Behavioral Change and Phenological Response in Captive Loggerhead Sea Turtles (Caretta Caretta): Seasonal Patterns and the Migra

BEHAVIORAL CHANGE AND PHENOLOGICAL RESPONSE IN CAPTIVE

LOGGERHEAD SEA TURTLES (CARETTA CARETTA):

SEASONAL PATTERNS AND THE MIGRATORY PROCESS

A Thesis

Presented to

The Faculty of the College of Arts and Sciences

Florida Gulf Coast University

In Partial Fulfillment

Of the Requirement for the Degree of

Master of Science

Amber Shaw

2018

Florida Gulf Coast University Thesis

APPROVAL SHEET

This thesis is submitted in partial fulfillment of the

requirements for the degree of

Master of Science

Amber Shaw

Approved: September 24th, 2018

Phil Allman, Advisor

Win Everham, Committee Member

Brian Bovard, Committee Member

The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline.

Acknowledgements The emotional cycle of completing a master’s thesis is the constant shift between feelings of excitement and achievement, followed by confusion and despair. The support and help provided by so many individuals throughout this process has been crucial to my success. Above all, I would like to thank the members of my thesis committee. I am grateful to Dr. Phil Allman, for agreeing to be my adviser. My situation was not typical of most graduate students, but he continually offered patience and guidance from a distance, and pushed me to work through the various challenges I faced. Dr. Win Everham and I spent many hours in the computer lab manipulating the environmental data. It was during these discussions that the idea of a “critical period” emerged as a way to elaborate phenological response. Dr. Brian Bovard’s assistance in the behavioral data analysis was invaluable every step (and misstep) of the way. I genuinely appreciate the time and effort all three of them made to coordinate committee meetings, exemplified by our first meeting in which they carpooled from Fort Myers to Sarasota to meet at my study site.

I owe a huge thanks to my amazing coworkers, Holly West and Lauren Miller. Holly and

I worked together for many years, and we had talked about investigating behavioral change in the captive loggerheads we cared for. Both she and Lauren, along with many wonderful interns, contributed to this study, conducting hundreds of observations and entering enormous amounts of data. They were both also willing to cover work shifts so I could return to school to pursue my graduate degree, and even more importantly, were sources of moral support throughout the entire process. Additionally, Dr. John Reynolds (although no longer with us), was a source of unwavering encouragement in my workplace. 4

I would also like to extend thanks to Mote Marine Laboratory and the Office of Research and Graduate Studies at Florida Gulf Coast University. Both institutions provided funding that helped support my pursuance of a graduate degree.

Although my distance from FGCU prevented me from spending a lot of time with the

Herpetology Research Lab, the group provided a sense of comradery for an exhausted graduate student, and it was motivating to see so much enthusiasm for reptiles and amphibians among the students. In particular, I want to thank Ivana Lezcano for taking an interest in my project and volunteering her time to go through some of the nocturnal video data for me.

Finally, I want to thank my family. My love of animals and the natural world started at a very young age, and my parents have always supported my interests, even when it meant disappearing into the jungle for a few months in Costa Rica to tag nesting turtles. I could not have done any of this without the love and support of my husband, Carlie, and my daughter, Elia.

They are my source of inspiration. I cannot express my gratitude to them enough for always being by my side and believing in me. <4.

This project would not have been possible without Montego and Shelley, the two best loggerheads I know. Thank you for being ambassadors for your species and helping educate the world about sea turtles.

Abstract Understanding vital life history processes is critical to the conservation and management of endangered species, especially in light of changing global climate conditions and the verging unknown impacts this will have on these mechanisms. The goal of this study was to employ direct observation methods on captive loggerhead sea turtles (Caretta caretta) to examine behavioral change as a proxy for the reproductive migratory process. Diurnal and nocturnal observations were conducted on two mature female loggerheads housed at Mote Marine

Laboratory in Sarasota, Florida, from December 2013 through December 2015. Both turtles displayed a reduced interest in food during the winter season each year, which lasted approximately 11 weeks in 2014 and 14 weeks in 2015, with a five week difference in timing between individuals. This time period corresponded with observed change in activity levels, swim patterns, interactions, and location preferences for both animals. There was a rise in diurnal resting, coinciding with a decrease in diurnal swimming as well as a decrease in interactions with their environment. Activity levels increased at night, with some nights of prolonged heightened swimming episodes. One of the turtles exhibited a swim pattern of fixated, directional movement into the wall at increased frequency during this time frame. Both animals spent the majority of their time in a small section of their habitat proximal to the open sky. Three environmental variables (air temperature, photoperiod, and lunar phase) were tracked during this study to investigate potential zeitgebers in sea turtles. Patterns indicate a possible response to day length and stage of the moon, though these findings are speculative and require further research.

Behavioral changes noted here suggest that reproductively mature sea turtles display a form of migratory restlessness in a captive setting, and turtles may utilize a nocturnal swimming strategy during the pre-nesting migration. This study highlights the importance of direct observations 7 when examining behavior and the benefits of using captive animals to help understand processes that are difficult to investigate in the wild. 8

Table of Contents

Introduction …………………………………………………………………………………… 11

Goals and Objectives …………………………………………………………………………. 25

Methods ……………………………………………………………………………………….. 26

Study Species and Site ………………………………………………………………… 26

Experimental Design ………………………………………………………………….. 27

Data Collection ……………………………………………………………………….. 29

Statistical Analysis ……………………………………………………………………. 31

Results ………………………………………………………………………………………… 33

Diurnal Behavior ……………………………………………………………………... 34

Nocturnal Behavior …………………………………………………………………… 39

Environmental Factors ……………………………………………………………….. 40

Discussion …………………………………………………………………………………….. 47

Behavioral Change …………………………………………………………………… 49

Environmental Factors ……………………………………………………………….. 59

Future Studies ………………………………………………………………………… 65

Management Implications ……………………………………………………………. 67

Conclusions …………………………………………………………………………... 68

Literature Cited ………………………………………………………………………………. 70

Appendices ………………………………………………………………………………….... 79

List of Figures

Figure 1. Conceptual diagram of a sea turtle’s life cycle ………………………………...... 19

Figure 2. Images of study subjects (Turtle M and Turtle S) …………………………………. 26

Figure 3. Habitat and study site …………………………………………………………….... 30

Figure 4. Diurnal activity levels for Turtle M and Turtle S over a two year period …………. 35

Figure 5. Frequency of swim pattern for periods of decreased food interest and normal food interest for Turtle M and Turtle S ……………………………………………………………. 36

Figure 6. Interactions with rocks for Turtle M and EEDs for Turtle S ……………………… 38

Figure 7. Percentage of time Turtle M and Turtle S spent in each location within the habitat during the time frame of decreased food interest …………………………………………….. 40

Figure 8. Schematic of permanent habitat for study subjects ………………………………... 42

Figure 9. Nocturnal behavioral patterns for Turtle M and Turtle S …………………………. 43

Figure 10. Environmental factor trends beginning in the late fall and ending in the late winter for the 2013-14 season and 2014-15 season …………………………………………………. 44

Figure 11. Swim patterns exhibited by study subjects ………………………………………. 53

Figure 12. Turtle’s interactions with objects within the environment ………………………. 58

List of Tables

Table 1. Description of behaviors listed in ethogram for diurnal and nocturnal observations… 28

Table 2. Summary of change in food interest for subjects from December 2013 through December 2015 ………………………………………………………………………………. 33

Table 3. SIMPER results showing the distance or dissimilarity between the proposed critical periods and the days outside of those periods, with respect to the environmental factors …... 41

Table 4. Environmental characteristics of the proposed critical periods for Turtle M and Turtle S ………………………………………………………………………………………. 45

Introduction

Migration is a fascinating phenomenon; ubiquitous in the natural world and an integral part of the structure and function of many ecosystems (Fryxell et al. 2011, Shaw 2016). It can be defined a number of ways, but in general it is commonly agreed upon that a migration has one or more of the following attributes: focused, directed movement; movement over a long-distance and duration of time; and movement back and forth between distinct home ranges (Russell et al.

2005, Fryxell et al. 2011). Migratory behavior is seen in multiple life stages of a variety of taxa and occurs for a number of reasons (Creswell et al. 2011). Organisms may migrate in order to exploit resources, to avoid predators, to seek safety from inclement environmental conditions, or to complete reproductive cycles (Heupel et al. 2003, Russell et al. 2005, Creswell et al. 2011,

Fryxell et al. 2011). It is often difficult to study these movements because animals can traverse great distances through aerial, terrestrial, and aquatic environments. The importance of migration in both the operation of ecosystems, as well as the life history of numerous species, necessitates a better understanding of the process and how it may be impacted as global climate conditions change (Chapman et al. 2014, Shaw 2016).

The process of migration has evolved multiple times through interactions between individual organisms, their genetic makeup, and the environment (Creswell et al. 2011, Chapman et al. 2014). A key benefit, thought to be a selective pressure favoring the development of migration, is the movement between resource rich habitats to evade both inter- and intra-specific competition. This scenario also requires a reproductive advantage resulting from the move to another environment, and these gains would have to exceed migratory costs. Successful migrations would eventually become longer and periodic, and in some instances, seasonal

(Creswell et al. 2011). A second hypothesis suggests that reliable, seasonal variation throughout 12 habitats can also incite shifts in movement amidst relatively sedentary populations; a pattern that resembles migratory behavior, as it is repeated over time. Again, motivators for movement include reproduction and food availability (Creswell et al. 2011, Chapman et al. 2014). A third hypothesis simply states that since some resources specific to a species are ephemeral, migration would have to evolve in response (Creswell et al. 2011). Many of these theories were developed by examining migratory patterns and behaviors in birds, with further supportive evidence from studies with insects (Creswell et al. 2011).

The decision to migrate presents a trade-off to the individual, but often the benefits outweigh the costs, making the process advantageous to the organism’s survival (Russell et al.

2005, Creswell et al. 2011, Chapman et al. 2014). An individual may benefit by migrating to another region in order to avoid competition over resources, to escape predators, and/or to expand home ranges (Alcock 2001, Creswell et al. 2011, Chapman et al. 2014). The most obvious costs come in the form of energetics; whether it is the storing up of fat deposits, developing a structure for transport (i.e. wings in certain insects), undergoing extreme physiological changes in order to shift between environments (as seen in diadromous fish), or the energy used en route to the new location (Sapir et al. 2011, Chapman et al. 2014). Additional potential costs include vulnerability to predators while traveling, expenses associated with reproduction (such as delayed reproduction or decreased fecundity), and the risk of not being able to find somewhere habitable (Creswell et al. 2011, Chapman et al. 2014).

Energy gain and expenditure must strike a balance in order for migration to be successful and beneficial (Sapir et al. 2011). These trade-offs can come in a number of forms. Fuel loading is a straightforward strategy in which an organism stores up energy by increasing food 13 consumption prior to departure (Southwood and Avens 2010). These stores are used to maintain the body’s general function during travel, especially when food may be scarce, and to power the muscular systems that make movement possible (Sapir et al. 2011). Many animal groups use this method, but there are limitations, and different species have found ways to adapt. Thrush nightingales (Luscinia luscinia), and many other small migratory bird species will take a break during their migration at one or more stopover sights in order to refuel (Fransson et al. 2001).

The use of stopover sights allows birds to store up small percentages of fat at a time, rather than a single large store that would hinder flight performance by increasing weight and drag (Sapir et al. 2011). Stored energy reserves can also be used after a migration to fuel reproductive activities upon arrival at mating grounds (known as capital breeding) and is common in reptiles

(Southwood and Avens 2010). The reproductive migration and subsequent deposition of eggs is so energetically costly for sea turtles and snakes that females will separate nesting seasons by multiple years (Southwood and Avens 2010, Witherington and Witherington 2015). Some bird species employ a highly developed digestive system, which may facilitate increased ingestion rates, the efficiency at which the food is processed, and allow for diet shifts so that an individual can feed on prey items specific to the ephemeral environment while traveling. However, a more developed digestive system would come at the cost of a large amount of extra body mass (Sapir et al. 2011). There are two general ways to accommodate this. Migratory flapping birds can opt to degrade their digestive systems in the days leading up to flight, build it back up at each stopover site for a period of time, then degrade it again before travel. Other animals may choose to bear the additional weight as long as they make use of the enlarged system continuously by feeding on the move (Sapir et al. 2011). Catharus thrushes, for example, fly at night and feed during the day, in order to maintain an extensive digestive system (Sapir et al. 2011). 14

Migratory species are able to locate their destination via remarkable navigational mechanisms. Some animals travel thousands of kilometers, at times through featureless landscapes, such as flat, desert terrains, or the open ocean (Creswell et al. 2011). It is likely that organisms use multiple cues to aid in orientation and direction finding (Southwood and Avens

2010). Songbirds utilize the Earth’s magnetic field and celestial bodies to maintain orientation during flight (Fransson et al. 2001). Birds are also able to use a setting sun to help them recalibrate their compass before a nocturnal migration (Bauer et al. 2011), and then use star patterns and rotation to guide them while traveling at night (Southwood and Avens 2010). These types of compass cues are particularly useful in covering expansive distances when landmarks are lacking (Southwood and Avens 2010). Many reptiles display impressive homing abilities as well. Magnetic and solar cues play an important role in the navigational abilities of crocodilians, turtles, and snakes (Russell et al. 2005, Southwood and Avens 2010). Box turtles and some species of freshwater turtles have displayed an impaired ability to home under an overcast sky, indicating an importance of solar cues when orienting for these animals (Russell et al. 2005,

Southwood and Avens 2010). Similarly, rattlesnakes (Crotalus atrox) placed in a test environment could only orient correctly when they had access to an open sky (Southwood and

Avens 2010). Alligators and sea turtles have the ability to detect and utilize the Earth’s magnetic field (Russell et al. 2005, Southwood and Avens 2010). Loggerhead sea turtle hatchlings

(Caretta caretta) have been shown to recognize two magnetic parameters: inclination angle

(Lohmann and Lohmann 1994) and intensity (Lohmann and Lohmann 1996). Additionally, juvenile green turtles (Chelonia mydas) have exhibited directional shifts when exposed to different magnetic fields in a laboratory setting (Lohmann et al. 2004). There is also some evidence that sea turtles respond to solar cues. Studies done with juvenile greens and 15 loggerheads, in which magnetic cues were disrupted, indicate turtles could still orient using the sun (Avens and Lohmann 2003, Mott and Salmon 2011). On a smaller spatial scale, chemoreception and olfaction may be a useful cue for freshwater turtles, snakes, and sea turtles

(Russell et al. 2010, Southwood and Avens 2010).

Environmental stimuli also influence the timing of many life cycle events, including migratory movements; a process known as phenology. In this situation, environmental cues act as zeitgebers, which entrain an individual’s internal biological clock rhythm (Tarrant and Reitzel

2013), and trigger the beginning of a migration (Coles and Musick 2000). Multiple factors, both internal and external, are involved in this complex process (Shaw 2016). It is commonly accepted that obligate migrants (characterized by predictable movements occurring at approximately the same time annually) respond to the combination of endogenous signals along with change in daylight (Ramenofsky et al. 2012). Day length has been noted as a common zeitgeber for many types of birds, cueing them to begin whichever of the bodily preparation strategies their species has adopted (Bauer et al. 2011). The rate at which day light changes is consistent each year, which makes photoperiod a reliable indicator of the time of year to an individual, and probably acts as a more general signal (Bauer et al. 2011, Shaw 2016).

Supplementary, or local factors (weather, air or water temperature) may drive the more specific time of departure (Bauer et al. 2011, Shaw 2016). Fluctuations in air temperature and rainfall have been suggested as seasonal cues for terrestrial mammalian herbivores (Alcock 2001, Bauer et al. 2011). Also crucial are adequate energy reserves and body preparation, which may manifest in a particular developmental stage and/or combination of hormone concentrations, acting as a final trigger to begin a migration (Bauer et al. 2011, Shaw 2016). 16

Research on wild bird populations suggest that air temperature plays a role in modifying departure time, flight speeds, and arrival at breeding grounds, or termination of migration. Forty years of mist net captures on several species of migratory birds in North America indicate birds adjust the timing and rate of a breeding migration in response to temperature, with warmer weather leading to earlier departure dates (Marra et al. 2005). A strong correlation was also observed between temperature and the arrival dates of male pied flycatchers (Ficedula hypoleuca) to breeding grounds, as well as between temperature and the onset of breeding with females, again with warmer temperatures reflecting an earlier initiation of both behaviors (Both et al. 2005). Although these examples demonstrate the significance of air temperature in shaping the timing of these behaviors, the general process of migration has an endogenous foundation in birds (Bauer et al. 2011). This was clearly demonstrated in a laboratory study done by Gwinner

(1977), in which birds were maintained in a controlled setting and exposed to a constant photoperiod for several years. Subjects progressed through the typical physiological process each year, indicating individuals were preparing themselves for a migration. This was further supported behaviorally by the noted restlessness displayed by the birds. While the overall schedule of migration follows a circannual rhythm, the specific timing of departure each year is flexible, thus allowing birds to fine-tune their migrations in response to the environment (Marra et al. 2005, Bauer et al. 2011).

Lunar cues have been shown to drive migratory behavior in some types of diadromous fish and eels (Sudo et al. 2014). Wild studies conclude peak numbers of migrating individuals occurring around either the full moon or new moon, depending on the species (Yako et al. 2002,

Kuparinen et al. 2009). Additionally, a more recent study conducted with silver-phase Japanese eels (Anguilla japonica) found captive individuals displaying an increase in activity levels 17 around the new moon phase during the months of November and December, corresponding with their seasonal migration in Japan (Sudo et al. 2014).

The various animal groups that migrate range in size, distribution, and taxonomy.

Migratory mechanisms have been noted in mammals, reptiles, amphibians, fish, birds, and invertebrates (Bauer et al. 2011, Shaw 2016). Fundamentally, reasons for migration remain similar across taxa, but migratory strategies and behaviors can differ greatly among these groups

(Chapman et al. 2014). For example, partial migration, when only part of a population migrates while the others remain stationary, is very common in a diverse array of taxa (Bauer et al. 2011,

Chapman et al. 2014, Shaw 2016). Similarly, differential migration between sexes occurs in birds and bats, with males and females traveling different distances for reproductive purposes

(Holbertson 1993, Bauer et al. 2011). Physical constraints exist for particular species as well; such as the depletion of energy reserves while migrating. Many species of birds address this issue by stopping for periods during their journey in order to rest and refuel (Fransson et al.

2001, Bauer et al. 2011, Sapir et al. 2011). Large, terrestrial herbivores tend to graze while migrating and do not require the same high energy stores for transport as other species.

Consequently, they are less restricted because they do not need to physically prepare their bodies in the same way that many birds, reptiles, fish, insects, and other mammals must (Bauer et al.

2011, Sapir et al. 2011). Amphibians tend to be very limited based on their physiology; vulnerability to both dehydration and extreme temperatures allows them only a narrow migratory range (Russell et al. 2005). Synchronous, mass movement is a strategy that can reduce predation risk and increase reproductive fitness, and has been observed in insects, amphibians, birds, fish, and mammals (Russell et al. 2005, Bauer et al. 2011, Creswell et al. 2011, Chapman et al. 2014).

Many aquatic reptiles need to find suitable terrestrial areas to nest, and will move between 18 aquatic and terrestrial environments readily when breeding (Russell et al. 2005, Southwood and

Avens 2010). Most notable, and well-studied, of reptilian migrants are marine turtles (Russell et al. 2005).

Marine turtles have a global distribution, and are capable of traversing thousands of miles through the open ocean (Witherington 2006). Long-distance migrations are a vital process that occur at multiple stages within the life history of all species of sea turtles (Spotila 2004, Russell et al. 2005, Southwood and Avens 2010). For example, along the coastline of southern Florida, loggerhead turtle hatchlings (Caretta caretta) travel from their natal beach via the Gulf Stream to a region in the Atlantic Ocean known as the Sargasso Sea (Witherington and Witherington

2015). After circulating within the North Atlantic gyre for many years, they leave the pelagic zone as juveniles and head for coastal waters in search of suitable foraging territory. Once they reach sexual maturity, adults journey to breeding areas, and after mating occurs, females return to a beach in their natal region where they will deposit multiple egg clutches over the span of several weeks. At the conclusion of nesting season, both sexes migrate back to foraging areas

(Plotkin 2003, Spotila 2004) (Figure 1).

Fully mature males and females make routine reproductive migrations between foraging habitats and mating grounds (Plotkin 2003). These long-distance movements have been studied for decades. Satellite and flipper tagging, along with mark-recapture studies, have revealed information on migratory corridors and ranges (Meylan et al. 1983, Nichols et al. 2000, Plotkin and Spotila 2002, Hawkes et al. 2007), navigational mechanisms (Papi et al. 2000), travel speeds

(Papi et al. 1997, Nichols et al. 2000, Bentivegna 2002), duration and patterns (Girard et al.

2009), and genetic populations (Plotkin and Spotila 2002). Most of this information is based on 19 post-nesting females, because they are easier to encounter than any other age class or group of sea turtle (Plotkin 2003).

Figure 1. Conceptual diagram of a sea turtle’s life cycle, displaying the various migrations at

different life stages. (Russell et al. 2005).

While post-nesting migrations have been extensively researched, less is known about sea turtle behavior before and during a reproductive migration to nesting grounds. Nichols et al.

(2000) used satellite telemetry to track an adult loggerhead that had been captured as a juvenile on west Pacific foraging grounds, held in captivity for 10 years, and then released. The turtle’s movements towards the eastern Pacific were congruent with its Japanese origin (determined by genetic testing), and it was presumed to be migrating back to its breeding grounds. This study found that the turtle used surface currents and maintained a swim speed of greater than 1 km/hr over the course of its journey. James et al. (2005) gathered information on the migratory movements of subadult and adult leatherbacks (Dermochelys coriacea), concluding that the migratory cycle consisted of five stages, characterized by travel rate, dive duration, and dive 20 behavior. However, many of the individuals sampled in this study were not of breeding age, so these conclusions may not necessarily apply to a reproductive migration. Additionally, the distinctiveness of leatherback ecology and physiology, when compared to the six species of hard- shelled sea turtles, precludes these findings as a generalization for all marine turtles.

Sea turtles have adaptations that may aid in the phenological process. It is believed sea turtles imprint to their natal beach, and the magnetic field is likely one of the cues they imprint on (Lohmann et al. 2013). Lohmann and Lohmann (1994) demonstrated that loggerhead hatchlings can detect and differentiate between magnetic inclination angles, helping them determine their latitudinal location. If this mechanism allows adults to discern their location, then sea turtles at a greater distance from their nesting grounds may start their migration earlier than turtles that are closer (Sherrill-Mix et al. 2007). Analysis of 19 years of loggerhead nesting on the east coast of Florida supports the geomagnetic imprinting hypothesis. Brothers and Lohmann

(2015) saw a strong relationship between the location of nesting densities and the unique magnetic signature (composed of an inclination angle and intensity) found along the coast. As magnetic signatures shift slightly over time, nest site selection is shadowing these movements.

These findings add more credence to the theory that turtles utilize geomagnetic cues in navigation and natal homing. Sea turtles also have a pineal gland, which is believed to be a light- sensitive organ through which day length can be recognized (Wyneken 2001). It has been suggested in leatherbacks, photoperiod, along with prey availability and fat stores, are a likely trigger for the migratory response (Davenport et al. 2014).

There are a few hypothesized zeitgebers for sea turtles, but the precise nature in which these variables interact, remains poorly understood. It is unknown how environmental stimuli accumulate in an effective signal for sea turtles to begin their voyage to mating grounds. While 21 sea surface temperature (SST) has been shown to potentially impact the departure date for sea turtles, with warmer temperatures correlating with earlier nesting dates in loggerheads

(Weishampel et al. 2004, Pike et al. 2006, Mazaris et al. 2009), the opposite effect was observed in D. coriacea. Neeman et al. (2015) found that the overall pattern exhibited by three populations of Caribbean leatherbacks was a delay in the onset of nesting with increased water temperature at the foraging grounds. Sherrill-Mix et al. (2007) used satellite telemetry to track 27 leatherbacks, and concluded that each individual’s location, as well as SST and chlorophyll a concentrations, influenced the timing of migration. In this study, it was thought that the SST and chlorophyll a levels may have been affecting the availability of the main food source of leatherbacks, jellyfish, and in turn this may have played a role in the departure time. Yet another study theorizes that day length plays a more important role than SST in phenology, and may be signaling leatherbacks to depart foraging areas in Northern Atlantic waters around Europe (Davenport et al. 2014).

Although conducting studies on wild populations of a migrating species has clear importance, there is also value in examining animals in a captive environment. A key advantage to this strategy is the ability to monitor behaviors closely and carefully, for an indefinite amount of time (Owens and Blanvillain 2013, Stuber and Bartell 2013). One particular characteristic especially relevant to the study of migratory species in captivity is increased activity, known as zugunruhe or migratory restlessness (Funnell and Munro 2005, Bauer et al. 2011). Zugunruhe can be used as a proxy to study aspects of a migration that are difficult to study in a wild scenario, and has been observed in a number of bird species (Holberton 1993, Funnell and

Munro 2005, Bauer et al. 2011, Stuber and Bartell 2013, Watts et al. 2016). In birds, this behavioral change in activity is often expressed as hopping between perches and/or wing- 22 whirring (Holberton 1993, Funnell and Munro 2005, Stuber and Bartell 2013, Watts et al. 2016).

To the author’s knowledge, migratory restlessness has never been scientifically documented in marine turtles, or even in any other animal species other than birds, with the exception of one instance in anguillid eels (Sudo et al. 2014). However, considering the well-known migratory capabilities of sea turtles and the necessity of this process to their life cycle, it is reasonable to hypothesize these animals may demonstrate migratory restlessness in a captive setting.

Technological advancements such as satellite tags, time depth recorders (TDRs), radio telemetry, Crittercams, and data loggers have been used to study different aspects of sea turtle behavior over the last 15 years (Renaud and Carpenter 1994, Heithaus et al. 2002, Taquet et al.

2006, Hatase et al. 2007, Blumenthal et al. 2009, Okuyama et al. 2013, Smolowitz et al. 2015).

Yet even with these modern advancements, monitoring the behaviors and physiological changes that occur specifically during the migration to nesting grounds in wild populations is difficult.

Direct observation provides a more fine-scale method of describing behavior than remote sources, and in captive situations, it allows a means of obtaining corresponding physiological information through access to animals for biological sampling (Rostal et al. 1998, Owens and

Blanvillain 2013). Few studies to date utilize this method because of the challenges of closely observing sea turtles in wild, semi-natural, or captive settings, and many of these reports are limited to being descriptive in nature (Layne 1952, Parrish 1958, Comuzzie and Owens 1990,

Rostal et al. 1998, Frick et al. 2000, Schofield et al. 2006).

Anecdotal evidence and preliminary observations on captive individuals suggest that sea turtles express a form of migratory restlessness (S. Boylan, pers. comm. 2014, D. Owens, pers. comm. 2014, A. Seladi, pers. comm. 2014, J. Seminoff, pers. comm. 2014, L. Wood, pers. comm. 2014). This project provides an assessment of long-term captive loggerhead sea turtle 23 behavior, by means of firsthand observation. In particular, it focuses on patterns of behavioral change associated with season and times of decreased food interest, a possible indicator of migratory restlessness. This method has not previously been employed for the purposes stated here. Three environmental stimuli (air temperature, day length, and lunar phase) are explored in order to evaluate their potential influence on any behavioral changes observed. Photoperiod has been identified as a plausible migratory trigger in leatherback turtles (Davenport et al. 2014); however, lunar phase and air temperature have yet to be evaluated. A review of phenological studies on other animal species led to the selection of these factors. Water temperature has been examined as a zeitgeber for marine turtles, but air temperature has been shown to influence many species of birds (Both et al. 2005, Marra et al. 2005). Birds evolved from a lineage of ancient reptiles, and with some modern-day reptiles and birds being very closely related (Gill 2003), it follows that avian studies could provide a practical baseline for marine turtle research.

The spawning migration in many species of fish may also be useful to consider in the analysis of breeding migrations in sea turtles. Marine species are exposed to a variety of environmental signals, some of which are unique to aquatic habitats. It is reasonable to hypothesize that different groups of marine organisms, although taxonomically distinct, may still respond similarly to environmental cues as a result of convergent evolution. Lunar cues appear to impact the migratory behavior of some fish species (Yako et al. 2002, Kuparinen et al. 2009, Sudo et al.

2014). With sea turtles, there is some discussion of nesting events tied to moon phase, but what is seen more often, is a relationship between tidal regime and nesting activity (Girondot and

Fretey 1996, Bernardo and Plotkin 2007). Since lunar stage influences the tide, it is possible that sea turtles may respond to either or both factors. 24

Due to the cosmopolitan nature of sea turtles, gaining a better understanding of their migratory process, including behavior, phenology, and physiology, is vital to the continued conservation efforts of these animals. Six of the seven extant species are listed on the IUCN Red

List with a conservation status of either vulnerable, endangered, or critically endangered (IUCN

2014). Their survival is threatened by a number of human induced activities, such as large-scale fisheries, coastal development, harvest of both adults and eggs, and waste and pollutants contaminating the oceans (Spotila 2004, Witherington 2006). Identifying which abiotic factors may be triggering sea turtles to begin their reproductive migration could provide insight on how changes to the environment could impact this important life process.

Goals and Objectives

Goal 1: Determine whether captive loggerhead sea turtles (C. caretta) exhibit a change in behavior seasonally.

Objective 1: Using an ethogram and direct observations, record diurnal behavior of two

captive individuals daily for a two year period, in order to establish any behavioral

patterns.

Objective 2: Using video cameras, record nocturnal behavior of two captive individuals

weekly for a one year period, in order to establish any behavioral patterns.

Goal 2: Investigate the environmental factors that are potentially influencing a seasonal change in behavior.

Objective 1: Track day length, lunar phase, and air temperature daily for a two year

period, and explore relationships with any observed behavioral changes in two captive

individuals.

Methods

Study Species and Site. The study subjects were two adult female loggerhead sea turtles housed at Mote Marine Laboratory and Aquarium (Mote), in Sarasota, Florida (Figure 2). Both were collected as eggs and hatched at the University of North Carolina in 1977. There they were raised with four other loggerheads also hatched that year. These six individuals were gathered from three different beaches along the eastern seaboard (Brevard County, Florida, and Onslow

County and Dare County in North Carolina) (Schwartz 1994). Although hatched in a laboratory, the study subjects rotated holding tanks between controlled, indoor housing and outdoor habitats exposed to weather and elements. They were part of several growth and development studies before relocating to Mote in 1998.

Figure 2. Turtle S (left) and Turtle M (right). Photos taken by author.

The subjects’ permanent habitat is a 17,500 gallon exhibit tank that is open to the outside environment. The area is mostly shielded from direct sunlight, but still exposed to natural light cycles and air temperature. Sea water is pumped in from Sarasota Bay, disinfected, and exchanged in the filtration system as needed each week. Water temperature is maintained at 26-

27ºC year round. The exhibit is entirely aquatic, with no area for the turtles to crawl out of the water to nest. Both animals receive approximately 1% of their body weight for their daily diet, comprised of capelin and squid, split into two feeds per day for each turtle. Additionally, they receive a multi-vitamin and calcium supplement daily. The two females participate in husbandry training sessions (feeding sessions designed to work on behaviors that facilitate the basic care of these animals) multiple times a week and have a rotating selection of environmental enrichment devices (EEDs).

Experimental Design. Behavioral ethograms were created for this study based on preliminary observations of the two subjects. An ethogram is a catalog of activities and/or behaviors observed for a particular animal (Martin and Bateson 2007). The following behaviors were recorded: resting, swimming, active (defined as awake and exhibiting movement other than swimming, i.e. playing, crawling), taking a breath, and interacting with rocks, EEDs, and each other (Table 1). Using these ethograms, observations were conducted on both animals over the course of two years. Additional information related to the turtle’s behavior was also monitored, which included the type of swimming pattern the turtle exhibited (divided into four classifications), what direction she was swimming in, where she was located in the habitat, where she was located in the water column, and the number of aquarium guests observing the turtle.

Table 1. Description of behaviors listed in ethogram for diurnal and nocturnal observations.

Behavior Description Diurnal Observations Resting Can be some head or flipper movement, but no body movement overall. Can be at surface or on bottom; includes drifting and floating. Swimming Using flippers to move through water. Active Awake and exhibiting movement other than swimming (i.e. crawling, shuffling, interacting with something in the environment). Into Wall Swim Facing directly into a wall and swimming forward. Along Wall Swim Swimming along wall with flipper in contact with wall. Off Wall Swim Swimming anywhere in the exhibit without touching the wall. Skimmer Swim Swimming with head in skimmer basket. Rock Interaction Picking up with mouth and/or moving rocks around. Breath Lifting head to breathe at surface of water. EED Interaction Biting at, pushing, swimming with, moving, scratching on, or sleeping under an EED. Conspecific Interaction Chasing, biting, bumping into, or trying to rub on other turtle.

Nocturnal Observations Corner Swim Facing directly into a corner and swimming forward. Pacing Repeatedly swimming back and forth along a wall. Floating Resting at surface. No body movement overall, but can include breathing and/or minimal flipper or head movement.

In order to evaluate the turtle’s behavior at night, three cameras (Night Owl L Series 8 channel HDR) were installed above the exhibit tank. The cameras were positioned so that as much of the habitat as possible was captured (Figure 3). This model is equipped with night vision capabilities, and was set to record to a DVR continuously over a 24 hour period. Video recordings occurred during the second year of the study. Three days per week were recorded for 29 an 18 week period during the winter and early spring (January through May) which correspond with intervals of reduced feeding behavior in the subjects, while two days per week were recorded for the remainder of the year. Employing both direct observations and video recordings can provide a more substantial catalog and complete description of each turtle’s behavior (Ruby and Niblick 1994, Schofield et al. 2006, Stuber and Bartell 2013).

Data Collection. Diurnal observations began in December 2013 and concluded in

December 2015. Focal-animal sampling method was employed. The behavioral observations were 10 minutes in length, divided into one minute intervals. Every behavior that occurred within each minute was recorded for that interval. Observations were conducted twice a day for both sea turtles. The time slots for each observation were haphazardly selected between two blocks: 8:00-12:30 (considered am) and 12:30-17:00 (considered pm), with each animal possessing an observation from each block. Three environmental factors were documented at the time of each observation (air temperature, lunar phase, and day length) in order to evaluate each as a potential phenological cue. Air temperature recordings began in the second year, and were collected using an Extech 39240 waterproof thermometer, placed over the exhibit. Air temperature readings for both years were downloaded from the National Oceanic and

Atmospheric Administration (NOAA), with recordings submitted from the nearest weather station to Mote Marine Laboratory. Regression analysis was used to evaluate whether NOAA air temperature values would be a good predictor of air temperature values at Mote, and yielded an equation to calculate air temperatures for the second year, based on the NOAA values [R² = 0.55,

F(1, 61) = 74.82, p <0.001]. Lunar phase and day length were logged from an online source

(http://aa.usno.navy.mil/data/docs/RS_OneDay.php). Comments on the current outdoor weather, 30 and the turtle’s interest in food and willingness to participate in training sessions were noted with each observation as well.

Figure 3. Permanent habitat of the study subjects’. Red circles indicate where cameras were

mounted.

For each video recording, the time frame between sunset and sunrise was sampled. Each minute, on the half hour mark, was reviewed and three behaviors were recorded: corner swimming, pacing, and floating or resting at the surface (Table 1). Due to an issue with the

DVR, the last two weeks of April were not sampled.

Statistical Analysis. Behavior occurrences were tallied for each observation, resulting in a total count for that behavior per day. In order to assess change within an individual over time, the counts for each behavior were pooled and split into two groups – occurring during time periods of normal food interest and time periods of decreased food interest. For the majority of behaviors, groups were compared using a t-test with an alpha of 0.05, employing transformations of the data where necessary. This was done to look for a significant increase or decrease in behaviors corresponding with changes in food interest. Behaviors analyzed in this way include resting, swimming, swim pattern, and EED interactions. The skimmer swim pattern was rarely observed, and is essentially an into wall swim pattern, therefore was combined with this pattern for analysis. In one instance (the rock interactions behavior), the non-parametric Wilcoxon rank- sum test was used when transformations could not meet assumption testing. A Chi-square goodness of fit test was used to examine where each individual spent time within the habitat. The objective was to identify preferred areas of their environment corresponding with changes in food interest. Each year was analyzed separately for each turtle. JMP software (v. 11.0) was used for all analyses run on behavior.

Multivariate methods in the software program Primer (v. 6.1.16) were used to explore for patterns between the abiotic factors, during the late fall/early winter season (Clarke and

Gorley 2015). This was done on both animals for both years. For the six week time frame before each turtle went off of her feed, the three environmental factors (air temperature, lunar phase, 32 and day length) were analyzed using multi-dimensional scaling (MDS). This method produces a visual representation of the data in 3-dimensional space, with distances between points signifying relative dissimilarities between the sampled days, based on the abiotic factors. Points

(days) that were grouped closely together in this space were, therefore, more similar than points that were far apart. The SIMPER function (similarity percentage test) in Primer was used to calculate the average squared distance between points within a grouping, as well as between two separate groupings. It also indicates which factor(s) contribute most to those distances, in other words, which factors are effectively driving the differences in these groupings. A series of

SIMPER analyses were run varying the time window (in number of days) to discover where differences between a potential critical period (where the turtles may be responding to environmental cues, triggering a migration), and data from days outside that period, are maximized.

Complications with the video recordings resulted in some inconsistencies in the data collection. For this reason, inferential statistics could not be run on these data. The video data are reported here graphically and with descriptive statistics, and serve as a supplement to the diurnal observations.

Results During the two year period the study subjects (hereon in Turtle M and Turtle S) were observed, both animals displayed a decrease in food interest beginning each winter season. A decrease in food interest was recorded for the day when one or more of the following behaviors were exhibited: chewing food, spitting food out, not eating food when offered, leaving the feeding station, or ignoring the feeding station altogether. Although the timeframe of diminished food interest was similar in length between the two animals each year, both turtles exhibited a substantially longer fasting phase (an increase of roughly 3.5 weeks) in the second year. In both years, Turtle M showed a loss of interest in food approximately five weeks before Turtle S

(Table 2).

Table 2. Summary of change in food interest for subjects from December 2013 through December 2015.

Difference in onset Dates of diminished food of diminished food Subject Total # of days interest interest between turtles (in days)

2013-14 (Year 1) Turtle M 1/16/2014 - 4/1/2014 76 35 Turtle S 2/20/2014 - 5/6/2014 76

2014-15 (Year 2) Turtle M 12/30/2014 - 4/9/2015 101 37 Turtle S 2/5/2015 - 5/12/2015 97

Diurnal Behavior

Both turtles showed a similar change in diurnal activity level over the course of the study

(Figure 4). The amount of time spent resting each year increased during times of diminished food interest. Turtle M went from spending 36/41 minutes resting (year 1/ year 2 respectively) when eating normally (average amount of the sampled minutes per week the behavior was observed) to

76/85 minutes when food interest decreased (year 1: t(27) = 9.16, p < 0.0001, year 2: t(24) =

7.19, p < 0.0001). Turtle S spent 50/59 minutes resting when eating normally, and this increased to 69/77 minutes during loss of food interest (year 1: t(14) = 3.18, p = 0.0031, year 2: t(22) =

2.77, p = 0.0056). Likewise, both turtles displayed a decrease in swimming during that same time frame across years. Turtle M spent 108/88 minutes swimming when displaying a normal food interest, versus 66/60 minutes swimming when food interest lessened (year 1: t(15) = -5.67, p < 0.0001, year 2: t(22) = -6.01, p < 0.0001). Turtle S spent 94/81 minutes swimming when eating normally, and 79/70 minutes swimming when food interest diminished (year 1: t(17) = -

2.59, p = 0.0096, year 2: t(26) = -1.71, p = 0.0494). The onset of these changes in activity level align very closely with the time that both animals showed less interest in feeding. Turtle M was relatively precise, initiating changes in late December (year 1) and mid-January (year 2). Turtle S showed a decrease in food interest beginning in February of both years, but began to display a shift in activity level a few months prior to decreased food interest in year 2 (Table 2).

Changes in swim pattern behaviors showed some similarities between subjects, but differences were noted as well (Figure 5). Turtle M displayed the same pattern over both years; amount of time exhibiting along wall and off wall swim patterns decreased while the animal was not feeding. The along wall swim pattern decreased by approximately 30 minutes both years 35

(a)

20 Resting 18 16 Swimming 14 12 10 8 6

Amount of time of Amount(minutes) time 4 2 0 Jul-14 Jul-15 Jan-14 Jan-15 Jun-14 Jun-15 Oct-14 Oct-15 Feb-14 Sep-14 Feb-15 Sep-15 Apr-14 Apr-15 Dec-13 Dec-14 Dec-15 Mar-14 Mar-15 Aug-14 Nov-14 Aug-15 Nov-15 May-14 May-15 Month

(b)

20 18 Resting 16 Swimming 14 12 10 8 6

Amount of time (minutes) time of Amount 4 2 0 Jul-14 Jul-15 Jan-14 Jan-15 Jun-14 Jun-15 Oct-14 Oct-15 Feb-14 Sep-14 Feb-15 Sep-15 Apr-14 Apr-15 Dec-13 Dec-14 Dec-15 Mar-14 Mar-15 Aug-14 Nov-14 Aug-15 Nov-15 May-14 May-15 Month

Figure 4. Diurnal activity levels for Turtle M (a) and Turtle S (b) over a two year period. Data are represented as the average amount of minutes each behavior was observed per day in a given week, out of a 20 minute observation period. Time periods of decreased food interest are designated by gray boxes. 36

(a)

100 90 Year 1 Diminished 80 Year 1 Normal 70 Year 2 Diminished 60 50 Year 2 Normal 40 30 20

Amount of Amount of time (minutes) 10 0 Into Wall Along Wall Off Wall Swim Pattern

(b)

100 90 Year 1 Diminished 80 Year 1 Normal 70 Year 2 Diminished 60

50 Year 2 Normal 40 30 20

Amount of time (minutes) 10 0 Into Wall Along Wall Off Wall Swim Pattern

Figure 5. Frequency of swim pattern for periods of decreased food interest and normal food interest for Turtle M (a) and Turtle S (b). Year 1 is designated by solid color and year 2 is designated by diagonal lines. Each column represents the average amount of time the swim pattern is exhibited per week (out of 140 minutes of observation), during either diminished or normal food interest. Standard error bars are shown. 37

(average amount of the sampled minutes per week the behavior was observed), while the off wall swim pattern decreased by 26/11 minutes (year 1/ year 2) [along wall year 1: t(18) = -4.96, p <

0.0001, along wall year 2: t(24) = -6.14, p < 0.0001, off wall year 1: t(19) = -8.26, p < 0.0001, off wall year 2: t(27) = -4.73, p < 0.0001]. No change in into wall swim pattern was observed; she displayed this behavior about 33 minutes of observation time per week year round for the first year, and 25 minutes of observation time per week for the second year (year 1: t(17) = 0.35, p = 0.7307, year 2: t(33) = 1.46, p = 0.1534). Turtle S also spent less time swimming along the wall both years while off feed, but the differences in time were not as prominent as Turtle M’s; only decreasing by 10/5 minutes (year 1/ year 2) [year 1: t(19) = -2.82, p = 0.0054, year 2: t(25)

= -2.04, p = 0.0258]. In contrast to Turtle M, Turtle S did demonstrate an increase in into wall swimming (16/8 minutes year 1/ year 2) when fasting (year 1: t(18) = 2.69, p = 0.0074, year 2: t(29) = 2.25, p = 0.0160), but only showed a decrease in off wall swimming of 13 minutes during the first year (year 1: t(19) = -4.62, p < 0.0001, year 2: t(19) = -1.28, p = 0.2152). In general, both turtles used the along wall pattern as their primary swim behavior throughout the year.

A pattern was observed for both animals between the amount of time spent interacting with objects within the environment and feeding behavior (Figure 6). Turtle M displayed a very clear interest in interacting with the rocks in her habitat the majority of the year, but that interest almost disappeared while off feed. During the first year she interacted with rocks 22 minutes

(average amount of the sampled minutes per week the behavior was observed) when eating normally, and was only observed with a rock a total of 3 minutes out of the entire 12 weeks she lost interest in food (Z = -4.84, p < 0.0001). Year 2 showed only a slight increase in rock interest when off feed; interacting with rocks an average of 3 minutes per week versus 20 minutes per 38

(a)

10 9 8 7 6 5 4 3 Amount of timeAmount (minutes) of 2 1 0 Jul-14 Jul-15 Jan-14 Jan-15 Jun-14 Jun-15 Oct-14 Oct-15 Feb-14 Sep-14 Feb-15 Sep-15 Apr-14 Apr-15 Dec-13 Dec-14 Dec-15 Mar-14 Mar-15 Aug-14 Nov-14 Aug-15 Nov-15 May-14 May-15 Month (b)

10 9 8 7 6

5 4 3 mut f timeAmount (minutes) of 2 1

0 Jul-14 Jul-15 Jan-14 Jan-15 Jun-14 Jun-15 Oct-14 Oct-15 Feb-14 Sep-14 Feb-15 Sep-15 Apr-14 Apr-15 Dec-13 Dec-14 Dec-15 Mar-14 Mar-15 Aug-14 Nov-14 Aug-15 Nov-15 May-14 May-15 Month

Figure 6. Interactions with rocks for Turtle M (a) and EEDs for Turtle S (b). Data are represented as the average amount of minutes each behavior was observed per day in a given week, out of a 20 minute observation period. Time periods of decreased food interest are designated by gray lines (a) and gray boxes (b).

39 week when eating normally (Z = -4.63, p < 0.0001). Turtle S was observed to interact with her environmental enrichment devices (EEDs) throughout the year, but those interactions decreased by 6/12 minutes per week (year 1/ year 2) during the time frame she exhibited a loss of food interest (year 1: t(16) = -1.92, p = 0.0360, year 2: t(32) = -4.25, p < 0.0001).

Both animals demonstrated a strong preference for a particular location within their habitat (Figure 7). The habitat is irregular shaped, with the four selected zones for the study differing in size (Figure 8). Over the course of the two year project, Turtle M was observed in zone 1 for 52% (year 1) and 60% (year 2) of the time, and Turtle S was observed in zone 1 for

46% (year 1) and 47% (year 2) of the time. For the surface area of that space compared to the rest of the exhibit, these percentages are much higher than expected [Turtle M year 1, year 2,

Turtle S year 1, year 2, respectively: χ²(3, N = 730) = 12603.45, 16556.22, 8785.98, 8877.73, p <

0.0001]. Additionally, when just looking at the period of diminished food interest, both animals showed an increase in preference for zone 1, compared to the yearly percentage. Turtle M was observed in this zone for 75% (year 1) and 84% (year 2) of the time, and Turtle S was observed in this zone for 62% (year 1) and 69% (year 2) of the time. Again, these percentages are significantly higher than expected [Turtle M year 1, year 2, Turtle S year 1, year 2, respectively:

χ²(3, N = 152, 202, 152, 194) = 5531.51, 9098.31, 3881.97, 6091.80, p < 0.0001.]

Nocturnal Behavior

The study subjects differed in their nocturnal behavioral patterns (Figure 9). Turtle M primarily displayed a pacing behavior during time periods of decreased food interest (63% of sampled nights), yet displayed an into wall swim pattern throughout the year, with the exception of approximately a ten week period during the summer season. Turtle S showed a very clear 40

90 Turtle M Year 1 80 Turtle M Year 2 70 Turtle S Year 1 Turtle S Year 2 60

Percentage oftime 30

0 1 2 3 4 Zone

Figure 7. Percentage of time Turtle M and Turtle S spent in each location within the habitat during the time frame of decreased food interest. Columns represent each turtle in a given year. The dashed line indicates the percentage of time the animals were expected to spend in

zone 1 (14%). pattern of high activity levels when interest in food lessened (corner swimming occurred on 40% of nights sampled; pacing occurred on 48% of nights sampled), and minimal activity levels when eating normally (5% of nights sampled). When swimming into the wall, the animals mainly focused on two corners; one facing a northwest direction and one facing a northeast direction

(Figure 8). On some nights, a turtle was observed to swim continuously into the wall in the same direction for anywhere between one to five hours, only taking brief, occasional breaks. Both turtles were observed consistently surfacing and floating while breathing throughout the year.

Environmental Factors

While nothing conclusive can be stated about which environmental factors are influencing changes in behavior and food interest in the study subjects, potential patterns emerged that will be mentioned, and later discussed (Figure 10). For Turtle S, SIMPER analysis suggested that the 13 day period prior to decreased food interest in year 1 was very similar 41

(average squared distance = 0.69), with respect to the three environmental variables, and could be a critical time period (Table 3). During this timeframe, air temperature ranged from 19º-24°C, day length increased by 17 minutes, and the lunar phase began at 59% waxing gibbous, went over a full moon, and was at 80% waning gibbous when she went off feed (Table 4). When comparing this period to the several weeks before she goes off feed, the average squared distance is 7.57, suggesting that these groupings of days are far apart in distance and less similar, with the distance being driven by day length (3.58/47.31%, see Appendix A). The second year yielded a similar pattern for Turtle S, even though the timeframe of diminished food interest occurred about two weeks earlier than year 1. The 12 day period prior to loss of food interest showed a closeness, or similarity (Table 3). Air temperature ranged from 17°-23°C, day length increased by 14 minutes, and the lunar phase began at 23% waxing crescent, went over a full moon, and was at 99% waning gibbous when she went off feed (Table 4). When comparing this period to the days outside of this time frame, the average squared distance is 9.57, and this distance is being driven by day length (4.75/49.70%, see Appendix B).

Table 3. SIMPER results showing the distance or dissimilarity (shown here as the average squared distance) between the proposed critical periods and the days outside of those periods, with respect to the environmental factors. The factor most responsible for the difference between these periods is listed, along with the contribution (%) of that factor to the total dissimilarity.

Average squared distance Driving environmental Average squared SubjectDates of critical period Contribution (%) between periods factor distance

2013-14 (Year 1) Turtle M 1/3/2014 - 1/15/2014 8.39 Day length 3.89 46.33 Turtle S 2/7/2014 - 2/19/2014 7.57 Day length 3.58 47.31

2014-15 (Year 2) Turtle M 12/13/2014 - 12/29/2014 6.65 Day length 2.66 39.92 Turtle S 1/24/2015 - 2/4/2015 9.57 Day length 4.75 49.7 42

Figure 8. Schematic of permanent habitat for study subjects. The four zones are marked, with dashed lines representing the boundaries of each region. The two arrows indicate which two corners the animals were observed to focus their directional swim patterns in during nocturnal observations.

(a)

80 Corner swimming Pacing 70

60 50

40 30

of observed minutes) of observedminutes) 20

10 0 Proportion of time exhibiting behavior (Percentage behavior exhibiting of time Proportion Jul-15 Jan-16 Jun-15 Oct-15 Feb-15 Sep-15 Feb-16 Apr-15 Dec-15 Mar-15 Aug-15 Nov-15 May-15 Month

(b)

70 Corner swimming Pacing

(Percentage of observedofminutes) (Percentage 10 ofbehavior time Proportionexhibitng 0 Jul-15 Jan-16 Jun-15 Oct-15 Feb-15 Sep-15 Feb-16 Apr-15 Dec-15 Mar-15 Aug-15 Nov-15

May-15 Month

Figure 9. Nocturnal behavioral patterns for Turtle M (a) and Turtle S (b). Data are represented as the proportion of time that the turtle was seen exhibiting the behavior and expressed as a percentage of the sampled data points. Time periods of decreased food interest are designated by gray boxes. Note that two partial periods of diminished food interest are displayed here.

(a)

50 140 Air temperature 45 Lunar phase 120 40 Turtle M Turtle S 35 100 C) ° 30 80 25 60 20 Lunar Phase(%) Lunar Air Temperature( 15 40 10 20 5

0 0 627 626 625 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 649 650 651 652 654 655 656 657 659 660 662 663 664 666 667 669 670 671 673 674 676 677 679 680 682 684 685 687 688 Day Length (minutes)

(b)

50 140 Air temperature 45 Lunar phase 120 40 Turtle M Turtle S 35 100 C) ° 30 80 25 60 20 Lunar Phase (%)Lunar

Air Temperature( 15 40 10 20 5 0 0 644 642 641 640 639 638 637 636 635 634 633 632 631 630 629 628 627 626 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 646 647 648 650 651 652 653 655 656 657 658 660 661 663 664 665 667 668 Day Length (minutes)

Figure 10. Environmental factor trends beginning in the late fall and ending in the late winter for the 2013-14 season (a) and 2014-15 season (b). Air temperature is shown on the primary y-axis and lunar phase is shown on the secondary y-axis. Day length is displayed on the x- axis. Day length values are given in minutes and represent when a change in light length occurs. The gray boxes indicate the proposed critical periods for Turtle M and Turtle S.

Table 4. Environmental characteristics of the proposed critical periods for Turtle M and Turtle S.

Change in Subject Dates of critical periodTotal # of days Air temperature (°C) Lunar phase day length (minutes)

2013-14 (Year 1) Turtle M 1/3/2014 - 1/15/2014 13 8 14-25 Ends on full moon Turtle S 2/7/2014 - 2/19/2014 13 17 19-24 Overlaps full moon

2014-15 (Year 2) Turtle M 12/13/2014 - 12/29/2014 17 2 21-26 Overlaps new moon Turtle S 1/24/2015 - 2/4/2015 12 14 17-23 Overlaps full moon

Since Turtle M displayed behavioral changes earlier than Turtle S both years, day length during the critical periods must differ between the two animals; however, lunar phase and air temperature have the possibility of being similar. For Turtle M, SIMPER analysis indicated the

13 day period preceding food interest loss in year 1 was found to be less similar (average squared distance = 8.39) to the several weeks before she goes off feed, when considering environmental influences (Table 3). Air temperature ranged from 14°-25°C, day length increased by 8 minutes, and lunar stage began at 8% waxing crescent and ended on a full moon when she stopped eating

(Table 4). Again, day length was found to be causing the difference between the two periods

(3.89/46.33%, see Appendix C). Year 2 for Turtle M contrasted from the others, and is somewhat of an anomaly. Onset of a diminished interest in food began very early in the season, on

December 30th. The prospective critical period was slightly longer at 17 days (Table 3). This time frame was less similar than the period of weeks before it, with an average squared distance of 6.65 (see Appendix D). All factors seemed to contribute somewhat equally to this difference, but day length contributes the most (2.66/39.92%). The overall change in day length was only 2 minutes, and transitioned from decreasing to increasing, because this period occurred over the winter solstice. The lunar stage also differed by beginning at 57% waning gibbous, going over a 46 new moon, and ending at 61% waxing gibbous at the time she went off feed. Air temperature ranged from 21°-26°C (Table 4).

Discussion Observations on long-term captive animals revealed seasonal patterns in loggerhead sea turtle behavior; patterns which may help researchers and conservationists understand the specifics of the migratory process in greater detail. While the current literature addressing migration in sea turtles focuses on post-nesting females (Dodd and Byles 2003, Girard et al.

2009) and primarily reports on aspects like migratory routes (Meylan et al. 1983, Nichols et al.

2000, Plotkin and Spotila 2002, Hawkes et al. 2007), swim speeds (Nichols et al. 2000,

Bentivegna 2002, Luschi et al. 2006), navigation and orientation (Lohmann and Lohmann 1996,

Lohmann et al. 2004, Brothers and Lohmann 2015), and dive duration and depths (Papi et al.

1997, Hays et al. 1999, Foley et al. 2013), the purpose of this study was to examine the behavioral ecology of mature sea turtles preceding, and potentially during, a reproductive migration.

The migratory process can be broken down into stages – preparation, departure, en route, and termination (Bauer et al. 2011). This study aimed to investigate behavior associated with the middle two phases, the departure and duration of a pre-nesting migration. Even though it is challenging to monitor these particular aspects in wild sea turtles, it is still a little easier to identify the beginning and end of these stages in wild individuals compared to captive, i.e. continuous movement away from nesting beach signaling the start of a post-nesting migration

(Foley et al. 2013). The day that Turtle M and Turtle S began to show a decrease in food interest was considered the departure phase, or onset of migration. This criterion was established due to the lack of food intake that occurs in wild female turtles when traveling to nesting grounds

(Southwood and Avens 2010, Bauer et al. 2011, Perrault et al. 2014). The signs indicating that 48 the two study subjects were becoming less interested in feeding included mashing and spitting food, not eating food, leaving the feeding station before a feed was finished, or not coming to the feeding station. Variation in the timing of these signs was seen between individuals. Turtle S would transition from a normal eating behavior to a daily decrease in food interest over the course of approximately 1.5 weeks. During this period she would alternate between one or more signs and normal feeding, until reaching a consistent disinterest in food, making it more difficult to pinpoint a probable departure time. Turtle M switched her feeding behavior much more quickly, displaying a stable decrease in feeding and interest within 1-2 days. Additionally, Turtle

M showed a loss of interest in food approximately five weeks before Turtle S each year. The duration, or en route stage, was classified as the length of time the animals displayed a decreased interest in food, and was concluded when they resumed normal eating habits.

These identifiers of migratory stage can be supported by the results of this study. Given that wild reproductive females do not eat while migrating to nesting grounds (Bauer et al. 2011,

Perrault et al. 2014), it would be reasonable to conclude Turtle M and Turtle S were going through the en route stage of the migratory process when exhibiting a diminished food interest.

Neither turtle showed a distinct diurnal behavioral change prior to or after the proposed departure stage, in fact there is an abrupt spike in resting and corresponding drop in swimming aligned with the same week that each turtle begins to show signs of decreased food interest. When looking at the nocturnal results, the pacing behavior appears about 3-4 weeks before the potential departure week and ends a few weeks before normal feeding resumes. This could suggest the en route stage was initiated while the turtles were still eating normally, and that perhaps food interest is not necessarily a marker for the departure stage. However, it seems more likely that 49 the change in food intake signals the departure time, and there may be another explanation for the early increased activity at night, such as response to an environmental factor.

Behavioral Change

It had been previously noted that the two research subjects went through a period of decreased food interest annually for at least four years prior to the initiation of this study. These observations prompted this investigation, and as predicted, both animals exhibited the same seasonal feeding pattern during this study. Based on the results of the diurnal observations, these two sea turtles generally became less active while off feed. A surprising finding related to their activity level when less interested in food was the increase in resting and decrease in swimming displayed by both turtles. This was the opposite of what was expected, since sea turtles swim great distances when migrating to and from nesting grounds (Nichols et al. 2000, Papi et al.

2000, Plotkin 2003, Witherington 2006), and considering the numerous examples of migratory restlessness demonstrated in captive bird studies (Holberton 1993, Funnell and Munro 2010,

Bauer et al. 2011, Stuber and Bartell 2013, Watts et al. 2016).

One possible explanation for the decrease in diurnal swimming may be related to the turtles’ nocturnal behavioral patterns. While Turtle M showed some level of activity throughout the year, the frequency of wall pacing behavior is concentrated around periods of decreased food interest, and is elevated while off feed. Turtle S displayed a clear increase in nocturnal activity beginning two weeks before, and continuing throughout, her period of diminished food interest.

If both animals are expending more energy at night during this timeframe, then this could support the observed rise in diurnal resting behavior. This potential trade-off may be a possible method of balancing energy use. Nocturnal migration is a strategy used in some bird species; 50 however, the cost is typically sleep loss (Chapman et al. 2014). The discovery of nocturnal activity was surprising for a few reasons. In general, most species of sea turtles (including C. caretta) are thought to mainly be active during the day (Fritsches and Warrant 2013). The eye of the sea turtle is adapted for vision in well-lit settings, promoting the day time occurrence of routine activities, such as foraging, predator avoidance, and mating (Fritsches and Warrant

2013). In accordance with this reasoning, studies have documented longer submergence times in turtles at night compared to day, suggesting these animals are relatively inactive and likely resting nightly, since submergence durations are believed to be correlated with activity levels

(Hays et al. 1999, Luschi et al. 2006). This theory was demonstrated by Turtle S, who was rarely observed at the surface of the water at night during the majority of the year, when she was feeding normally.

It is unclear whether the belief sea turtles are active during the day and inactive at night carries over to the migratory phase; however, nocturnal nesting indicates at least some activity is occurring at night during this stage. Most tracking studies are directed at obtaining information which describes location, specific swim paths, and position in water column (Nichols et al. 2000,

Bentivegna 2002, Dodd and Byles 2003, Girard et al. 2009), often with no distinction in the time of day that the animals are swimming. A study conducted by Foley et al. (2013), in which satellite tags capable of recording depth were used to track post-nesting female loggerheads during their return migrations from Florida nesting grounds, found these individuals spent more time at the surface throughout the day time while migrating, and stayed near the bottom at night.

However, there is no clarification on whether the animals were moving at night, but as previously mentioned, long submergence times would indicate they were not particularly active. 51

Another study conducted on C. mydas in Hawaii found migrating males and females traveled at shallow depths during the day, but were performing very deep dives at night (Rice and Balazs

2008). Since the animals in this study were only equipped with basic TDRs, it is unknown whether they were swimming at night. The authors speculate on the purpose for these nocturnal dives, commenting on behaviors such as resting, evading predators, and searching for food.

Reports of nocturnal swimming during the migration period are uncommon in the literature.

Researchers that followed a few post-nesting female loggerheads off the coast of Mozambique did elaborate on the timing of migratory swimming during a 24 hour period, stating that similar swim speeds were noted both day and night (Papi et al. 1997), and this was also seen in one post- nesting green turtle in the South China Sea (Papi et al. 1995). The nocturnal swimming seen in both Turtle S and Turtle M suggests that this behavior may also occur in wild turtles, and warrants further investigation.

An interesting aspect of the nocturnal swimming was a steady orientation exhibited by both animals. There were instances where a turtle spent several hours at night swimming steadily into the wall, only disrupting this pattern on occasion to swim back and forth along the wall once before resuming the into wall swim behavior again. This occurrence was seen in both turtles, on multiple occasions, and only during times of decreased food interest for Turtle S, while Turtle M did demonstrate the behavior at times throughout the year. It is possible that this behavior could be an example of manifested migratory restlessness in sea turtles, similar to how wing-whirring and perch hopping are noted in captive birds (Holberton 1993, Funnell and Munro 2005, Stuber and Bartell 2013, Watts et al. 2016). Direct movements with a relatively fixed heading have been documented during both pre-nesting and post-nesting migratory trips of C. caretta and C. mydas 52

(Nichols et al. 2000, Papi et al. 2000, Girard et al. 2009), and the attempted directed movements by the study subjects may represent this same process in captive turtles. During these swim episodes, the animals were focused in either a northeast direction or northwest direction; however, it is unknown whether the direction seen here is meaningful. There did not appear to be a pattern between animals, direction, or time of year. If direction were important, it could be expected these turtles would orient and maintain a direction that would lead them back to their natal beach, or in the instance of these two specific animals, where they perceive their natal beach to be located. The background of these individuals is discussed in greater detail later on.

The into wall swim pattern behavior was noted during diurnal observations as well. Of the three general swim patterns the turtles could exhibit (swimming into the wall, swimming along the wall, and swimming off of the wall) (Figure 11), Turtle S demonstrated an increase in into wall swimming when less interested in food, while Turtle M, again, displayed this swim pattern at times throughout the year. In general, both animals showed a decrease in the other two swim patterns during the time of diminished food interest. These focused swims into the wall did not last as long as the episodes at night, but could easily last a full observation period. Often when staff walked by the habitat within the next hour of an observation, the animal was no longer swimming into the wall. In addition to the same northeast and northwest directions observed at night, the turtles were also noted to concentrate in a southwest direction. Since these directed movements were seen both night and day, and primarily during times of decreased food interest, it is a behavioral change that may signify migratory restlessness in captive turtles, and deserves further investigation. Anecdotally, this behavior has been noticed in other captive sea turtles, and was observed in a mature female loggerhead (Seminoff, personal communication) in 53 the years leading up to her release and subsequent tracked migration to breeding grounds

(Nichols et al. 2000).

A B

Figure 11. Swim patterns exhibited by study subjects. (A) Into wall; (B) along wall; (C) off wall.

It is worth noting that the nocturnal swimming documented in this study may be a new finding due to differences between pre-nesting and post-nesting migrations in sea turtles. Female sea turtles need to accumulate a significant storage of fat in order to prepare their bodies for migration to mating grounds and egg production (Miller and Limpus 2003), and typically do not feed during a pre-nesting migration (Bauer et al. 2011, Perrault et al. 2014). This is reinforced by physiological evidence found in leatherbacks at nesting grounds, indicating sea turtles are capital breeders, meaning they use stored energy reserves to fuel reproduction (Perrault et al. 2016). 54

Furthermore, direct observations of loggerheads in the wild during breeding season yield inactive females, with minimal attempts at feeding (Schofield et al. 2006). If there is no need to search for food while migrating to mating grounds, then perhaps swimming at night is advantageous, as it may be a better time to cover ground as quickly as possible while avoiding predators. On the contrary, previous studies have proposed that post-nesting turtles may be foraging during their return migration (Dodd and Byles 2003, Foley et al. 2013). Since these animals need energy to recover from a reproductive episode (Miller and Limpus 2003), it is logical for them to seek food, especially if they are traveling hundreds of miles during a post-nesting migration. Diurnal surface swimming, as well as short submergences and traveling at shallow depths, has been documented in several post-nesting studies (Luschi et al. 2006, Girard et al. 2009, Foley et al.

2013), and would be a strategic method of searching for food during a long-distance movement.

The specific timing and duration of the behavioral changes noted in the study subjects appears to match with what is known about migration schedules in wild loggerheads.

Loggerhead turtles begin a breeding migration anywhere from several weeks to months before the start of nesting season (Plotkin 2003); with individuals having further to travel presumably requiring more time to reach their destination. Females are able to mate in the month prior to nesting (Spotila 2004). Turtle M and Turtle S are from nesting beaches on either the east coast of

Florida or North Carolina; however, were incubated in sand from their natal beach and reared in water from North Carolina (Schwartz 1994). It is unknown whether or not these two are from the same nest or from two completely different nesting populations. The nesting season for loggerheads in these regions runs from April/May through September (FWC 2017, USFWS

2017), so a departure in January (Turtle M) and February (Turtle S) followed by a termination in 55

April (Turtle M) and May (Turtle S) is supported by local wild populations. Satellite tagging of reproductive females in Florida Bay provides an idea of pre-nesting migration departure times and lengths for the nesting population on the east coast of Florida for comparison. In 2003, three reproductive females were tagged at their foraging grounds in Florida Bay and tracked through the nesting season; two of these females successfully made the journey to and from their natal grounds, near Vero Beach, Florida, to nest. Both left in early April, but one arrived in two weeks while the other arrived in four weeks. Both were documented nesting at the end of May. They both began their return migration in August, and arrived back in Florida Bay approximately one month later (Schroeder et al. 2003). Another female loggerhead from this population was tagged in 2013. She made a reproductive journey to the same nesting grounds, taking two weeks to swim there, and three weeks to return to Florida Bay at the end of season (Witherington and

Witherington 2015). These findings are comparable to the timing and duration seen in this study, when considering Turtle M and Turtle S are located in Sarasota County on the west coast of

Florida, and may be directed to somewhere in the stretch between Brevard County, Florida and

Dare County, North Carolina. Further statistics have been documented in Hawaiian green turtles; post-nesting C. mydas have been recorded making an 800-1100 km trip in 20-50 days, at an average swim speed of 1.5 – 2 km/hr (Rice and Balazs 2008). Although the swim speed of Turtle

M and Turtle S were not tracked in this study, the swim speed reported in Hawaiian greens has been demonstrated in numerous studies with loggerheads (Sakamoto et al. 1997, Nichols et al.

2000, Bentivegna 2002, Luschi et al. 2006).

In wild sea turtles, the termination of the pre-nesting migration is followed by mating, and then a nesting period (Spotila 2004). Female loggerheads typically oviposit four clutches of 56 eggs every two weeks during a nesting season, and tend to reproduce about every three years. At the conclusion of the nesting period, they begin their return migration to foraging grounds

(Plotkin 2003). Turtle M and Turtle S displayed an en route process for approximately 11 weeks

(year 1) and 14 weeks (year 2), before resuming normal feeding behavior. Since these two animals do not breed, but instead reabsorb the lipids from mature follicles (Manire et al. 2008), it can be argued that they do not go through a nesting process, and explains why normal feeding and behavior recommence by early May. Although some studies of post-nesting loggerheads suggest foraging during their return migration (Dodd and Byles 2003, Foley et al. 2013), aside from eating normally, no evidence was seen in Turtle M and Turtle S that they went through a post-nesting migration process. All behaviors monitored seemed steady throughout the remainder of the year. Additionally, it is not surprising that Turtle M and Turtle S consistently exhibit behavioral changes every year, when wild loggerheads only go through the process every

2-3 years (Plotkin 2003). Captive animals benefit from having access to a reliable food supply year round, allowing a migratory animal, such as a sea turtle, to store away the necessary energy reserves with ease year after year. Moreover, the retention and absorption of lipids in place of the formation of eggs is a huge energy savings for these two individuals (Manire et al. 2008), and facilitates body preparation for the same process the following year. This would imply that such physiological characteristics are key in enabling a migration, since wild turtles are exposed to the same seasonal factors each year, yet are not cued by abiotic stimuli unless these bodily parameters are met (Hamann et al. 2003, Bauer et al. 2011).

Aside from shifts in activity level, food interest, and swim pattern, the other noticeable change in behavior was related to interactions within the study subjects’ environment. The 57 personalities of the two turtles in this study differ in many ways, one of which is the particular objects in their environment they choose to interact with, individually. Turtle M shows a very fervent interest in biting at and moving the small rocks placed throughout the habitat. This behavior is completely absent during the time that she exhibits a decreased food interest. Turtle S displays a preference towards the environmental enrichment devices (EEDs) that are rotated and placed into the habitat daily. These enrichment items are designed to encourage natural behaviors of sea turtles, providing surfaces for scratching or rubbing on, or objects to push around, bite at, and sleep under. (See Schofield et al. 2006 for image catalog of wild C. caretta exhibiting many of these behaviors for reference) (Figure 12). Turtle S has been observed exhibiting all of the aforementioned behaviors with EEDs regularly, yet these interactions significantly decreased coinciding with diminished food interest. These behavioral changes were expected, because it was assumed the animals would spend most of their time swimming while off of feed, simulating a reproductive migration. While the opposite was actually observed during the day (increased resting and decreased swimming), the turtles still appeared to have a specific focus during that timeframe, which resulted in less attention towards their typical interests displayed the rest of the year.

Lastly, the animals expressed a preference for a particular section of their habitat, and this preference strengthened markedly when the turtles became less interested in food. This section

(referred to as zone 1) is fairly small and enclosed, especially when compared to the larger section that it is directly attached to. This preference is probably not related to food access because the turtles are target trained (and associate a shape and color with food rather than a location in the exhibit) and are fed at multiple locations around their habitat. It is also unlikely 58 that it is due to enrichment interests, because small rocks can be found in three areas of the habitat, and EEDs are introduced in different spots every day, and can be moved around by the animals and water currents. While the entire habitat is covered, zone 1 is the closest to an open sky, has better air circulation, and is more exposed to the elements, which may help explain the strong attraction. Interestingly, the northeast corner in zone 1 is one of the favored spots for the directional swimming observed by both animals. If they are responding to an environmental influence, then it is logical for them to spend the majority of their time in an area where they may be best able to detect these variables.

A B

C D

Figure 12. Interactions with objects within the environment. (A) Biting at rocks; (B) resting under EED; (C) swimming with and biting at EED; (D) scratching on EED. 59

Environmental Factors

An objective of this study was to explore the environmental stimuli that may aid in the mechanism that signals a sea turtle to begin a reproductive migration to mating grounds. It is not fully understood what triggers this phenological response, but it is likely a combination of external, abiotic variables in conjunction with a level of internal, physiological preparedness

(Hamann et al. 2003, Shaw 2016). Sea surface temperature (SST) is often a suggested zeitgeber in sea turtles. In general, studies have found a correlation between warmer water temperatures and earlier nesting dates in loggerheads (Weishampel et al. 2004, Pike et al. 2006, Mazaris et al.

2009), implying earlier departure dates to nesting grounds. The opposite has also been seen though, with leatherbacks exhibiting later nesting dates with increased water temperatures

(Neeman et al. 2015). In addition to SST, chlorophyll a concentrations (as a proxy for food availability) and photoperiod have been discussed as potential influences in migratory departure times of D. coriacea (Sherrill-Mix et al. 2007, Davenport et al. 2014). It should be considered; however, that the overall lifestyle of the leatherback differs a great deal when compared to the six other species of sea turtle (Russell et al. 2005), and so behavior and phenology in this species may not be representative of any other sea turtle. Water temperature was able to be controlled in this study, thus was kept at a constant 26-27°C throughout the year to eliminate it as a trigger.

Therefore, other accessible variables within the habitat of the study subjects’ were investigated as potential influences, and day length, lunar phase, and air temperature were selected.

Although the exact timing of behavioral change differed between individuals, and displayed a shift between years, the general timing of these changes was consistent on a seasonal basis. It is known that sea turtles possess a pineal gland, which is associated with the regulation 60 of biological rhythms (Wyneken 2001). In leatherbacks, it has been demonstrated that light can pass through the top of the skull, reaching this gland, and this may effectively relay photoperiod information to the brain (Davenport et al. 2014). While there is no evidence of this same light pathway in cheloniid turtles, the organ may still be used to aid in perceiving changes in day length. The ability of the two research subjects to reliably display the same behavioral shifts seasonally each year supports the concept of day length acting as a broad signal; a reliable gauge of time of year (Bauer et al. 2011, Shaw 2016). Day length was also shown to be the driving variable between periods of time (selected groupings of days in the SIMPER analyses) in the weeks leading up to the departure stage of each turtle, signaling its importance in phenological response. If photoperiod were used as a more precise cue, then the timing of the behavioral shifts would be expected to be more exact (i.e. occurring around the same date every year), at least within the individual.

If day length is utilized to convey the general time of year, then other factors, either more local or ephemeral in nature, must be responsible for manipulating the exact time of departure

(Bauer et al. 2011, Shaw 2016). Temperature is thought to be influential to different groups of migratory animals, including birds, fish, insects, and amphibians (Shaw 2016), as well as sea turtles (Weishampel et al. 2004, Pike et al. 2006, Mazaris et al. 2009, Neeman et al. 2015). Air temperature during the winter months in Sarasota, Florida is around 22°C as an average high, while the average high is 29°C during the rest of the year (US Climate 2017). Air temperature tracked at the study site varied throughout the winter season, ranging from 14°-27°C, and while this range is cooler than that of the remainder of the year, no general trend was seen during the days leading up to the departure stage for either animal, either year. Additionally, the SIMPER 61 results indicated that air temperature was the factor that fluctuated the most prior to the departure stage. This is not surprising, since day length and lunar phase are both cyclical, so change over time should be more predictable and steady. Many studies that have linked warming SST to earlier nesting seasons in C. caretta use long-term data sets (Weishampel et al. 2004, Pike et al.

2006, Mazaris et al. 2009), and their conclusions are used to infer the impacts of climate change on these important life events of sea turtles. This information is different from the purpose of this investigation, which was to identify potential zeitgebers and explore patterns sea turtles may be responding to. It is possible that the overall cooler air temperatures recorded in the winter months may act as an added broad signal to day length, indicating the general time of year and season.

Celestial bodies are an abiotic variable identified as playing a role in important life history events in sea turtles (Lohmann et al. 2004), making it a reasonable factor to examine in this study. It has been hypothesized that lunar cycles may be used by sea turtles to coordinate another stage in the reproductive process, the initiation of nesting activities (Girondot and Fretey

1996, Bernardo and Plotkin 2007). The timing between the departure stages of the study subjects was very similar for both years (35 and 37 days, respectively) with Turtle M displaying signs before Turtle S. This is very close in alignment with the length of one full lunar cycle, which lasts just about 30 days (McDowall 1969). In looking at the days just prior to the departure stage for each animal in more detail, the moon is in a waxing gibbous stage and either just crosses over or terminates on a full moon at the departure stage (with the exception of Turtle M in year 2, which will be discussed later on). Although not conclusive, there appears to be a pattern. With a steady interval that is extremely close to a complete lunar cycle between animals each year, and the departure stage synchronized with a full moon, the turtles may be using lunar phase as a 62 proximate cue. As mentioned earlier, sea turtles are believed to use celestial bodies to aid in other processes, such as navigation and nesting events (Girondot and Fretey 1996, Lohmann et al. 2004, Bernardo and Plotkin 2007, Brothers and Lohmann 2015); however, the function of these bodies in these activities is more apparent. For example, since lunar cycles affect tidal phases (McDowall 1969), gravid sea turtles can select optimal times to emerge from the ocean to nest, based on the phase of the moon (Barik et al. 2014). The benefit of beginning a reproductive migration during a particular lunar phase, the full moon in this case, is unclear. Light and tide are the two main environmental factors influenced by the moon (McDowall 1969), and while both impact nesting activities in sea turtles (Chacón et al. 1996, Barik et al. 2014), there is no obvious explanation as to how either of those two factors would facilitate an advantage at the onset of migration. In nature, there tends to be a correlation between rhythmic cycles and recurring environmental conditions (McDowall 1969), so over evolutionary time, a synchronization may have developed between the lunar cycle and endogenous rhythms in sea turtles.

When examining environmental variables and phenological response, the interaction and timing of factors is probably quite involved. One aspect that contributes to this complexity may be an accumulation of signal(s) per individual. This idea suggests an animal is cued after a particular amount of change in an environmental variable(s) has occurred, such as a general increase in temperature or day length, over a period of time. Based on this, the author proposes the notion of a critical period; a time frame defined by the amount of change in an environmental variable(s) that initiates a phenological response in an individual, triggering the onset of migration. This study suggested an approximate 12-13 day critical period prior to the departure stage of each turtle. The environmental characteristics of this proposed critical period for Turtle 63

S are very similar both years, and these periods (followed directly by the departure stage) differ by approximately 2 weeks between years, with both phases shifted forward in the second year.

The overall pattern for Turtle S suggests that she may use an increase in day length in the range of 14 – 17 minutes as a broad signal of time of year, in conjunction with a waxing gibbous lunar stage as a proximate cue, with departure occurring around the full moon. It is also possible the local air temperature may act as an additional broad signal, with a cooler range of 17°C -23°C indicating time of year.

Turtle M displayed the same shift of phases in year 2, but it is difficult to compare her proposed critical periods. She exhibits behavioral changes very early in the calendar year, and in the second year, her critical period crosses over the winter solstice. As a result, day length stays relatively constant, only decreasing by one minute at the beginning of the period and then increasing by one minute at the end. The other major difference is that although the critical period for year 1 follows the same lunar stage as Turtle S, year 2 occurs over a new moon. So while Turtle M’s critical period in the first year is similar to the overall pattern seen in Turtle S, the environmental characteristics of year 2 are somewhat of an anomaly. The proposed period is even a few days longer than the 12 – 13 day period seen in the other three instances. The only abiotic factor that falls into a similar range is the air temperature, at 21°C - 25°C. Some species of migratory birds that winter in the northern hemisphere respond to change in photoperiod directly before or after the winter solstice (Ramenofsky et al. 2012), but since this was not seen in year 1 for either turtle, and likewise in year 2 for Turtle S, it still seems the environmental factors observed in year 2 for Turtle M could be considered an outlier. The explanation for why this period contrasts from the others is unknown; however, it is likely the critical period would 64 align with an individual’s physical body preparation, such as sufficient fat reserves and supportive hormone levels (Bauer et al. 2011, Shaw 2016).

Environmental factors were chosen in an attempt to investigate potential signals that these two sea turtles had access to, and that have been recognized as a zeitgeber in other migratory species or as a cue for other important events in the life history of sea turtles.

Additional environmental variables that these animals were exposed to that may have either a direct or indirect involvement in the process include weather patterns and barometric pressure. A drop in barometric pressure can be an indicator of approaching inclement weather systems, and has been documented as a signal in blacktip sharks to retreat to deeper waters during an incoming storm (Heupel et al. 2003). In one study with loggerhead turtles, barometric pressure was the only environmental parameter to consistently change at the times turtles were departing breeding grounds (Schofield et al. 2010). This suggested meteorological cues, such as barometric pressure, may be at least partially triggering their movements, and may be a means of promoting favorable reproductive fitness. Although barometric pressure, air temperature, and changes in the weather are all connected, an animal’s response to any one of these variables does not necessarily imply that the animal would respond to either or both of the other two factors. Air temperature was suggested to be a broad signal of time of year to the turtles in this study, but it may be worth investigating barometric pressure in a future endeavor. Additionally, when changes in weather patterns result in dark or cloudy skies, it is possible that this could impact sea turtles’ ability to detect light levels, which in turn may affect their interpretation of either photoperiod or moon phase. Since it is likely that multiple variables are involved with a sea turtle’s decision to begin a reproductive migration, even though barometric pressure and specific 65 weather variables were not assessed here, they may only be part of the phenological response process, and not the key component.

Future Studies

One aspect that was intended to be a part of this study, but could not be completed due to permitting timelines, was hormonal trends, in which monthly blood samples were to be taken on both sea turtles in order to monitor fluctuations in testosterone, estradiol, and progesterone. Such data may have helped corroborate that observed behavioral changes were associated with a migratory process. The results of both captive and wild studies have shown general hormonal patterns in reproductively mature nesting female sea turtles (Blanvillain et al. 2011). Although the two individuals in this study are not able to mate and nest, and therefore may not display the exact same patterns in hormones as a nesting female, tracking the changes in their hormones would still provide information on their reproductive status for comparison with changes in behavior. Other physiological characteristics that could be measured for added indication of reproductive stage are vitellogenin levels (Smelker et al. 2014) and follicular development

(Manire et al. 2008).

Another variable that would have been very interesting to pursue in this project is genetic background of the study subjects’. Both animals were collected as eggs in July of 1977 and incubated in sand from their natal beach until hatch. They were reared with four other hatchlings of the same year class in water from Bogue Sound, and housed at the Institute of Marine

Sciences at Morehead City, Carteret County, North Carolina. Of these six turtles, pairs came from each of three beaches: Melbourne Beach in Brevard County, Florida, Onslow Beach in

Onslow County, North Carolina, and Pea Island National Wildlife Refuge in Dare County, North 66

Carolina (Schwartz 1994). Turtle M and Turtle S were exposed to both indoor and outdoor habitats and conditions during their time there, and were used in a number of growth and physiological studies until being relocated to Mote Marine Laboratory in Sarasota, Florida, in

1998. It is unknown which beach they were laid on (and consequently which sand they were incubated in), and whether or not they are genetically related. Determining these two factors may provide an explanation to some of the findings in this study. For example, what is known about natal imprinting in sea turtles suggests there may be a chemical and geomagnetic component to the process (Lohmann et al. 2013). These two individuals were reared in the same water source and magnetic field parameters, but potentially in different sand sources with different genetic makeup. These factors could play a role in the difference in timing of behavioral changes seen between the two turtles each year or the preferred directions displayed by both animals when exhibiting an into wall swim pattern. Perhaps a dissimilarity in genetic background would lend insight to the unusual critical period seen in Turtle M during the second year. It is also possible that if the two are related, a mere variation between individuals could still explain physiological differences, and therefore behavioral changes.

Further investigation of the directional swimming behavior may elucidate the migratory process in sea turtles as well. Lohmann et al. (1994) demonstrated that loggerhead hatchlings were able to distinguish between magnetic inclination angles; an ability that may explain how adult turtles are able to return to their natal beach. Examining directional preference, temporal changes, and manipulating magnetic fields on reproductively mature sea turtles of known background would strengthen this argument. It would be particularly interesting to compare these two turtles with turtles that had matured and migrated in the wild prior to being housed in a 67 captive setting. There is a need to increase the sample size in this study to see if patterns documented here can be representative of a larger group or population. Since Turtle M and

Turtle S have been hand reared since hatch, similarities in behavioral patterns between these two and wild raised turtles may indicate an innate nature to parts, or the entirety, of the migratory and phenological process. It would also be informative to continue examination of the high activity levels and directional swimming seen at night with these two animals; whether it exists in wild turtles or other captive turtles, and if present, determining what the ecological advantage to this migratory behavior may be.

This study was limited in that environmental parameters were tracked and recorded in an effort to look for patterns concurrent with behavioral changes in the turtles. The findings here are speculative, and provide suggestions on how the animals may be responding to environmental stimuli. The next step would be to individually manipulate variables in order to look for a significant relationship between a change in one abiotic factor and a change in behavior. As mentioned earlier, other variables that could also be considered in future studies include barometric pressure and weather patterns.

Management Implications

Migration is a necessary and vital behavior in the life history of sea turtles. Gaining a better understanding of this ecological process is critical to the conservation efforts of the species. Since sea turtles often cross international borders, it is especially important for program managers all over the world to work together in order for conservation endeavors to be successful. The results of this study offer a baseline of the pre-nesting migratory behavior; a foundation for future research to investigate the many aspects of this process in greater detail. 68

The reproductive period, or length of time during which a sea turtle is capable of reproducing, is unknown for all species. Modelling techniques have projected a time span of 19 years in Australian female green turtles (Chaloupka and Limpus 2005), but this is a difficult number to confirm. Long-term nesting programs may be able to estimate this period, based on age of maturity and reoccurrence of a tagged female returning to her natal beach to nest.

However, this method cannot account for the disappearance of a female at a nesting beach due to a decline in fertility versus the death of the individual. If the behavioral changes documented in this study can act as a proxy for reproductive ability, then continued observations of these study subjects may lead to a gauge on the reproductive lifespan of female sea turtles, by monitoring for a decrease or disappearance in these behaviors as they age.

As environmental conditions change, conservationists need to consider how these changes may impact the migration process. Selective pressures have been shown to influence a variety of migratory species, modifying migration schedules and duration, even resulting in the cessation of the movement itself (Shaw 2016). Global climate change can alter sea turtle habitat primarily through shifting weather patterns, rising sea levels, and increased water and air temperatures (Hamann et al. 2003). A greater understanding of how sea turtles will respond to an altering environment is essential to their preservation. Identifying not only which variables sea turtles use in their phenological mechanism, but also how they will adapt as one or more of these variables shift, could direct the survival of species.

Conclusions

The behavioral changes described here suggest mature sea turtles can display a form of migratory restlessness in a captive setting, and previous exposure to the open ocean is not a 69 prerequisite for these patterns to manifest. It is likely turtles are responding to multiple variables within the environment to initiate this process. Temperature seems to be of less importance than previously thought, while photoperiod and lunar cycle may play a role in the phenological response. The examination of captive animals can be a useful method when studying ecological processes that are challenging to observe in the natural environment, and was used here in an attempt to make inferences about the pre-nesting migratory mechanism in wild populations of sea turtle. The behavior and process deserve further investigation, and analysis of multiple species, as well as comparison between wild raised and captive raised animals, are needed to analyze migratory restlessness and phenological response in greater depth.

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Appendix A Image from multi-dimensional scaling (MDS) done in SIMPER on the environmental data for Turtle S during year 1. Blue triangles represent days in the proposed critical period, while green triangles represent days outside this period.

Appendix B Image from multi-dimensional scaling (MDS) done in SIMPER on the environmental data for Turtle S during year 2. Blue triangles represent days in the proposed critical period, while green triangles represent days outside this period.

Appendix C Image from multi-dimensional scaling (MDS) done in SIMPER on the environmental data for Turtle M during year 1. Blue triangles represent days in the proposed critical period, while green triangles represent days outside this period.

Appendix D Image from multi-dimensional scaling (MDS) done in SIMPER on the environmental data for Turtle M during year 2. Blue triangles represent days in the proposed critical period, while green triangles represent days outside this period.