms of these two variables are themselves independent. Stephenson et al (2000) showed that the circadian rhythm in the respiratory response to inhaled C0 2 occurs in the absence of circadian oscillations in the metabolism of humans under "constant" conditions. Except for this study, however, there is no evidence to differentiate between the rhythms in these two variables. Pseudemys scripta in the present study showed remarkably similar trends in daily changes of both ventilation and metabolism (Figure 4.2). The modulation of circadian rhythms in any physiological variable can be explained by the "principle of physiological hierarchies" (Menaker 1982). Since circadian rhythms in multiple physiological and biochemical processes occur simultaneously, an overall temporal organization of the various rhythms is required in order to be advantageous to the organism. It has been suggested that some primary variables may be directly organized by the circadian clock, while others are driven by these hierarchically superior rhythms (Mortola, 2004). The circadian rhythm of ventilation might be the result of the interaction of several other physiological circadian cycles, with metabolism being the main determinant (Mortola and Seifert, 2002; Mortola, 2004). 113 Role of environmental cues in entraining circadian rhythms If daily changes in ventilation are driven and modulated by rhythms in metabolism, the effects of periodic environmental cues on ventilation are likely to be similar to those on metabolism. As for the circadian rhythms of metabolism of Pseudemys scripta, there is no doubt that both temperature and photoperiod were important zeitgebers in entraining the circadian patterns of breathing. Increases and decreases in ventilatory rates occurred prior to sunrise flights on) and sunset Qights off)- This suggests that the rhythm was entrained by the changing seasonal photoperiod. As well, daily changes in ambient temperature were required for the rhythm to be expressed since indoor turtles chronically exposed to constant conditions did not show day-night changes in ventilation in any season. Furthermore, when outdoor turtles were acutely exposed to constant conditions the circadian rhythms in ventilation were largely lost in the fall and winter. As with metabolism, long-term exposure to changing environmental cues was necessary to express the rhythm, since turtles acutely exposed to seasonal cues did not show daily changes in ventilation. Thus, I found evidence for roles of both changes in temperature and photoperiod in regulating these cycles. However, two differences between daily changes in ventilation and metabolism gave evidence for inherent rhythms in respiration that were independent of the circadian pattern in metabolism (Stephenson et al, 2 0 0 0 ) . First, unlike metabolism, daytime ventilation during the fall was higher than in the summer season, despite a 6 °C cooler water temperature. Nighttime ventilation was also elevated, but not higher than summer nighttime values. This difference may be due to the effects of hormones associated with reproduction on breathing. Red-eared sliders normally mate during the spring and sometimes in the fall as well (Cagle, 1950; Moore and Lindzey, 1992) . Furthermore, spermiogenesis and vitellogenesis start in the late summer or fall (Kuchling, 1999; Duvall et al, 1982 ) . Steroid hormones are known to influence breathing in mammals (Mortola, 2 0 0 4 ) . Progesterone and estrogen increase hypoxic chemosensitivity in mammals by 114 acting at peripheral chemoreceptors and central sites (Hannhart et al, 1990; Tatsumi et al, 1997). Hormones active during reproduction in the fall may have produced the large increase in ventilation in Pseudemys scripta. The antigonadotropic effect of melatonin might have suppressed the effects of these hormones at night (Lutterschmidt er al, 2003). Second, turtles acutely exposed to constant indoor conditions showed circadian rhythms in ventilation during the summer, but did not show such cycles in metabolism. The same turtles did not show differences in day-night ventilation under chronic exposure to seasonal cues during the same season despite the almost identical water temperatures between treatments. This suggests that long photoperiods suppress the rhythms, rather than warmer temperatures, as proposed with metabolism. Rismiller and Heldmaier (1987) also demonstrated that exposure of Lacerta viridis to a short photoperiod induced greater day-night differences in body temperature selection. Endogenous rhythms Although the circadian rhythm in ventilation results from the interaction of other physiological changes (Seifert and Mortola, 2002a), in order to be a true circadian rhythm ventilation should oscillate under constant light or dark conditions. Endogenous rhythms in respiration have been shown in Thamnophis elegans under constant darkness and in humans under "routine conditions" (Hicks and Riedesel, 1983; Stephenson et al, 2000). Indoor turtles chronically exposed to constant indoor conditions showed robust rhythms under constant darkness, suggesting that daily oscillations of breathing in red-eared sliders are under inherent control. However, turtles chronically exposed to outdoor seasonal conditions did not show day-night differences in ventilation when acutely placed in constant darkness. The lack of significant day-night differences was the result of a shift in the rhythm to earlier in the day (Figures 4.2). To obtain mean "day" and "night" values under constant darkness I averaged values of ventilation that 115 corresponded to the daytime (7:00 to 17:00) and nighttime (17:00 to 7:00) hours in the fall season under natural photoperiod. This obscured the rhythm as the period of elevated ventilation was now shared half in each phase. Circadian rhythms in the breathing pattern One difficulty with studying circadian rhythms in the respiration of reptiles is that the respiratory system serves various functions including buoyancy control, thermoregulation, oxygen stores for diving, courtship and defense to name a few. Studies get even more complicated when working with episodic breathers, such as Pseudemys scripta. The breathing pattern is more variable and a few respiratory variables, such as apnea length, breathing episodes and breaths per episode are added to the picture. Furthermore, various components of the breathing pattern such as tidal volume and duration of the ventilatory and non-ventilatory periods may be under separate control (Milsom, 1988). In addition, the mechanism determining the length of the ventilatory period still remains unknown. Although a number of studies have recently focused on circadian rhythms in respiration, the majority have been carried out on mammals that breathe continuously (rats and humans) (Peever and Stephenson, 1997; Stephenson et al, 2000; Seifert et ah, 2000; Seifert and Mortola 2002a, 2002b). Thus, information on circadian rhythms in breathing in ectotherms, hibernators and diving mammals and birds are scarce. Interestingly, Pseudemys scripta in the present study, as well as Thamnophis elegans (Hicks and Riedesel, 1983) showed day and night differences in the breathing pattern. Breathing frequency and its components Ventilation can increase by modifying either breathing frequency, tidal volume or both. Furthermore, episodic breathers can vary the breathing frequency by means of changing the length of non-ventilatory and ventilatory periods, by varying the number of breathing episodes and/or the number of breaths in each episode. The elevated daytime ventilation of Pseudemys scripta was achieved primarily by 116 increases in breathing frequency during the day in all seasons. These results are consistent with findings in Terrapene ornata and Thamnophis elegans, which showed diurnal rhythms in ventilation due to the daytime increases in breathing frequency (Glass et al, 1979; Hicks and Riedesel, 1983). Daily changes in breathing frequency were the result mainly of changes in the number of episodes per hour rather than the number of breaths per episode. Fewer breathing episodes per hour during the night with slightly more breaths per episode led to longer apneas at night. These results agree with other studies in reptiles in which the duration of the non-ventilatory period was the primarily regulated variable (Milsom and Jones, 1980). Longer apneas at night occurred more often in outdoor than indoor turtles, even at times of acute exposure to outdoor or indoor conditions. This suggests that long-term exposure to natural cues is important in determining daily changes in the length of the NVP. Increased surfacing during the day in fall and summer has also been reported in Rheodytes leukops in the field. The authors attributed this behaviour to the increase in activity periods in these seasons (Gordos, et al, 2003), but the results of the present study suggest this is not necessarily the case. While activity will undoubtedly enhance this rhythm, changes in breathing pattern that promote longer dive durations can occur under resting conditions. Tidal volume Tidal volumes of red-eared sliders were the same during the day and night in all of the seasons. These results agree with other studies in reptiles (Hicks and Riedesel 1983; Glass et al, 1979) but contrast with mammals that show circadian rhythms in tidal volume and breathing frequency (Seifert and Mortola, 2002a, 2002b). Even though I found no significant differences between day and night tidal volumes, both groups of turtles showed slightly higher nighttime values under outdoor seasonal and constant indoor conditions. Larger tidal volumes at night also allow for longer apneas (Milsom and Chan, 1986) since the lung is the main 117 site for oxygen stores in reptiles (Wood and Lenfant, 1976; Butler and Jones, 1982; Burggren, 1988). Role of environmental cues in establishing the circadian rhythms in the components of the breathing pattern Daily changes in breathing frequency occurred under all sets of conditions. Circadian rhythms in tidal volume or in the number of breaths per episode were not expressed under any condition except for a significant day and night difference in the number of breaths per episode of outdoor turtles in the winter and tidal volume of indoor turtles in the summer. The level of ventilation preset by the circadian clock was achieved mainly by means of changes in breathing frequency. No other studies of which I am aware have explored the effects of zeitgebers on breathing frequency and tidal volume. Respiratory mechanics Changes in the breathing pattern of red-eared sliders imply daily changes in control mechanisms, as suggested by Hicks and Riedesel (1983). It has been suggested that the oxidative cost of breathing in reptiles is high relative to their low metabolism (Milsom and Vitalis, 1984a; Milsom, 1984b; Vitalis and Milsom, 1986; Milsom, 1989). The muscles recruited in respiration perform work to overcome elastic forces (opposing the inflation) and non-elastic forces (flow-resistance) associated with the expansion of the lungs and chest (Milsom, 1989). Thus, increasing lung volume is quite costly. Increases in breathing frequency on the other hand, will decrease the work to overcome elastic forces, while that used to overcome non-elastic forces (flow-resistance) will increase. Nevertheless, increasing breathing frequency is less costly than increasing tidal volume, but enhances the ventilatory dead space. Therefore, animals have an optimum combination of tidal volume and instantaneous breathing frequency for each level of ventilation, and increases in ventilation are achieved mainly by regulation of the non-ventilatory period (Milsom, 1988), as shown in Pseudemys scripta. 118 Interestingly, recent studies performed on humans have shown circadian rhythms in some of the mechanical properties of the respiratory system. Airway resistance increases at night and pulmonary resistance and compliance were higher in the morning than in the afternoon in children (Mortola and Seifert, 2002; Mortola, 2004). Studies on the circadian rhythms of mechanical properties of the respiratory system in reptiles are lacking and it will be quite interesting to conduct such studies in reptiles. Circannual rhythms in total ventilation I found evidence of seasonal changes in the ventilation of Pseudemys scripta exposed to seasonal conditions. Seasonal rhythms in ventilation followed those of metabolism to some extent and were highly determined by the environmental temperature. However, temperature seemed to have slightly different effects on both variables, primarily in the winter. Furthermore, red-eared sliders chronically exposed to seasonal cues showed an elevated fall ventilation, which was not significantly different from that observed in the summer, despite the difference in seasonal temperatures. High ventilation values during the fall were also evident under constant indoor conditions, in both groups of turtles. These results suggest the existence of an endogenous rhythm. Fall and summer seasons correspond with reproductive stages of the circannual cycle of Pseudemys scripta. These turtles mate in the spring and fall (Cagle, 1950). Therefore, increased metabolism is expected during these seasons which results in ventilation increases (Duvall et al, 1982; Rismiller and Heldmaier, 1991; Kuchling, 1999). As mentioned previously further increases in ventilation may be caused by the effects that hormones, such as progesterone and estrogen, have on ventilation (Hannhart et al., 1990; Tatsumi, et al., 1997). Ventilation of Pseudemys scripta chronically exposed to seasonal cues was extremely reduced during the winter. This is not at all surprising since this season 119 corresponds to the period of dormancy. Therefore, ventilation falls because of the large reduction in metabolism during this season. Furthermore, it appears that ventilation reduction in winter is under endogenous control, since it was still evident under constant indoor conditions. These results agree with those from studies carried out in reptiles and mammals, which demonstrated a robust decrease in ventilation independent of temperatures in the winter (McArthur and Milsom, 1990; Andrade and Abe, 1999). Circannual rhythms in the ventilation components Seasonal changes in the ventilation of red-eared sliders were achieved by changes in breathing frequency, rather than in tidal volume. In fact, significant differences in tidal volume were only observed between summer and winter in outdoor turtles acutely exposed to constant indoor conditions. Tidal volumes have been shown to remain relatively constant during hibernation in Terrapene ornata, Spermophilus lateralis and Tupinambis merianae (Glass et al, 1979; McArthur and Milsom, 1991; Andrade and Abe, 1999). Circannual rhythms in frequency components Seasonal changes in the breathing frequency of red-eared sliders were differentially caused by either changes in the number of episodes per hour or the number of breaths per episode. Outdoor turtles chronically exposed to seasonal conditions decreased frequency in the winter by decreasing the number of breaths in each breathing episode, while increased breathing frequency in the summer was the result of a larger number of episodes per hour. Acute exposure of indoor turtles to seasonal conditions caused a large increase in the number of episodes per hour in the summer and small, but significant changes in winter and fall. Taken together, these results suggest that at higher temperatures turtles tend to change breathing frequency by modifying the number of episodes per hour, while changes in the number of breaths per episode become more important at colder 120 temperatures. This is consistent with results reported by Funk and Milsom (1987) on turtles, where they found that change in temperature from 10 to 30 °C (big temperature change) caused an increase in the number of episodes, while a change in temperature from 20 to 30 °C (small temperature change) caused the number of breaths per episode to increase. Changes in the respiratory variables described above led to seasonal changes in the apnea length in both groups of red-eared sliders, but only under seasonal conditions. Apneas were longer in the winter, which coincides with studies on hibernators and diving birds and mammals. The breathing pattern of ground squirrels and Tupinambis merianae change upon entrance into hibernation from a continuous and uniform ventilatory pattern, respectively, to an episodic breathing pattern (McArthur and Milsom, 1991; Andrade and Abe, 1997). Furthermore, southern elephant seals and macaroni penguins showed longer dives in the winter season (Bennett et al., 2001; Green et al., 2005). Finally, Rheodytes leukops show lower surfacing frequencies in the winter (Gordos, et al., 2003). Conclusions Since ventilation provides oxygen to satisfy metabolic demands (Shelton et al, 1986), higher daytime ventilatory rates are expected in diurnal species such as Pseudemys scripta that exhibit higher metabolic rates during the day (Cagle, 1950; Boyer, 1965; Chapter 3: circadian and circannual rhythms in the metabolism of red-eared sliders). The tight correlation between ventilation and metabolism poses difficulties in determining whether cycles in respiration are independent of metabolism. Both displayed remarkably similar circadian patterns. For instance, like the circadian rhythm in oxygen consumption, the phase and amplitude of daily changes in ventilation were also influenced by the interaction of temperature and photoperiod. Long-term exposure to seasonal changes in both environmental cues were required for the rhythm to be expressed since indoor turtles that were 121 acclimatized to constant conditions did not show daily cycles in ventilation, and acute exposure to seasonal conditions was insufficient to establish the rhythms. Interestingly, daily changes in ventilation resulted from different day and night breathing patterns, leading to changes in diving profiles. Increases in daytime ventilation were the result of changes in breathing frequency, while tidal volume remained the same. Breathing frequency increased due to higher number of episodes per hour, while breaths per episode remained constant throughout the 24 hours, leading to longer apneas at night. Breathing pattern also varied seasonally. Although seasonal changes in ventilation were also caused by changes in breathing frequency, the importance of breaths per episode and episodes per hour differed between seasons. Decreases in breathing frequency during winter were mainly caused by changes in the number of breaths per episode, while large increases in episodes per hour resulted in the elevated summer breathing frequency. Like circadian rhythms, seasonal changes resulted in longer apneas during the winter. Longer apneas at night and in the winter may be advantageous at times when the metabolic rate is low. It has been proposed that the oxidative cost of breathing in reptiles is high relative to their low metabolism (Vitalis and Milsom, 1986b; Milsom, 1989). Thus at night and in the winter when metabolism is low, ventilation should decrease correspondingly. The cost of breathing is minimized by regulating the non-ventilatory period, while instantaneous breathing frequency and tidal volume remain constant. Therefore longer apneas at night and in the winter will maximize energy conservation. In addition fewer trips to the surface will further assist in energy conservation. 122 C H A P T E R 5: G e n e r a l D i s c u s s i o n 7 Circadian rhythms This study found evidence of circadian rhythms in the metabolism and ventilation of Pseudemys scripta under natural temperature and photoperiod. At night, both oxygen consumption and total ventilation decreased accordingly as indicated by the air convection requirement (ACR) (Figure 5.1). The ACR is the ratio of ventilation to oxygen consumption (VE/vo2)or the volume of air inspired in order to supply each ml of oxygen (Jackson et al, 1974). Changes in ACR are accompanied by opposite changes in oxygen extraction (EQi, ml 0 2 consumed/ml O 2 inspired). In general ACR and oxygen extraction remained relatively constant throughout the 24 hours in all the seasons, indicating that cycles in metabolism and ventilation are tightly coupled. Daily changes in the ACR and oxygen extraction of turtles chronically acclimatized to seasonal cues were only observed in the fall (P<0.05) as the result of the elevated daytime ventilation (Figure 4.3). Although the cause of daytime hyperventilation in the fall is unknown, it is likely that reproductive hormones that increase metabolism during the mating season (Rismiller and Heldmaier, 1991; Kuchling, 1999) further affect ventilation. Levels of progesterone and testosterone in female turtles increase during the mating season (Whittier and Tokarz, 1992). Additionally, it has been demonstrated that progesterone enhances chemoreceptor responses in mammals (Hannhart et al, 1990; Tatsumi et al, 1997). Hormones active in reproduction may have similar effects on the ventilation of reptiles during the mating season. At night, however, the effects of such hormones might be blunted due to the antigonadotropic effects of melatonin which is produced and secreted at night (Lutterschmidt er al, 2003). 7 Mean daytime and nighttime measurements for all the physiological variables referred to in this chapter are shown in Tables D1-DD4. P values obtained with statistical tests performed on the physiological variables referred to in this chapter are shown in Table D5-D12 in Appendix D. 123 250 200 o 150 UJ > or Q 100 < 50 Circadian C h a n g e s in Air Convect ion Requirement (ACR-Air) Acclimatization to Seasonal Cues Acute Exposure to Seasonal Cues I M A Fall Winter Summer No cues (Fall) Fall Winter Summer 250 Acute Exposure to Constant Conditions Acclimatization to Constant Conditions Fall Winter Summer Season * I minim Fall Winter Summer No cues (Summer) Season Figure 5.1: Mean air convection requirement (ACR) ± SEM during different seasons. Figures show the day (open bars) and night (filled bars) values of ACR for turtles chronically acclimatized to seasonal and indoor conditions. Day and night ACR are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05). 124 C i r c a d i a n C h a n g e s in Oxygen Ext rac t ion (% Eo 2 -A i r ) Accl imatization to Seasonal Cues Acute Exposure to Seasonal Cues Fall Winter Summer No cues (Fall) Fall Winter Summer Acute Exposure to Constant Condit ions Acclimatization to Constant Condit ions Fall Winter Summer Season Fall Winter Summer No cues _ (Summer) Season Figure 5.2: Mean oxygen extraction ± S E M during different seasons. Figures show the day (open bars) and night (filled bars) values of oxygen extraction for turtles chronically acclimatized to seasonal and indoor conditions. Day and night oxygen extraction are also shown for turtles acutely exposed to seasonal and indoor conditions (n = 6-8). See Table 2.1 for details. Significant differences between day and night values are indicated by (*) (P<0.05). 125 In addition to the circadian rhythms in metabolism and ventilation, daily changes in breathing pattern were also observed. Breathing frequency decreased at night due to increases in the non-ventilatory period. The number of episodes per hour decreased, while the number of breaths within each episode remained constant. Both the decrease in metabolic rate and number of episodes per hour allow for prolonged dive times at night. Daily cycles were endogenous as they occurred independent of activity and without day-night temperature changes, and persisted under constant darkness. However, long-term entrainment by seasonally changing temperature and photoperiod was required for the rhythms to be expressed. Circadian rhythms in these physiological variables may confer an advantage to red-eared sliders by conserving energy during night-time inactivity (Bennett and Dawson, 1976). Fewer surfacing periods at night reduce costs of locomotion, as well as the risk of predation since most predators of P. scripta are nocturnal (Cagle, 1950). Furthermore the endogenous nature of this rhythm produces matching increases in metabolism and ventilation in anticipation of daytime active periods. Circannual rhythms Seasonal rhythms also occurred in the metabolism and ventilation of Pseudemys scripta. These rhythms were largely due to seasonal changes in temperatures although increases in the fall metabolic rate and ventilation seemed to be due to changes in photoperiod as well. I also found endogenous increase in metabolism and ventilation in the fall and suppression in the winter when turtles were kept at constant indoor temperatures. 126 Circannual Changes in Air Convection Requirement (Air) Acute Exposure to Seasonal Cues 2 0 0 A O 1 5 0 A K. 1 0 0 \ o < 5 0 A Acclimatization to Seasonal Cues B 8 . 8 C , 2 0 . 8 C 1 3 . 6 C B 2 0 . 8 C S u m m e r F a l l W i n t e r 2 0 0 A •5 LU < 5 0 A S u m m e r F a l l W i n t e r N o c u e s ( F a l l ) Acute Exposure to Constant Conditions Acclimatization to Constant Conditions D c -T 1 9 . 6 C j 1 9 6 C - 2 2 . 4 C * 1 9 . 6 C a 1 9 . 6 C ^?z'*ciiSM S u m m e r F a l l W i n t e r 2 0 0 O 1 5 0 or 100 o < 5 0 A Acute Exposure to Constant Conditions T 1 9 ; 6 C 1 9 . 6 - C 1 9 . 6 C T 1 9 . 6 C C o r r e c t e d • S u m m e r F a l l W i n t e r No c u e s ( S u m m e r ) Acclimatization to Constant Conditions 1 9 . 6 C C o r r e c t e d a B19.6C a 1 9 . 6 C ^ b a 1 9 . 8 C S u m m e r F a l l W i n t e r S e a s o n S u m m e r F a l l W i n t e r N o c u e s ( S u m m e r ) S e a s o n Figure 5.3: Seasonal changes air convection requirement (ACR) ± S E M . Figures show the seasonal values for ACR for turtles chronically exposed to seasonal and constant conditions. Seasonal values of ACR are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (P<0.05) are indicated by different letters or (B) (P=0.055 and 0.053, for outdoor and indoor turtles respectively) 1 2 7 Circannual Changes in Oxygen Extraction (Air) 20 i 15 H x 10 H Acclimatization to Seasonal Cues 5^ T 2 0 a C T 14.7-C .8C 13.6C B Acute Exposure to Seasonal Cues a J 20.8 C Summer Fall Winter 20 n Summer Fall Winter No cues (Fall) Acute Exposure to Constant Conditions Acclimatization to Constant Conditions 19.8C Summer Fall Winter Summer Fall Winter No cues (Summer) 2 0 15 Acute Exposure to Constant Conditions Acclimatization to Constant Conditions 19.6C ~ T Corrected mm * 10- T 1 9- 6 C I M 9 . 6 C • nil -19.6C 19.6 C ^ . 1 9 - « t .6Cf TCorrected T 19.6-C H II Summer Fall Season Winter Summer Fall Winter No cues (Summer) Season Figure 5.4: Seasonal changes in oxygen extraction ± SEM. Figures show the seasonal values for oxygen extraction for turtles chronically exposed to seasonal and constant conditions. Seasonal values of oxygen extraction are also shown, for turtles acutely exposed to seasonal cues and constant conditions (n = 6-8). See Table 2.1 for details. Significant differences between seasons (PO.05) are indicated by different letters. 128 The metabolic suppression in winter was also reflected in a large Qi0. Interestingly, ACR increased during the winter, indicating that metabolism decreased more than ventilation (P=o.055 and 0.053, for outdoor and indoor turtles respectively) (Figure 5.3). Similar findings have been reported in other studies performed on freshwater turtles (Jackson et al, 1974; Kinney et al, 1977; Glass et al, 1985; Funk and Milsom, 1987). Further evidence for endogenous changes in ventilation and metabolism that are independent of the effects of temperature comes from ACR results. Temperature effects on the ACR of red-eared sliders chronically exposed to seasonal cues were not observed between fall and summer (Figure 5.3, panel A). Furthermore turtles chronically exposed to constant indoor conditions showed differences in winter ACR despite similar water temperatures between seasons (Figure 5.3, panel F). Although oxygen extraction was low during the winter in turtles chronically acclimatized to seasonal and constant conditions, significant differences were only observed in those acutely exposed to seasonal conditions (P<o.os) (Figure 5.4, panel B). These data demonstrates the occurrence of seasonal changes in the ACR that are independent of temperature. Circannual rhythms in ventilation were due to changes in breathing frequency. The role that the number of episodes per hour and breaths per episode played in changing breathing frequency differed between seasons. In the summer a large increase in the number of episodes per hour resulted in higher breathing frequency, while breathing frequency decreased during winter due to a reduction in the number of breaths per episode. Thus metabolic suppression and the large reduction in the number of breaths per episode during winter allowed for longer apneas. Longer apneas may enhance survival by reducing the cost of breathing and surfacing at times when metabolism is suppressed. In reptiles the oxygen cost of breathing is high relative to their metabolic rate. Studies on the mechanical work of breathing in reptiles have suggested that maintaining the length and depth 129 of individual breaths, while changing the non-ventilatory period may minimize the cost of breathing (Vitalis and Milsom, 1986a, 1986b; Milsom, 1989). This will be advantageous during the winter, when the metabolism is reduced. Furthermore, longer apneas will reduce the number of surfacing episodes, minimizing the cost of locomotion. One of the most interesting results from this study was the indication of an interaction between circadian and seasonal rhythms in metabolism and ventilation. Stronger circadian rhythms occurred in cooler seasons. Although daily oscillations in these physiological variables aid in energy conservation, it is likely that reduction in metabolism and ventilation at night is most important during cold seasons to maximize energy conservation. Additionally daily and seasonal changes in photoperiod and temperature were required to entrain circadian rhythms. As mentioned in chapter 3 the interaction of both biological rhythms is expected since they seem to be controlled by the same mechanisms. Possible mechanisms underlying circadian and circannual rhythms It has been proposed that the pineal organ in reptiles serves as a neuroendocrine transducer of the changes in environmental conditions, and mediates its effects by release of melatonin (Tosini et ah, 2001). External factors can modulate the rhythm of melatonin synthesis, and it is known that both photoperiod and temperature influence the phase and amplitude of synthesis respectively (Lutterschmidt et al, 2003). The amplitude, duration and phase of the melatonin cycle may convey environmental information such as day duration to the system, influencing behavioural as well as physiological processes. For instance, this hormone plays roles in reproduction (Underwood, 1985; Lutterschmidt et al, 2003), thyroid function, activity, thermoregulation and behavioural temperature selection of reptiles (Bennett and Dawson, 1976; Rismiller and Heldmaier, 1987; Underwood, 1992; Seebacher and Franklin, 2005). In fact, Rismiller and Heldmaier (1982,1987) demonstrated that both short photoperiods and melatonin 130 injections during long photoperiods induced patterns of body temperature selection similar to the ones seen under natural photoperiod in the fall in Lacerta viridis. Studies on Iguana iguana suggested that melatonin, which is synthesized at night, could suppress heat production, and therefore basal metabolic rate, or increase heat loss (Tosini and Menaker, 1998). The effects of seasonal changes in photoperiod and temperature on melatonin synthesis might be reflected in circannual rhythms as well. Melatonin exerts effects on a number of variables that directly or indirectly affect metabolism. As mentioned previously, melatonin decreases thyroid activity (Bennett and Dawson, 1976; Gregory 1982; Underwood, 1992; Lutterschmidt et al, 2003) and has adverse effects on some gonadotropins (Lutterschmidt et al, 2003). Lynn (1970) suggested that thyroxine plays the same role in metabolic rate maintenance and stimulation in reptiles as it does in mammals. Evidence for this is the increase in metabolism of lizards, such as Anolis carolinensis and Sceloporus cyanogenys, after thyroxine injections, although only at these reptiles' preferred temperature (Bennett and Dawson, 1976). Increased thyroxine levels corresponded with the beginning of vitellogenesis of Lepidochelys kempi females (Rostal et al, 1998) and increased thyroid hormone was observed during gonad development of Rana perezi (Gancedo et al, 1995). Therefore, interactions between thyroid and steroid hormones could affect the metabolism of Pseudemys scripta during summer and early fall. Reduced metabolism during winter is the result of a number of physiological and behavioural adjustments. Shorter photoperiods will presumably extend the duration of melatonin synthesis at night, which will induce selection of colder body temperatures (Rismiller and Heldmaier, 1982,1984,1987) and reduce thyroid activity, ultimately reducing oxygen consumption during hibernation (Gregory, 1982). In conclusion, hormonal outputs timed by the pineal gland could regulate seasonal cycles of reproduction, growth, metabolism and hibernation. 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Journal of Thermal Biology 24:137-142. 146 APPENDIX A Air Temperature 45 n -5 J Water Tempera tu re 30 n 2003 2004 Figure A1: Ambient air and water temperature. Temperature values were recorded every hour from October 2003 to November 2004. 147 Table A1: Meteorological variables measured daily throughout the experiment. Ardent Terrperature (°Q EarorremcFressure Ressure Rainfall Hiridrty Ctte Max. Ma (rrmrfc) (kPa) (nrrt Sunrise Sunset 1501-03 1Q2 55 750.57 100 128 88 7:33 1824 1601-03 14.1 88 7436 10Q6 85 93 7:34 1822 17-01-03 166 133 749.97 100,9 558 98 7:33 1820 1&Q1-03 166 135 751.03 10Q8 Q6 75 7:37 1818 1901-03 17.8 121 75346 101 6 92 7:39 1816 2QO1-03 122 9.4 74597 10Q6 254 94 7:41 1815 21-01-03 17.9 124 76024 1023 4.4 96 7:42 1813 22-01-03 17.2 1Q6 74818 101.1 4.2 90 7:44 1811 23Q1-03 131 59 76323 103.2 0 67 7:45 17:41 2401-03 166 135 769.31 103.4 Q6 87 7:47 1807 2501-03 124 53 7689 101 0 92 7:39 1816 2501-03 11.1 54 76582 103 0 100 650 17:46 27-01-03 131 58 76313 1026 0 97 652 17:02 2&O1-03 134 83 75296 101.1 1Q4 93 653 17:00 2901-03 11.2 22 75216 101.5 0 71 655 1658 3001-03 1Q2 29 760.44 1022 0 43 657 1655 31-01-03 69 -0.7 760.36 103.1 0 37 653 1655 1-NLv03 62 -0.7 760.63 1022 0 64 7:00 1653 2-NvOS 88 -1.5 757.47 101.7 0 80 7:01 1651 3TNCV-03 7.7 -22 759.27 102 0 81 7:03 1650 4Nv03 7.3 -36 761.85 1026 0 52 7:05 1648 &Nv03 59 AA 76588 1025 0 88 7:06 1647 6-NbvOS 58 -4.6 764.43 1028 0 94 7:03 1645 7-Nbv03 7.1 A.2 759.44 102 0 91 7:09 1643 &ND/C3 9.8 25 76227 101.9 Q2 63 7:11 1642 atsbv-CB 1Q3 -1.4 760.37 101.4 0 69 7:13 1641 10Nv03 9.4 35 75516 101.5 6 97 7:14 1639 H-Nb/03 1Q4 5 757.15 1026 0 78 7:16 1638 12-Ntv03 1Q7 29 767.32 1022 0 85 7:17 1633 13ND^03 9.3 -1.4 75876 101.4 0 79 7:19 1635 14NV03 127 21 75505 101.5 83 7:21 1634 15Rv03 11.2 69 751.37 10Q8 88 95 7:22 1632 148 Terrperature(°Q Barorretric Pressure Pressxe Rainfall KfTidty Die iVtoc M a (rrmhg) (rrm) Sunrise Sunset 1&N3/-03 9.6 7.2 7386 90 14.2 88 7:24 1631 17-ND*03 1Q4 4.5 75246 101 19 87 7:25 1630 1 8 ^ 0 3 1Q3 33 75296 101.1 37 83 7:27 1629 19N><B 7.8 28 750.66 10Q9 7.2 84 7:28 1628 54 -0.8 75S05 101.7 0 84 7:30 1627 21-Ntx/-03 4.4 -1.6 761.03 1026 0 57 7:31 1623 22-NaMB 2 1 -1.1 764.43 1029 Trace 73 7:33 1625 23rHx-CQ 67 1.3 760.34 1023 4.2 93 7:34 1624 24N>/-03 67 1.7 764.98 101 4.4 89 7:36 1623 25Abv-03 8 1.1 74588 10Q1 32 82 7:37 1622 2o^b/-03 9.2 -1.2 760.66 101.5 0 91 7:39 1621 27-M>03 82 24 760.54 1022 ? 84 7:40 1620 2&Na/-03 9 55 749785 100.5 626 93 7:42 1620 2&MV-03 83 32 759.1 1021 0 81 7:43 1619 3CH\b/03 69 -1.6 75994 1022 0 77 7:44 1618 1 - D 3 0 0 3 56 -1.5 75363 101.8 3 72 7:46 1618 2-D3O03 11.6 4.1 754.93 101 1Q8 8 3 7:47 1617 3Da>03 82 26 764.33 1028 0 80 7:48 1617 4Dao03 11.3 -0.7 74867 10Q4 4.2 38 7:49 1616 5Da>03 98 68 74531 10Q1 66 85 7:51 1616 6Dao03 747.6 10Q5 79 7:52 1616 TDaXB & O E D G3 69 4 761.56 1023 1 90 7:54 1615 9Oa>03 4.5 -1.7 757.97 101.9 0 88 7:55 1638 1CrD3Cr03 6? 32? 759.63 101.2 56 7:56 1615 11-Dao03 12-Dao03 89 26 751.866 101 7.6 88 7:58 1615 13D&XB 7.7 4.1 744.19 100 34 91 7:59 1615 1 4 D 3 O 0 3 7.7 22 749.695 100.6 24 87 8 0 0 1615 15DE&03 7.7 Q1 759.27 1023 Trace 79 801 1615 16Oa>03 753065 101.1 83 802 1615 17-DaXB 760.14 106 72 ao2 1615 13vJsr>04 9.6 62 769.24 1022 54 93 a w 1639 14Jar>04 11.9 86 757.05 101.7 166 95 a o s 1640 15Ja>04 11.2 38 755153 101.4 22 95 a o s 1641 149 Terrp2rature(°Q EaronriricFnssure ftessure Rainfall hUricfty Dste Wax. Mn. (rrmhg) (nrrrt Sunrise Sunset 16\Jar>04 a3 33 759.14 102 0 95 802 1643 17Jar>04 9.6 35 759.34 101.9 26 88 801 1644 18Jar>04 ai 67 •75594 101.8 17.2 95 800 1646 19Jan04 9.5 35 759.24 1022 4.8 93 800 1647 2DJar>04 7.4 3 759.44 1028 0 92 7:59 1649 21^ Jar>04 7.8 4.4 7707 1031 0 83 7:58 1650 22-Jar>04 7.6 5 76233 1022 56 83 7:57 1652 230ar>04 7.3 54 75S15 101.6 1.8 83 7:55 1653 24Jar>04 64 1.6 747.58 10Q4 52 73 7:55 1655 250sr>O4 58 1.5 757.25 101.8 0 63 7:54 1653 2SJ=i>04 7.3 2 757.45 101.1 7 95 7:53 1658 27-Jan04 54 1.9 759.05 101.4 34 95 7:51 17:00 2&Jsr>04 9.8 24 75565 101 64 88 7:50 17:01 29Jar>04 87 55 750.07 10Q9 21.2 93 7:49 17:03 30Jar>04 94 22 75555 10Q8 1 71 7:48 17:04 31 a^r>04 52 0.2 75874 101.3 4.4 91 7:46 17:06 1-Feb04 69 1.8 75535 101.6 Trace 80 7:45 17:08 2feb04 749.97 10Q9 55 7:44 17:09 3Feb04 66 28 751.77 52 4fer>04 7.9 1.1 7627 101.8 56 83 7:41 17:13 5Feb04 54 -0.8 764.53 1028 26 81 7:39 17:14 &Feb04 1Q1 53 754.93 101.5 11.4 87 7:38 17:16 7-Feb04 8 4.4 765936 1029 Q2 75 7:36 17:18 &Feb04 77381 1035 83 7:35 17:19 &fer>04 66 -01 769.41 0 10Feb04 74 26 771.6 103.3 0 83 7:31 17:23 11-Feb04 89 Q3 76375 1027 0 82 ^ 7:30 17:24 12-febC4 1Q1 -0.7 75954 1022 0 92 7:28 17:26 13Feb04 1Q2 -1.6 75551 101.6 0 84 7:26 17:28 14-Feb04 9.1 59 757.25 101.7 1Q4 95 7:25 17:29 15Fefc>04 7.6 64 75585 101.7 7 97 7:23 17:31 16-Fer>04 9.8 35 75Q57 100.6 1Q2 66 7:21 17:33 17-Feb04 9.5 27 75216 101 58 74 7:19 17:34 1&Feb04 9.5 61 761.37 101.5 84 SO 7:17 17:33 19-Feb04 1Q9 4.9 75S86 1025 0 87 7:16 17:38 150 Terrperature(°q Barometric Pressure Pressure Rainfall Hurricfty Date Max. M n (rrmHg) (kPa) (rrm) Sunrise Sunset 2&feb04 a6 4. 76598 1023 0 85 7:14 17:39 21-Feb04 -0.4 764.38 101.2 0 95 7:12 17:41 22-Fer>04 1Q7 Q5 75635 10Q7 0 69 7:10 17:42 23Fet>04 124 66 75525 10Q8 Q2 60 7:03 17:44 24Feb04 751.84 10Q4 65 7:03 17:43 25Feb04 1Q1 29 Q8 2&F<±>04 9.9 32 754.95 100.1 Q8 88 7:02 17:49 27-FebO*" 9.1 4 75916 101.4 9.4 95 7:00 17:51 2&feb04 9.6 47 760.54 1023 Q2 90 658 17:52 2&Fefc>04 9.8 55 75944 101.9 1.8 68 653 17:54 1-Mar04 9.3 51 757.1 101.8 0 83 654 17:55 2-Mr-Ot 76593 1024 65 652 17:57 5Mr-04 9.8 4.9 74603 10Q3 68 88 646 1802 6JVbr-04 83 0.5 76315 1027 4.6 63 644 1803 7-Jvfer-W 9.4 5 764.52 103 14.4 95 642 1805 SMr-04 14.8 84 767.591 1026 1.6 85 640 1803 9Mjr-04 11.7 7.2 76597 103 1.6 75 638 1808 IOMr-04 9.8 25 78657 103.2 0 85 636 1810 11-M=r04 1Q6 25 75907 102 0 90 634 1811 12M=r04 761.96 1025 78 632 1813 14Mr-04 138 61 763058 Q2 15Mr-04 1Q3 4.4 764.45 1029 Trace 81 625 1817 16Mr-04 10 67 76325 1025 22 90 623 1819 17-M=r-04 757.97 102 75 621 1820 19M=r04 9.4 32 76318 103 0 80 615 1825 204vfer-O4 10 1.4 7659 1032 0 66 615 1825 21-M3T-04 136 32 760.69 1022 0 53 613 1827 22-Mar04 163 84 75639 101.6 Q2 71 611 1828 23^ Vbr-04 138 7.4 75391 101.4 24 75 606 1830 24-M3T-04 9.8 66 75222 10Q9 56 73 603 1831 25Mr-04 9.9 64 761.27 10Q2 128 82 604 1833 2&M=r-« 75625 100.9 77 602 1834 3OMr-04 14.9 7.2 75903 101.3 11.4 87 554 1849 31-M=r04 10.6 1.4 764.93 1023 . 0 71 551 1842 112 0.5 754.98 1029 0 58 549 1843 151 Terrperature(°q Barometric Pressure Pressure Rainfall Hurridty Date Max. Mn. (rrrnHg) (kPa) (rrm) Sunrise Sunset 2-Ax-W 128 1.2 760.89 1023 0 60 547 1845 3AX-04 7522 101.2 59 545 1846 126 8 0 5Apr-04 135 4.9 76257 101.8 0 89 641 1949 6/DT-04 159 5 759193 1021 0 90 639 1951 7-Apr-04 158 83 761.49 1024 1.2 87 637 1952 SVHr-04 4.5 4.2 761.089 1023 0 80 633 1955 9A7-04 155 5.9 757.1 1025 0 78 631 19.57 10AT-O4 169 51 7537 101.9 0 79 629 1959 H-Aa-04 101 60 627 2000 12/pr-04 157 10 Trace 13Acr-04 157 9.5 754.28 101.3 0.8 64 625 20.02 14Acr-04 751.62 10Q9 83 623 2003 17-Acr-04 14.9 7.4 0.2 1&Ap"-04 153 5.9 101.7 0 63 615 2Q09 1&AT-04 15 7.3 75655 101 4.2 43 613 2011 20AO--04 14.7 84 754.4 101 1.6 62 611 2012 21-A?r-04 152 67 757 101.8 0 64 609 20.14 22-AO--04 189 63 760.96 1024 0 38 607 2Q15 23Apr-04 14.9 9.2 1023 36 55 605 2Q17 24AT-04 1028 43 603 2018 25A3T-04 162 5.9 0 26AT-04 19.3 7 102.5 0 68 600 2021 27-Acr-04 15 9.7 101.5 Q2 70 528 2023 28Apr-04 17.8 36 1023 0 71 553 20.24 29Apr-04 185 62 101.9 0 57 554 2026 30Aa--04 102 55 552 2Q27 1-M 0^4 24.3 1Q7 Trace 2-M3/-04 191 124 1024 28 76 549 20.30 168 1Q9 101.9 1 57 547 2Q32 155 1Q1 101.1 Q8 61 546 2Q33 5My04 158 88 101.5 Q4 62 544 20.35 6JVter/04 166 6 101.5 0 63 542 2Q33 7-MyOI 17.7 83 101.7 1.4 84 541 2038 101.8 59 539 2Q39 152 Ternperature (°Q Pressure Rainfall Humidity Date Max. Mia (kPa) (mm) (°/<) Sunrise Sunset 10M3/-O4 145 10.6 1.4 H-Msy-04 18.6 8.5 101.5 1.4 89 5:35 20:43 12-Msr/-04 15.9 7.7 102 0 69 5:33 20:45 13^ vlay-04 18.9 8.6 1021 0 60 5:32 20:45 14-May-04 1021 57 5:31 20:48 1&Jvfey-04 19 8.8 0 16-May-04 17.6 9.7 101.7 0 67 5:28 20:50 17-May-04 20.2 10.4 101.6 Trace 74 5:27 2052 1cMvTay-04 20.7 11.3 101.3 0 71 5:25 20.53 19May-04 18.7 123 1021 0 65 5:24 20:54 2CMvlay-04 20.4 11.2 101.7 0 69 5:23 20.56 21-May-04 101.4 73 5:22 20.57 23May-04 20.5 9.3 0 24May-04 20.4 10 101.7 0 48 5:19 21:00 25JVtey-04 16.5 10.6 101.2 128 95 5:17 21:03 2&May-04 19.6 123 100.7 0 87 5:17 21:03 27-Ma/-04 15 11.2 100.9 14.2 92 5:16 21:04 2oMvby-04 19 9.7 100.9 0.2 70 5:15 21:05 2&tvb/-04 101.8 61 5:14 21:06 3OMay-04 18.4 11.1 0.6 31-May-04 17.5 10.2 1021 1.8 71 5:13 21:08 Wun-04 18.4 11 101.9 1 85 5:12 21:09 2-Jur>04 19.3 10 1021 0 75 5:11 21:10 3-Jur>04 227 9.8 101.5 0 56 5:11 21:11 4-Jur>04 25.5 121 101.6 Trace 45 5:10 21:12 5-Jur>04 101.3 76 5:10 21:13 6-Jur>04 19.6 11.7 Trace 7-Jur>04 17.4 11.2 101.4 1 73 5:09 21:15 8-Jun-04 223 10.8 100.9 0 68 5:08 21:15 9-Jur>04 19.2 13.6 101 0 73 5:08 21:16 10JunO4 15.5 124 101.6 22 78 5:08 21:17 1Wur>04 16.8 9.9 102 3.6 59 5:07 21:17 12-Jun04 16.6 8.8 101.6 5 84 5:07 21:18 13-Jur>04 1028 55 5:07 21:19 14-Jur>04 19 11.4 Trace 153 Ternperature (°Q Pressure Rainfall Humidity Date Max Min. (kPa) (mm) (°/<) Sunrise Sunset 15>Jun04 19.2 8 1029 0 65 5:07 21:20 1&Jur>04 22 10.3 10.5 0 71 5:07 21:20 17Jun04 19.8 11.2 102 0 61 5:07 21:21 18-Jur>04 101.7 45 5:07 21:21 20Jun04 28.1 13.1 0 2Wurv04 101.6 67 5:08 21:22 22-JUT>04 27.2 14.6 0 23-Jur>04 227 14.3 101.7 0 63 5:08 21:22 24-Jun04 21.8 10.9 102 0 73 5:08 21:22 25-JUTV04 1022 64 5:09 21:22 2fckJur>04 23.3 14.2 0 29-Jurv04 23.8 14.6 101.5 0 65 5:11 21:22 30-Jurv04 24.1 13 101.5 0 52 5:11 21:22 Ud-04 101.7 49 5:12 21:21 23-Jul-04 101.3 49 5:34 21:05 25Jul-04 23.2 17 102 0 65 5:36 21:02 2&Jul-04 23 13.9 102 0 65 5:37 21:01 27-Jul-04 23.1 183 101.7 0 66 5:39 21:00 2&Jul-04 229 15.8 101.5 0 63 5:40 2058 29-Jii-04 25.2 16 101.4 0 59 5:41 20.57 3O0ul-04 24.5 16.6 101.4 0 63 5:43 2056 31-Jul-04 24.5 16.6 101.2 0 60 5:44 2054 1^ Aug04 223 13.3 101.2 0 60 5:44 2054 2 A g 0 4 25.4 13.2 101.2 0 62 5:47 2051 3Aig04 221 15.4 101.7 3.6 91 5:48 20:50 4-AUO/04 223 14.7 101.5 0.2 70 5:49 20:48 5A#04 101.4 69 5:51 20:47 6Ajg-04 19 15.3 15.2 7-£UQM 1024 73 5:54 20.43 8-A1XJ04 24.2 13.8 0 27.9 14.7 101.8 0 58 5:56 2040 IOAJO/04 29.8 15.6 102 0 41 5:28- 2038 H-Aug/04 26.4 14.9 101.8 0 59 5:59 2036 12-Aag04 24.7 15.1 101.5 0 69 6:01 2035 13A^04 101.5 64 6:02 2033 154 Temperature (°Q Pressure Rainfall Humidity Date Max Ma (kPa) (mm) (°/c) Sunrise Sunset 15A^04 26.8 18.2 0 16AJC>04 26.6 15.9 101.7 0 59 6:06 2Q27 17Ag04 25 15.1 102 0.8 79 6:08 2026 18Aig-04 24.8 15.6 102 0 77 6:09 2024 19-Aug-04 24.9 15.4 101.7 0 72 6:11 2022 101.6 61 6:12 2020 21,Aug-04 19.1 16.4 19.8 22Ajg04 100.4 77 6:15 2016 24Ajg-04 18.3 15.4 132 25Ajg-04 18.8 14.8 100.2 19.2 87 6:19 20:10 26Ajg-04 21.2 15.1 101.6 0.2 90 6:21 2008 27-Aug-04 20.2 14.5 1023 1.8 85 6:22 20.03 28-Aug04 18.6 15.2 1021 Trace 89 6:23 2004 29-Aug-04 228 14.6 101.8 0 78 6:25 2002 30-AugO4 24.3 14.2 101.7 0 66 6:26 2Q00 31-Aug04 24.6 14.9 101.6 Trace 62 6:28 1958 1-Sep-04 19.1 13.2 101.6 3.2 66 6:29 1956 2-Sep04 101.8 74 6:31 19.54 6-Sep-04 18 10.2 0 7-Ser>04 224 123 101.7 0 44 6:38 1943 8-Ssp04 18.5 13.3 101.3 6.2 89 6:39 1941 9-Sep04 221 137 101.4 0 62 6:42 1937 10-Ser>04 100.8 85 6:42 19.37 12-Sep04 19.5 11.5 0 13-Sep04 15.4 11.4 100.9 6.8 84 6:46 1930 14-Ser>04 17.7 11.1 101.5 6 76 6:48 1928 15-Seo£4 15.3 10.2 100.8 7 95 6:49 1926 1&Ssp-04 101 83 6:51 1924 17-Ser>04 17.5 9.7 4.6 18-Sep04 100.4 83 6:54 1920 19-Ser>04 15.6 9.8 3.4 20-Ser>04 16 7.6 1021 0 89 6:57 915 21-Ser>04 17.1 9.7 1028 0.4 68 6:58 19.13 22-Ser>04 15.1 12 1022 5.2 74 6:59 1911 23-Ssr>04 16.5 11.7 1023 0.4 91 7:01 1909 1 5 5 Ternperature(°C) Pressure Rainfall Humidity Date Max Win. (kRa) (mm) 09 Sunrise Sunset 24-Sep-04 16.4 8.8 1021 0 84 7:02 19.07 25-Sep-04 101.8 SO 7:04 1904 2&Ser>04 17.1 8.9 0 27-Ser>04 17.3 9.7 1022 0 97 7:07 1900 28-SepW 17.2 11.6 101.5 0 81 . 7:08 18:58 29-Sep-04 101.6 80 7:10 18:56 cVCct-04 14.4 11.3 100.8 21 91 7:23 18:37 9-Oct-04 101.4 88 7:25 18:35 1C-Cct-04 15.6 11.7 0.2 11-0ct-04 16.3 123 1025 0.4 73 7:28 18:31 12-Oct-04 103 83 7:29 18:29 17-0*04 99.2 90 7:37 18:19 19-Oct-04 99.4 85 7:40 18:15 20-Cct-04 121 82 0.8 21-Cct-04 121 7.8 101 0.4 83 7:43 18:11 22-Oct-04 124 8.2 100.4 14.6 85 7:45 18:09 23-Cct-04 101.1 56 7:46 18:07 27-Oct-04 101.2 83 7:53 18:00 280*04 9 S3 24 29-Cct-04 102 87 7:56 17:57 4-NCV-C4 8.9 1.2 1028 0 74 7:06 16:47 5-NCV-04 1023 78 7:07 16:45 8-Na/-04 101.5 95 7:12 16:41 156 APPENDIX B: Metabolism Table B1: M e a n s ± S E M of physiological variables for turtles chronically accl imatized to s e a s o n a l c u e s for each s e a s o n . SEASONS VARIABLES D A Y NIGHT TOTAL (24 hours) Fa l l O2 consumption (ml 02/min-kg) 0.32 ±0.057 0.18 ±0.02 0.24 ±0.03 C02 production (ml C02/min-kg) 0.32 ±0.056 0.18 ±0.02 0.24 ±0.03 RER 1.02 ±0.008 1.01 ±0.008 1.02 ±0.007 % Time inactive 89.48 ±2.6 94.41 ±2.1 92.38 ±1.89 Winter O2 consumption (ml 02/min-kg) 0.13 ±0.02 0.06 ±0.008 0.085 ±0.011 Co 2 production (ml C02/min-kg) 0.13 ±0.023 0.06 ±0.008 0.08 ±0.01 RER 1.02 ±0.005 1.02 ±0.013 1.02 ±0.009 % Time inactive 94. 9 ±2.25 98.59 ±0.56 97.20 ±1.05 Summer O2 consumption (ml 02/min-kg) 0.38 ±0.06 0.301 ±0.03 0.35 ±0.049 C02 production (ml C02/min-kg) 0.39 ±0.061 0.30 ±0.03 0.36 ±0.049 RER 1.02 ±0.006 1.02 ±0.007 1.02 ±0.006 % Time inactive 94.07 ±0.99 96.54 ±1.32 94.89 ±0.49 No cues O2 consumption (ml 02/min-kg) 0.18 ±0.02 0.12 ±0.02 0.16 ±0.02 C02 production (ml C02/min-kg) 0.18 ±0.02 0.13 ±0.02 0.16 ±0.02 RER 1.04 ±0.015 1.06 ±0.026 1.05 ±0.01 % Time inactive 94.86 ±1.109 98. 7 ±1.11 96.14 ±1.04 157 Table B2: Means ± S E M of physiological variables for turtles chronically acclimatized to constant conditions (seasonal cues absent) for each season. SEASONS VARIABLES D A Y NIGHT TOTAL (24 hours) Fal l O2 consumption (ml 02/min-kg) 0.69 ±0.09 0.53 ±0.06 0.61±0.042 C02 production (ml C02/min-kg) 0.70 ±0.09 0.54 ±0.06 0.62 ±0.042 RER 1.01 ±0.002 1.02 ±0.006 1.02 ±0.003 % Time inactive 89.43 ±2.50 88.41 ±3.84 89.00 ±2.16 Winter 0 2 consumption (ml 02/min-kg) 0.20 ± 0.04 0.22 ±0.06 0.21 ±0.05 C02 production (ml C02/min-kg) 0.21 ±0.04 0.22 ±0.06 0.21 ±0.05 RER 1.03 ±0.009 1.04 ±0.008 1.03 ±0.008 % Time inactive 98.24 ±0.59 95.48 ±3.14 96.85 ±1.55 Summer O2 consumption (ml 02/min-kg) 0.24 ±0.02 0.28 ±0.053 0.26 ±0.02 C02 production (ml C02/min-kg) 0.25 ±0.02 0.28 ±0.05 0.26 ±0.02 RER 1.02 ±0.004 1.01 ±0.003 1.02 ±0.003 % Time inactive 97.41 ±0.41 90.36 ±5.88 93.89 ±2.99 No cues O2 consumption (ml 02/min-kg) 0.41 ±0.04 0.29 ±0.03 0.35 ±0.03 C02 production (ml C0 2/minkg) 0.42±0.04 0.30 ±0.03 0.36 ±0.03 RER 1.02 ±0.003 1.02 ±0.002 1.02 ±0.002 % Time inactive 96.58 ±1.54 96.62 ±2.64 96.6 ±1.94 158 Table B3: Means ± S E M of physiological variables for turtles acutely exposed to seasonal cues. SEASONS VARIABLES D A Y NIGHT TOTAL (24 hours) Fa l l O2 consumption (ml 02/min-kg) 0.17 ±0.02 0.15 ±0.03 0.16 ±0.02 C02 production (ml C0 2/min-kg) 0.18 ±0.02 0.15 ±0.03 0.16 ±0.02 RER 1.02 ±0.006 1.03 ±0.006 1.02 ±0.005 % Time inactive 88.31 ±3.07 92.41 ±3.59 90.74 ±3.18 Winter O2 consumption (ml 02/min-kg) 0.06 ±0.007 0.09 ±0.02 0.08 ±0.01 C02 production (ml C0 2 /minkg) 0.056 ±0.006 0.092 ±0.02 0.079 ±0.012 RER 1.02 ±0.01 1.03 ±0.014 1.02±0.009 % Time inactive 97.72 ±0.92 94.14 ±1.82 95.48 ±1.12 Summer O2 consumption (ml 02/min-kg) 0.42 ±0.06 0.35 ±0.03 0.39 ±0.041 Co 2 production (ml C0 2/min-kg) 0.42 ±0.06 0.36 ±0.03 0.40 ±0.04 RER 1.02 ±0.004 1.01 ±0.003 1.019 ±0.004 % Time inactive 92.65 ±2.02 95.65 ±1.88 93.65 ±1.69 159 Table B4: Means ± S E M of physiological variables for turtles acutely exposed to constant conditions (seasonal cues absent). SEASONS VARIABLES D A Y NIGHT TOTAL (24 hours) Fa l l O2 consumption (ml 02/min-kg) 0.73 ±0.19 0.50 ±0.06 0.63±0.13 C02 production (ml C02/min-kg) 0.74 ±0.19 0.51 ±0.06 0.63 ±0.13 RER 1.02 ±0.007 1.019 ±0.002 1.02 ±0.004 % Time inactive 82.96 ±5.79 90.96 ±2.29 86.88 ±3.23 Winter O2 consumption (ml 02/min-kg) 0.19 ±0.04 0.18 ±0.03 0.19 ±0.04 C02 production (ml C02/min-kg) 0.21 ±0.04 0.18 ±0.03 0.19 ±0.04 RER 1.07 ±0.028 1.07 ±0.03 1.07 ±0.03 % Time inactive 93.20 ±3.86 98.54 ±0.81 95.87 ±2.30 Summer 0 2 consumption (ml 02/min-kg) 0.45 ±0.08 0.34 ±0.06 0.4 ±0.06 C02 production (ml C02/min-kg) 0.46 ±0.08 0.35 ±0.06 0.41 ±0.06 RER 1.07 ±0.03 1.06 ±0.03 1.07 ±0.03 % Time inactive 95.41 ±1.53 96.5 ±1.32 96.04 ±0.73 160 Statistic Tables: Circadian rhythms Table B5: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles chronically exposed to seasonal cues. RER, refers to the respiratory exchange ratio. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES CHRONICALLY EXPOSED TO SEASONAL CUES (Paired t-test) Season Variable t P df n Fall 0 2 consumption 3.248 0.014 7 8 C O 2 production 3-250 0.014 7 8 RER 0.842 0.427 7 8 % Time inactive -1.831 0.110 7 8 Winter 0 2 consumption 3-197 0.015 7 8 C0 2 production 3.214 0.015 7 8 RER -0.492 0.638 7 8 % Time inactive -2.490 0.042 7 8 Summer 0 2 consumption 1.966 0.106 5 6 C0 2 production 2.021 0.099 5 6 RER 0.891 0.414 5 6 % Time inactive -1.166 0.296 5 6 No cues 0 2 consumption 2.504 0.054 •5 6 C0 2 production 2.491 0.055 5 6 RER -0.997 0.365 5 6 % Time inactive -4-653 0.006 5 6 161 Table B6: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles chronically exposed to constant conditions. RER, refers to the respiratory exchange ratio. The (*) indicates times when the non-parametric Wilcoxon CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES CHRONICALLY EXPOSED TO CONSTANT CONDITIONS (Paired t-test) Season Variable t P df n Fall 0 2 consumption 1.244 0.254 7 8 C0 2 production 1.228 0.259 7 8 RER -1-175 0.278 7 8 % Time inactive O.213 0.837 7 8 Winter 0 2 consumption -0.400 0.705 5 6 C0 2 production -0.379 0.720 5 6 RER -0.493 0.643 5 6 % Time inactive *W=-i.oo 1.00 5 6 Summer 0 2 consumption -0.598 0.576 5 6 C0 2 production -0.584 0.585 5 6 RER 2.327 0.067 5 6 % Time inactive 1.249 0.267 5 6 No cues 0 2 consumption 2.485 0.056 5 6 C0 2 production 2.532 0.052 5 6 RER 0.340 0.748 5 6 % Time inactive -0.848 0.435 5 6 162 Table B7: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles acutely exposed to seasonal cues. RER, refers to the respiratory exchange ratio. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES ACUTELY EXPOSED TO SEASONAL CUES (Paired t-test) Season Variable t P df n Fall 0 2 consumption 1.160 0.284 7 8 C0 2 production 1.127 0.297 7 8 RER -1.003 0.349 7 8 % Time inactive -1.721 0.129 7 8 Winter 0 2 consumption -1.678 0.154. 5 6 C0 2 production -1-735 0.143 5 6 RER -0.527 0.621 5 6 % Time inactive 1.625 0.165 5 6 Summer 0 2 consumption 0.800 0.460 5 6 C0 2 production 0.826 0.447 5 6 RER 1.783 0.135 5 6 % Time inactive -1-394 0.222 5 6 163 Table B8: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to the metabolism of turtles acutely exposed to constant conditions. RER refers to the respiratory exchange ratio. The (*) indicates times when the non-parametric Wilcoxon CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES ACUTELY EXPOSED TO CONSTANT CONDITIONS (Paired t-test) Season Variable t P df n Fall 0 2 consumption 1.207 0.267 7 8 C0 2 production 1.240 0.255 7 8 RER *W=-4.oo 0.844 7 8 % Time inactive *W=8.oo 0.641 7 8 Winter 0 2 consumption 0.964 0.379 5 6 C0 2 production 1.048 0.342 5 6 RER 0.330 0.754 5 6 % Time inactive -1.694 0.151 5 6 Summer 0 2 consumption 2.224 0.077 5 6 C0 2 production 2.416 0.060 5 6 RER *W=3.oo 0.844 5 6 % Time inactive -0.449 0.672 5 6 164 Statistic Tables: Circannual rhythms Table B9: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles chronically Circannual rhythms i n the physiological variables of turtles chronically exposed to seasonal cues (One-way repeated measures A N O V A ) Variable F P n 0 2 consumption 33404 <o.ooi 7 C0 2 production 34.887 <0.001 7 R E R 1.665 0.212 7 % Time inactive 3-462 0.040 7 Table B10: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles chronically exposed to constant conditions. RER refers to the respiratory exchange ratio. Circannual rhythms in the physiological variables of turtles chronically exposed to constant conditions (One-way repeated measures A N O V A ) Variable F P n 0 2 consumption 14.190 <0.001 8, fall; 6 winter & summer 0 2 consumption corrected. 6.712 0.004 8, fall; 6 winter & summer C0 2 production 14.530 <0.001 8, fall; 6 winter & summer R E R 4.162 0.025 8, fall; 6 winter & summer % Time inactive 4.429 0.020 8, fall; 6 winter & summer 165 Table B11: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles acutely exposed to seasonal cues. RER, refers to the respiratory exchange ratio. Circannual rhythms in the physiological variables of turtles acutely exposed to seasonal cues (One-way repeated measures A N O V A ) Variable n 0 2 consumption 48.663 <0.001 8, fall; 6 winter & summer C 0 2 production 37-356 <o.ooi 8, fall; 6 winter & summer R E R 0.222 O.805 8, fall; 6 winter & summer % Time inactive 1.948 0.193 8, fall; 6 winter & summer Table B12: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to the metabolism of turtles acutely exposed to constant conditions. RER, refers to the respiratory exchange ratio. Circannual rhythms in the physiological variables of turtles acutely exposed to constant conditions (One-way repeated measures A N O V A ) Variable F P n 0 2 consumption 12.465 0.002 8, fall; 6 winter & summer 0 2 consumption corrected 10.412 0.004 8, fall; 6 winter & summer C 0 2 production 13-005 0.002 8, fall; 6 winter & summer R E R 1.851 0.207 8, fall; 6 winter & summer % Time inactive 5.382 0.026 8, fall; 6 winter & summer 166 Table B13: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of differences in Q 1 0 values of turtles chronically exposed to seasonal cues. Differences between the temperature coefficients calculated for: fall-winter, summer-fall, summer-winter, no cues VARIABLE F P N Qio (day) 0.634 0.644 6 Q«, (night) 0-554 0.698 6 Qio total (24 hours) 1.267 0.316 6 Table B14: Statistic values for the One-way repeated measures ANOVA performed to determine the existence of differences in Q 1 0 values of indoor turtles acutely exposed to seasonal cues. Differences between the temperature coefficients calculated for: fall-winter, summer-fall, summer-winter, no cues (summer)-fall and no cues (summer)-winter were determined. See table B15 for pairwise multiple comparisons. VARIABLE F P N Qio (day) 0.487 0.745 6 Qio (night) 12.356 <O.OOl* 6 Qio total (24 hours) 2.815 0.053 6 Table B15: Pairwise multiple comparisons (Holm-Sidak) for night temperature coefficients* of indoor turtles acutely exposed to seasonal cues. VARIABLE t Unadjusted P NC (summer)-Fall vs. Fall-Winter 5-993 0 . 0 0 0 0 0 7 3 6 Summer-Fall vs. Fall-Winter 4 - 8 3 5 0 . 0 0 0 1 0 0 NC(summer)=Fall vs. NC(summer)-Winter 4-347 0 . 0 0 0 3 1 3 NC (summer)-Fall vs. Summer-Winter 4 - 293 0 . 0 0 0 3 5 5 Summer-Fall vs. NC(summer)-Winter 3-19 0 . 0 0 4 6 0 Summer-Fall vs. Summer-Winter 3-135 0 . 0 0 5 2 1 Summer-Winter vs. Fall-Winter 1-7 0 .105 NC(summer)-Winter vs. Fall-Winter 1.646 0.115 NC(summer)-Fall vas. Summer-Fall 1-157 0 .261 Summer-Winter vs. NC(summer)-Winter 0 . 0 5 4 2 0 . 957 167 Table B16: Statistic values for the Two-way repeated measures A N O V A performed to determine the existence of differences between Q 1 0 values of turtles chronically exposed to seasonal cues and indoor turtles acutely exposed to seasonal cues. Pair of seasons: fall-winter, summer-fall and summer-winter. *Significant differences between outdoor and indoor turtles were found for Q10 (night) values calculated between fall-winter (P=0.011) and summer-fall (P=0.019). • Significant differences between outdoor and VARIABLE Turtle Pair of seasons Interaction (group x pair of seasons) Q 1 0 (day) P=o.oo3 P=0.205 P=0.905 F=i4.803 F=i.7i5 F=0.lOO Q, 0 (night) P = O . 8 8 Q P=0.346 P=0.001* F=0.0204 F=1.120 F=9.244 Q i o total (24 hours) P=o.405 P=6o6 P=.oo8A F=o.756 F=0.514 Table B17: Statistic values for the Two-way repeated measures ANOVA performed to determine the existence of differences between day and night Q 1 0 values of turtles chronically exposed to seasonal cues and indoor turtles acutely exposed to seasonal. Only statistics for the difference between outdoor TURTLE GROUP F P Outdoor turtles 9-925 O.025 Indoor turtles 1-733 0.245 168 APPENDIX C: Ventilation Table C1: Means ± S E M of physiological variables for turtles chronically acclimatized to seasonal cues for each season. SEASONS VARIABLES DAY NIGHT TOTAL (24 hours) Fall Ventilation (ml/min/kg) 20.73 ±4.34 9.21 ±1.196 13.82 ±2.3 Breathing frequency (breaths/min) 2.88 ±0.350 1.53 ±0.197 2.07 ±0.23 Tidal volume (ml/kg) 6.70 ±0.789 5.70 ±0.365 6.11 ±0.49 Breaths/Episode 27.19 ±13.6 12.53 ±2.61 18.28 ±6.83 Episodes/hour 15.89 ±1.93 10.31 ±1.67 12.54 ±1.64 % Apnea 92.39 ±1.12 95.95 ±0.56 94.54 ±0.68 Apnea length 51.27 ±1.39 57.06 ±0.44 54.7 ±0.61 Winter Ventilation (ml/min/kg) 12.26 ±2.6 3.46 ±0.58 6.76 ±0.98 Breathing frequency (breaths/min) 1.46 ±0.11 0.47 ±0.12 0.84 ±0.06 Tidal volume (ml/kg) 5.39 ±0.88 3.27 ±0.75 4.06 ±0.6 Breaths/Episode 5.27 ±0.845 2.62 ±0.54 3.61 ±0.41 Episodes/hour 14.03 ±2.30 5.017 ±1.49 8.39 ±1.61 % Apnea 94.14 ±0.59 98.009±0.59 96.56 ±0.3 Apnea length 55.8 ±0.50 58.78±0.36 57.66 ±0.22 Summer Ventilation (ml/min/kg) 16.89 ±2.31 12.89±0.84 15.55 ±1.7 Breathing frequency (breaths/min) 3.13 ±0.35 2.14 ±0.13 2.8 ±0.27 Tidal volume (ml/kg) 5.51 ±0.64 6.28 ±0.71 5.77 ±0.63 Breaths/Episode 8.82 ±0.86 7.78 ±0.75 8.47 ±0.79 Episodes/hour 22.37 ±2.91 17.20 ±1.43 31.44 ±3.34 % Apnea 92.54 ±0.93 94.48 ±0.41 93.19 ±0.75 Apnea length 54.04 ±0.81 56.68 ±0.25 54.92 ±0.58 No cues Ventilation (ml/min/kg) 9.85 ±0.67 7.99 ±1.69 9.23 ±0.68 Breathing frequency (breaths/min) 1.41 ±0.18 1.004±0.16 1.27 ± 0.12 Tidal volume (ml/kg) 6.61 ±0.38 8.816 ±0.75 6.68 ±0.48 Breaths/Episode 8.42 ±0.899 7.92 ±1.36 8.25 ±0.95 Episodes/hour 11.60 ±2.73 7.19 ±0.93 10.12 ±2.10 % Apnea 95.62±0.60 9.68 ±0.58 95.97 ±0.44 Apnea length 57.35 ±0.36 57.99±0.35 57.56 ±0.26 169 Table C2: Means ± S E M of physiological variables for turtles chronically acclimatized to constant conditions. . SEASONS VARIABLES DAY NIGHT TOTAL (24 hours) Fall Ventilation (ml/min/kg) 29.96 ±4.97 21.84 ±2.95 25.91 ±2.77 Breathing frequency (breaths/min) 4.16 ±0.53 2.71 ±0.34 3.45 ±0.39 Tidal volume (ml/kg) 6.9 ±0.64 8.05 ±0.72 7.44 ±0.56 Breaths/Episode 10.68 ±1.26 11.02 ±1.92 10.87 ±1.51 Episodes/hour 24.04 ±2.08 15.69 ±2.24 19.97 ±1.87 % Apnea 90.67 ±1.43 93.1 ±0.78 91.87 ±0.97 Apnea length 53.05 ±0.93 52.82 ±1.12 52.96 ±0.82 Winter Ventilation (ml/min/kg) 11.75 ±2.63 10.73 ±2.94 11.32 ±2.75 Breathing frequency (breaths/min) 2.60 ±0.57 1.86 ±0.41 2.28 ±0.47 Tidal volume (ml/kg) 4.82 ±0.72 5.92 ±1.05 5.33 ±0.87 Breaths/Episode 9.36 ±1.38 8.94 ±1.09 9.19 ±1.25 Episodes/hour 16.5 ±2.67 11.5 ±2.71 14.22 ±2.55 % Apnea 93.43 ±1.48 94.87 ±1.23 94.03 ±1.28 Apnea length 55.78 ±0.84 55.15 ±1.16 55.5 ±0.83 Summer Ventilation (ml/min/kg) 10.14 ±1.44 10.68 ±1.82 10.35 ±1.36 Breathing frequency (breaths/min) 2.59 ±0.24 1.72 ±0.26 2.16 ±0.21 Tidal volume (ml/kg) 4.07 ±0.68 6.46 ±1.04 5.23 ±0.83 Breaths/Episode 13.72 ±1.65 10.7 ±0.97 12.31 ±0.64 Episodes/hour 11.72 ±1.16 10.97 ±1.9 11.26 ±1.02 % Apnea 93.18 ±0.89 94.87 ±0.82 94.02 ±0.79 Apnea length 54.84 ±0.71 56.92 ±0.49 55.87 ±0.52 No cues Ventilation (ml/min/kg) 16.71 ±1.16 11.06 ±1.13 13.89 ±0.68 Breathing frequency (breaths/min) 3.22 ±0.29 1.94 ±0.14 2.58 ±0.17 Tidal volume (ml/kg) 5.6 ±0.64 5.74 ±0.49 5.67 ±0.44 Breaths/Episode 20.83 ±3.13 12.18 ±0.71 16.51 ±1.65 Episodes/hour 11.47 ±1.34 10.75 ±1.56 11.11 ±1.24 % Apnea 90.69 ±1.64 94.14 ±1.01 92.42 ±1.3 Apnea length 53.7 ±1.56 56.03 ±069 54.87 ±1.06 170 SEASONS VARIABLES DAY NIGHT TOTAL (24 hours) Fall Ventilation (ml/min/kg) 13.92 ±2.90 9.89 ±1.46 11.52 ±1.69 Breathing frequency (breaths/min) 2.36 ±0.3 1.43 ±0.24 1.81 ±0.25 Tidal volume (ml/kg) 6.13 ±1.9 7.09 ±1.91 6.7 ±1.88 Breaths/Episode 9.9 ±1.36 9.77 ±1.73 9.83 ±1.5 Episodes/hour 14.33 ±1.72 9.49 ±1.7 11.48 ±1.57 % Apnea 93.24 ±0.87 95.48 ±0.8 94.55 ±0.78 Apnea length 51.44 ±1.25 56.83 ±0.75 54.6 ±0.89 Winter Ventilation (ml/min/kg) 5.42 ±1.11 6.55 ±1.99 6.12 ±1.43 Breathing frequency (breaths/min) 1.07 ±0.215 0.969±0.257 1.007 ±0.2 Tidal volume (ml/kg) 4.82 ±0.17 6.21 ±1.01 5.7 ±0.63 Breaths/Episode 9.68 ±1.607 5.9 ±1.18 7.33 ±0.99 Episodes/hour 7.36 ±1.26 9.67 ±1.70 8.85 ±1.31 % Apnea 95.85 ±0.8 95.3 ±1.62 95.47 ±1.27 Apnea length 56.44 ±0.94 57.11 ±0.96 56.87 ±0.9 Summer Ventilation (ml/min/kg) 15.14 ±2.68 11.67 ±3.14 13.98 ±2.45 Breathing frequency (breaths/min) 2.82 ±0.49 1.87 ±0.25 2.5 ±0.33 Tidal volume (ml/kg) 5.51 ±0.96 5.98 ±0.72 5.67 ±0.86 Breaths/Episode 9.33 ±1.82 7.79 ±0.68 8.81 ±1.3 Episodes/hour 19.31 ±2.77 15.54 ±3.11 27.94 ±4.35 % Apnea 92.57 ±1.39 94.22 ±1.36 93.12 ±1.18 Apnea length 53.89 ±0.75 55.98 ±0.94 54.59 ±0.68 171 Table C4: Means ± SEM of physiological variables for turtles acutely exposed to constant conditions (seasonal cues absent). SEASONS VARIABLES DAY NIGHT TOTAL (24 hours) Fall Ventilation (ml/min/kg) 27.89 ±6.77 21.28 ±3.08 24.73 ±4.77 Breathing frequency (breaths/min) 3.55 ±0.43 2.53 ±0.24 3.06 ±0.31 Tidal volume (ml/kg) 7.31 ±0.95 7.91 ±0.68 7.6 ±0.76 Breaths/Episode 8.83 ±0.68 9.34 ±1.22 9.08 ±0.88 Episodes/hour 24.77 ±2.78 16.46 ±1.4 20.79 ±1.75 % Apnea 91.87 ±1.10 93.7 ±0.67 92.74 ±0.83 Apnea length 53.08 ±1.26 55.17 ±0.58 54.1 ±0.65 Winter Ventilation (ml/min/kg) 11.26 ±1.15 10.37 ±1.92 10.82 ±1.48 Breathing frequency (breaths/min) 2.07 ±0.2 1.55 ±0.29 1.81 ±0.23 Tidal volume (ml/kg) 5.76 ±0.62 6.65 ±0.8 6.21 ±0.69 Breaths/Episode 7.72 ±0.94 7.8 ±1.49 7.76 ±1.18 Episodes/hour 17.3 ±2.24 13.36 ±1.31 15.3 ±1.72 % Apnea 95.01 ±0.44 95.95 ±0.44 95.48 ±0.4 Apnea length 54.82 ±1.15 57.56 ±0.27 56.19 ±0.63 Summer Ventilation (ml/min/kg) 23.82 ±2.16 17.3 ±2.16 20.54 ±1.79 Breathing frequency (breaths/min) 3.67 ±0.37 2.56 ±0.47 3.11 ±0.4 Tidal volume (ml/kg) 6.43 ±0.41 7.03 ±0.55 6.74 ±0.46 Breaths/Episode 10.85 ±1.68 11.1 ±1.06 10.99 ±1.29 Episodes/hour 23.6 ±4.8 15.22 ±3.24 19.33 ±3.99 % Apnea 91.26 ±0.74 93.04 ±1.28 92.16 ±0.94 Apnea length 54.75 ±0.44 55.42 ±0.78 55.08 ±0.57 172 60 40 20 10 15 20 2 5 10 15 2 0 2 5 Hour Hour 60 4 0 20 10 15 2 0 2 5 10 15 2 0 Hour Hour Figure C1: Number of breaths per episode ± S E M over a 24 hour period. Plots on the top show the circadian changes in breaths/episode for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in breaths/episode with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 173 W inter —A— Fall — • — S u m m e r No cues Seasonal Cues Outdoor An ima l s No Seasonal Cues Ep i sodes /Hou r (Air) Seasonal Cues 40 30 21 10 0 40 30 20 10 Indoor An ima ls No Seasonal Cues 10 15 20 25 10 15 20 25 10 15 20 25 10 15 20 25 Hour Hour Hour Hour Figure C2: Number of breathing episodes per hour ± S E M over a 24 hour period. Plots on the top show the circadian changes in episodes/hour for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in episodes/hour with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 174 W inter A Fall m Sum mer No cues % Time in Apnea (Air) Seasonal Cues Outdoor Animals No Seasonal Cues Seasonal Cues Indoor Animals No Seasonal Cues 100 95 90 85 80 100 95 9G 85 80 1C0 95 90 85 80 100 95 90 85 80 10 15 20 25 10 15 20 25 10 15 20 25 10 15 20 21 Hour Hour Hour Hour Figure C3: Apnea duration ± S E M over a 24 hour period. Measured as the proportion of time turtles were apneic. Plots on the top show the circadian changes in the proportion of time in apnea for each season (winter, fall, summer). Open symbols indicate the photophase and filled symbols indicate the scotophase. Plots on the bottom compare the daily rhythm in the proportion of time turtles spend in apnea with no external cues with the corresponding season (summer for indoor and fall for outdoor turtles). See Table 2.1 for details. 175 Statistic Tables: Circadian rhythms Table C5: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles chronically exposed to seasonal cues. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES CHRONICALLY EXPOSED TO SEASONAL CUES (Paired t-test) SEASONS Season Variable df n Fall Ventilation (ml/min/kg) 3-27 0.014 Breathing frequency (breaths/min) 5-10 O.OOl Tidal volume (ml/kg) I.74 0.125 Breaths/Episode I.79 0.1l6 Episodes/hour 3-88 0.006 % Apnea -3.81 O.OO7 Apnea length 36.OO 0.008 7 8 8 8 8 8 8 8 Winter Ventilation (ml/min/kg) 3-19 0.015 Breathing frequency (breaths/min) 4.70 0.002 Tidal volume (ml/kg) 1-95 0.093 Breaths/Episode 2.42 O.046 Episodes/hour 4-93 0.002 % Apnea -3-71 0.008 Apnea length -4.02 0.005 7 7 7 8 8 8 8 8 8 8 Summer Ventilation (ml/min/kg) 1.99 0.103 Breathing frequency (breaths/min) 3-73 0.014 Tidal volume (ml/kg) -I.78 O.136 Breaths/Episode 2.08 0.093 Episodes/hour 2.72 O.042 % Apnea -3-27 0.022 Apnea length -3-54 0.017 No cues Ventilation (ml/min/kg) O.98 O.371 Breathing frequency (breaths/min) I.46 0.203 Tidal volume (ml/kg) -0-39 O.708 Breaths/Episode O.46 O.661 Episodes/hour 2.25 O . O 7 4 % Apnea -I.24 O.27I Apnea length -I.24 O.271 176 Table C6: Table 3.10: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles chronically exposed to constant conditions. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES CHRONICALLY EXPOSED TO CONSTANT CONDITIONS (Paired t-test) Season Variable t P df n Fall Ventilation (ml/min/kg) 1-359 0.216 7 8 Breathing frequency (breaths/min) 3-543 0.009 7 8 Tidal volume (ml/kg) -1.522 0.172 7 8 Breaths/Episode -0.287 0.782 7 8 Episodes/hour 38.72 0.006 7 8 % Apnea -1-957 0.091 7 8 Apnea length 0.183 0.860 7 8 Winter Ventilation (ml/min/kg) 0.992 0.367 5 6 Breathing frequency (breaths/min) 1.801 O.132 5 6 Tidal volume (ml/kg) -2.001 0.102 5 6 Breaths/Episode O.848 0-435 5 6 Episodes/hour 3.532 0.017 5 6 % Apnea -1-349 0.235 5 6 Apnea length 0.639 0.551 5 6 Summer Ventilation (ml/min/kg) -0.306 0.772 5 6 Breathing frequency (breaths/min) 3-016 0.030 5 6 Tidal volume (ml/kg) -4.076 0.010 5 6 Breaths/Episode 1.233 0.272 5 6 Episodes/hour -9.00 * 0.438 5 6 % Apnea -2-559 0.051 5 6 Apnea length -3.190 0.024 5 6 No cues Ventilation (ml/min/kg) 3052 0.028 5 6 Breathing frequency (breaths/min) 4.282 0.008 5 6 Tidal volume (ml/kg) -0.183 0.862 5 6 Breaths/Episode 2.768 0.039 5 6 Episodes/hour 0.477 0.653 5 6 % Apnea -4-035 0.010 5 6 Apnea length -2.009 0.101 5 6 177 Table C7: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles acutely exposed to seasonal cues. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES ACUTELY EXPOSED TO SEASONAL CUES (Paired t-test) Season Variable t P df n Fall Ventilation (ml/min/kg) I.389 O.207 7 8 Breathing frequency (breaths/min) 4.845 0.002 7 8 Tidal volume (ml/kg) -I.423 0.198 7 8 Breaths/Episode 2.00 * 0-945 7 8 Episodes/hour 3.401 0.011 7 8 % Apnea -3.884 0.006 7 8 Apnea length -6.022 <0.001 7 8 Winter Ventilation (ml/min/kg) -O.57O 0.593 5 6 Breathing frequency (breaths/min) 0.391 0.712 5 6 Tidal volume (ml/kg) -I.328 0.241 5 6 Breaths/Episode 1.976 0.105 5 6 Episodes/hour -1.266 0.261 5 6 % Apnea 0.438 0.680 5 6 Apnea length -I.O46 0.343 5 6 Summer Ventilation (ml/min/kg) 1.138 0.307 5 6 Breathing frequency (breaths/min) 1-655 0.159 5 6 Tidal volume (ml/kg) -1-195 0.286 5 6 Breaths/Episode 0.883 0.418 5 6 Episodes/hour 1.606 0.169 5 6 % Apnea -1.098 0.322 5 6 Apnea length -2.209 0.078 5 6 178 Table C8: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables related to ventilation of turtles acutely exposed to constant conditions. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES ACUTELY EXPOSED TO CONSTANT CONDITIONS (Paired t-test) Season Variable t p df n Fall Ventilation (ml/min/kg) 1.007 0.347 7 8 Breathing frequency (breaths/min) 3-00 0.020 7 8 Tidal volume (ml/kg) -0.878 0.409 7 8 Breaths/Episode -0.592 0.573 7 8 Episodes/hour 2.957 0.021 7 8 % Apnea -2.333 0.052 7 8 Apnea length -1.420 0.199 7 8 Winter Ventilation (ml/min/kg) 0.805 O.457 5 6 Breathing frequency (breaths/min) 2.682 O.O44 5 6 Tidal volume (ml/kg) -2.467 0.057 5 6 Breaths/Episode -0.103 0.922 5 6 Episodes/hour 3-078 0.028 5 6 % Apnea -2.547 0.051 5 6 Apnea length -2.518 0.053 5 6 Summer Ventilation (ml/min/kg) 2.683 O.O44 5 6 Breathing frequency (breaths/min) 3426 0.019 5 6 Tidal volume (ml/kg) -1.849 0.124 5 6 Breaths/Episode -0.233 O.825 5 6 Episodes/hour 4.421 0.007 5 6 % Apnea -1.952 0.108 5 6 Apnea length -1.199 O.284 5 6 179 Statistic Tables: Circannual rhythms Table C9: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to ventilation of turtles chronically exposed to seasonal cues. Circannual rhythms in the physiological variables of turtles chronically exposed to seasonal cues (One-way repeated measures A N O V A ) Variable F P n Venti la t ion (ml/min/kg) 10.667 < O . O O l 7 Breathing frequency (breaths/min) 33-351 < 0.001 7 Tida l volume (ml/kg) 2.080 0.141 7 Breaths/Episode 6.046 0.005 7 Episodes/hour 32.781 < 0.001 7 % Apnea 15-579 < 0.001 7 Apnea length 9-859 < 0.001 7 Table C10: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles chronically exposed to constant conditions. ^ ^ = = ^ = = = = = = = ^ = = = ^ ^ ^ ^ = = = ! = = = ^ ^ = Circannual rhythms in the physiological variables of turtles chronically exposed to constant conditions (One-way repeated measures A N O V A ) Variable F P n Ventilation (ml/min/kg) IO.053 < 0.001 8, fall 6 winter, summer & NC Breathing frequency (breaths/min) 2-743 O.080 8, fall 6 winter, summer & NC Tidal volume (ml/kg) 3.678 0.036 8, fall 6 winter, summer & NC Breaths/Episode 4-673 0.017 8, fall 6 winter, summer & NC Episodes/hour 4-933 0.014 8, fall 6 winter, summer & NC % Apnea 1.842 O.183 8, fall 6 winter, summer & NC Apnea length 2-553 0.094 8, fall 6 winter, summer & NC 180 Table C11: Statistic values for the O n e - w a y repeated m e a s u r e s A N O V A performed to determine the existence of c ircannual rhythms in physiological variables related to the ventilation of turtles acutely e x p o s e d to s e a s o n a l cues. Circannual rhythms in the physiological variables of turtles acutely exposed to seasonal cues (One-way repeated measures ANOVA) Variable F P n Ventilation (ml/min/kg) 5-571 0.024 8 fall; 6 winter & summer Breathing frequency (breaths/min) 12.12 0.002 8 fall; 6 winter & summer Tidal volume (ml/kg) 0.l8l 0.837 8 fall; 6 winter & summer Breaths/Episode O.65 0.542 8 fall; 6 winter & summer Episodes/hour I76.I < O . O O l 8 fall; 6 winter & summer % Apnea 5.165 0.029 8 fall; 6 winter & summer Apnea length 5.24 0.028 8 fall; 6 winter & summer Table C12: Statistic values for the One-way repeated m e a s u r e s A N O V A performed to determine the existence of c ircannual rhythms in physiological variables related to the ventilation of turtles acutely e x p o s e d to constant conditions. Circannual rhythms in the physiological variables of turtles acutely exposed to constant conditions (One-way repeated measures ANOVA) Variable F P n Ventilation (ml/min/kg) 10.711 0.003 8 fall; 6 winter & summer Breathing frequency (breaths/min) 5.258 O.028 8 fall; 6 winter & summer Tidal volume (ml/kg) 1.695 O.232 8 fall; 6 winter & summer Breaths/Episode 7446 0.010 8 fall; 6 winter & summer Episodes/hour 3.002 0.095 8 fall; 6 winter & summer % Apnea 7.064 0.012 8 fall; 6 winter & summer Apnea length 1.251 0.327 8 fall; 6 winter & summer 181 APPENDIX D: Air convection requirement and Sensitivity of Ventilation Table D1: Means ± SEM of physiological variables for turtles chronically acclimatized to seasonal cues for each season. SEASONS V A R I A B L E S D A Y NIGHT T O T A L (24 hours) Fall Air convection requirement 64.09 ±5.31 51.88 ±7.07 56.83±6.28 % Oxygen extraction 8.61 ±0.94 11.79 ±1.6 10.52±1.28 Winter Air convection requirement 101.52±42.1 83.05±22.2 1.83.77±52.5 % Oxygen extraction 7.28±1.39 9.03±2.35 8.75±1.72 Summer Air convection requirement 45.7±1.86 46.02±5.33 45.8±2.78 % Oxygen extraction 10.9±0.42 11.16±1.097 10.99±0.6 No cues Air convection requirement 56.83±6.92 73.51±11.46 62.25±7.62 % Oxygen extraction 11.39±2.13 8.52±1.4 10.46±1.79 Table D2: Means ± SEM of physiological variables for turtles chronically acclimatized to constant conditions. SEASONS V A R I A B L E S D A Y NIGHT T O T A L (24 hours) Fall Air convection requirement 44.03±3.64 40.19±3.3 42.19±3.28 % Oxygen extraction 11.87±0.68 12.84±1.02 12.34±0.81 Winter Air convection requirement 58.32±7.14 56.19±4.49 57.6±5.53 % Oxygen extraction 9.08±0.98 9.22±0.65 9.15±0.76 Summer Air convection requirement 40.63±3.57 39.19±5.07 39.96±3.87 % Oxygen extraction 12.5±0.89 13.28±1.43 12.88±1.13 No cues Air convection requirement 42.36±3.57 37.73±3.82 40.04±3.61 . % Oxygen extraction 11.85±0.8 13.53±1.31 12.69±1.02 182 Table D3: Means ± S E M of physiological variables for turtles acutely exposed to seasonal cues. SEASONS V A R I A B L E S D A Y NIGHT T O T A L (24 hours) Fall Air convection requirement 74.92±9.39 68.76±9:63. 70.79±8.87 % Oxygen extraction 9.26±1.97 8.19±0.87 8.58±1.13 Winter Air convection requirement 95.29±10.10 83.72±11.60 87.97±9.09 % Oxygen extraction 7.5±1.13 10.59±2.58 9.55±1.69 Summer Air convection requirement 36.92±5.78 30.94±5.62 34.92±5.68 % Oxygen extraction 14.6±1.49 17.36±1.97 15.52±1.63 Table D4: Means ± S E M of physiological variables for turtles acutely exposed to constant conditions (seasonal cues absent). SEASONS V A R I A B L E S D A Y NIGHT T O T A L (24 hours) • Fall Air convection requirement 41.9±2.52 40.6±2.40 41.32±1.85 % Oxygen extraction 12.43±0.72 12.89±0.67 12.64±0.58 Winter Air convection requirement 16.53±17.3 66.12±10.99 69.92±13.73 % Oxygen extraction 8.42±1.38 9.17±1.55 8.8±1.38 Summer Air convection requirement 76.46±19.5 67.7±16.74 71.9±17.97 % Oxygen extraction 8.73±1.4 9.51±1.43 9.15±1.35 183 Statistic Tables: Circadian rhythms Table D5: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles chronically exposed to seasonal cues. The (*) indicates times when CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES CHRONICALLY EXPOSED TO SEASONAL CUES (Paired t-test) Season Variable t P df n Fall Air convection requirement 4 - 3 0 8 0 . 0 0 4 7 8 % Oxygen extraction W=36 0 . 0 0 8 * 7 8 Winter Air convection requirement 0 . 6 7 7 0-535 4 5 % Oxygen extraction -1 .062 O . 3 2 3 7 8 Summer Air convection requirement 0 . 2 3 5 O . 8 2 3 5 6 % Oxygen extraction -0 .311 0 . 7 6 8 5 6 No cues Air convection requirement -1 .858 0 .122 5 6 % Oxygen extraction 1.807 O.131 5 6 Table D6: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles chronically exposed to constant conditions. The (*) indicates times CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES CHRONICALLY EXPOSED TO CONSTANT CONDITIONS Season Variable t P df n Fall Air convection requirement I . 6 0 7 0 .152 7 8 % Oxygen extraction - I . 6 9 4 O . 1 3 4 7 8 Winter Air convection requirement O . 3 6 0 0 . 7 3 3 7 8 % Oxygen extraction - O . 1 6 2 O . 8 7 8 7 8 Summer Air convection requirement O . 4 6 8 0 . 6 6 0 5 6 % Oxygen extraction -I.O59 O . 3 3 8 5 6 No cues Air convection requirement 2 . 8 9 O . 0 3 4 5 6 % Oxygen extraction - 2 . 3 0 6 O . 0 6 9 5 6 184 Table D7: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles acutely exposed to seasonal cues. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF I TURTLES ACUTELY EXPOSED TO SEASONAL CUES (Paired t-test) Season Variable t P df n Fall Air convection requirement 2.153 0.068 7 8 % Oxygen extraction 8.00 0.641* 7 8 Winter Air convection requirement 0.900 0.409 7 8 % Oxygen extraction -1.006 0.361 7 8 Summer Air convection requirement 3-737 0.013 5 6 % Oxygen extraction -3.940 0.011 5 6 Table D8: Statistic values for the paired t-test performed to determine the existence of circadian rhythms in physiological variables of turtles acutely exposed to constant conditions. The (*) indicates times when the non-parametric Wilcoxon Signed Rank Test was used. CIRCADIAN RHYTHMS IN THE PHYSIOLOGICAL VARIABLES OF TURTLES ACUTELY EXPOSED TO CONSTANT CONDITIONS (Paired t-test) Season Variable t P df n Fall Air convection requirement 0.392 O.707 7 8 % Oxygen extraction -O.589 0.574 7 8 Winter Air convection requirement O.818 O.45O 7 8 % Oxygen extraction -O.77O O.476 7 8 Summer Air convection requirement 1.538 O.185 5 6 % Oxygen extraction -O.942 0.390 5 6 1 8 5 Statistic Tables: Circannual rhythms Table D9: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to ventilation of turtles chronically exposed to seasonal cues. Circannual rhythms in the physiological variables of turtles chronically exposed to seasonal cues (One-way repeated measures ANOVA) Variable F P n Air convection requirement 3-092 0.055 7 % Oxygen extraction 0470 O.707 7 Table D10: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles chronically exposed to constant conditions. Circannual rhythms in the physiological variables of turtles chronically exposed to constant conditions (One-way repeated measures ANOVA) Variable F P n Air convection requirement 7.504 0.003 8, fall; 6 winter, summer & NC % Oxygen extraction 7-532 0.003 8, fall; 6 winter, summer & NC Table D11: Statistic values for the One-way repeated measures A N O V A performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles acutely exposed to seasonal cues. Circannual rhythms in the physiological variables of turtles acutely exposed to seasonal cues (One-way repeated measures ANOVA) Variable F P n Air convection requirement 18.506 <0.001 8 fall; 6 winter & summer % Oxygen extraction 6.231 0.017 8 fall; 6 winter & summer 186 Table D 1 2 : Statistic values for the One-way repeated measures ANOVA performed to determine the existence of circannual rhythms in physiological variables related to the ventilation of turtles acutely Circannual rhythms in the physiological variables of turtles acutely exposed to constant conditions (One-way repeated measures ANOVA) Variable F P n Air convection requirement 2.815 0.107 8 fall; 6 winter & summer % Oxygen extraction 5.487 0.025 8 fall; 6 winter & summer 187 

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