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STRESS, CORTICOSTERONE, AND THE SOCIAL BEHAVIOR OF THE PRAIRIE VOLE MICROTUS OCHROGASTER:
AN INTEGRATIVE APPROACH
By
DIMITRI VINCENT BLONDEL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2013
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© 2013 Dimitri Vincent Blondel
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To my parents, Linda and Pierre Blondel, for all of their support throughout my life
and throughout the intense journey of graduate school
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ACKNOWLEDGMENTS
I thank my major advisor, Steven Phelps, and my co-advisor, Colette St. Mary,
for their tireless and generous efforts and advice in the adventure of earning this
degree. I thank the rest of my committee, H. Jane Brockmann, David Reed and Clive
Wynne, for their expert feedback and comments. I thank my parents, Pierre and Linda
Blondel, for all of their support throughout grad school, and without which this
dissertation would not have been possible. Gerard Wallace helped greatly with Illinois
fieldwork and with behavioral trails and colony logistics in Austin. Stefanie Calderone
and Marija Gorenshteyn went above and beyond as dedicated field assistants and were
valuable contributors to many aspects of our Illinois experiments. Ron Pulcher was a
great landlord for our Illinois enclosures and also generously contributed to the project
in many ways. Geoffrey Dahl, Joyce Hayen, Tao Sha, Izabella Thompson, and Ana
Monteiro at UF Animal Sciences provided equipment and lab space for the
radioimmunoassays and cheerful help with anything I needed. Maureen Doran at
Southern Illinois University was a life-saver in providing dry ice in an area that turns out
to be the “Burmuda Triangle” of dry ice. Nilmini Jayasena was my patient
radioimmunoassay guru. Ondi Crino provided helpful advice on fecal hormone
extractions and radioimmunoassays. Adrian Duehl and Karen Scott provided freeze-
dryers for samples. Lou Guillette, Keith Choe, Marta Wayne, Charlie Baer, Kaoru
Kitajima, Iske Larkin, Rebecca Kimball, Gigi Ostrow, Matt Salomon, Cedric Worman,
Sarah Phelps, and Emily Wang provided equipment and supplies for experiments. Alex
Ophir, Polly Campbell, Andreas George, Bret Pasche, Julie Allen, Samantha Hilber, Jill
Mateo, Mike McCoy, Jake Ferguson, Ping Huang, Todd Peterson, Carol Chaffee,
Martijn Slot, and Jorge Pino provided help and advice in the lab, field, and on my
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dissertation document and exit seminar. Alejo Berrio generously provided a place to
stay and acted as host on our Austin vole-fetching trips. I thank my brother Emile
Blondel for visits and needed R&R between experiments. Thanks to Mark Buetow and
Marc Melvin and his family for providing a “home away from home” in southern Illinois,
some home-cooked meals, and good times boardgaming. Thanks to Mike Jones and
Neil Edge and their families for being awesome boardgaming hosts in Gainesville, and
for making holidays away from my own family less lonely. Thank you to UF Animal
Care Services for taking care of our colony animals over the years. Finally, last but not
least, I thank the undergraduate volunteers who helped in both lab and field over the
years, from both the University of Florida and Southern Illinois University: Girard Cua,
Karen Olsen, Gerthy Eugene, Dania Gutierrez, Bert Ketchum, Chace Nacke, Tuesday
Lee, Ruth Kelly, Nicole Szczepanik, Rebecca Filippini, and Kelley Waldschmidt.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 11
ABSTRACT ................................................................................................................... 12
CHAPTER
1 INTRODUCTION .................................................................................................... 14
2 CORTICOSTERONE-FACILITATED SOCIAL PREFERENCE IN THE PRAIRIE VOLE: MONOGAMOUS PAIR BOND OR COPING BEHAVIOR? .......................... 20
Background ............................................................................................................. 20 Methods .................................................................................................................. 23
Subject Animals ................................................................................................ 23 Treatments ....................................................................................................... 24
Procedure ......................................................................................................... 24 Unmated treatments .................................................................................. 24
Mated treatments ....................................................................................... 26 Data Analysis ................................................................................................... 26
Results .................................................................................................................... 27
Discussion .............................................................................................................. 28
3 STRESS COPING STRATEGIES AND FECAL CORTICOSTERONE ASSAY VALIDATION IN THE PRARIE VOLE, MICROTUS OCHROGASTER: STRESS-REACTIVE INDIVIDUALS SHOW LESS, NOT MORE, GLUCOCORTICOIDS ............................................................................................. 37
Background ............................................................................................................. 37 Methods .................................................................................................................. 40
Subject Animals ................................................................................................ 40
Fecal Hormone Assay Validation ..................................................................... 41
Test for assay linearity ............................................................................... 41 Swim challenge .......................................................................................... 42 Fecal CORT extraction and radioimmunoassays (RIAs): ........................... 43
Open-Field Test ................................................................................................ 44 Results .................................................................................................................... 45
Fecal Hormone Assay Validation ..................................................................... 45 Test for assay linearity ............................................................................... 45
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Radioimmunoassays .................................................................................. 46
Swim challenge .......................................................................................... 47 Open-field Trials ............................................................................................... 48
Discussion .............................................................................................................. 49
4 CORTICOSTERONE, STRESS-REACTIVITY AND SOCIAL BEHAVIOR: EFFECTS OF POPULATION DENSITY AND INDIVIDUAL DIFFERENCES AMONG MALE PRAIRIE VOLES ........................................................................... 59
Background ............................................................................................................. 59
Methods .................................................................................................................. 61 Experimental Design Overview ........................................................................ 61 Subject Animals ................................................................................................ 62 Open-Field Test ................................................................................................ 62
Fecal CORT Extraction and Measurements ..................................................... 63 Semi-Natural Enclosures and Density Manipulation ......................................... 63
Data Analysis ................................................................................................... 66 Results .................................................................................................................... 67
CORT Quantification ........................................................................................ 67 Open-Field Trials .............................................................................................. 68 Semi-Natural Enclosures .................................................................................. 69
Discussion .............................................................................................................. 72 Individual and Population Density Differences in Field Space-Use .................. 73
Effects of Population Density on Fecal CORT .................................................. 74 Stress-Reactivity in Lab and Field .................................................................... 76 Interactions Between Population Density and Individual Differences ............... 79
Anogenital Distance and Field CORT ............................................................... 79 Future Directions .............................................................................................. 80
Summary .......................................................................................................... 81
5 CONCLUSIONS ................................................................................................... 101
LIST OF REFERENCES ............................................................................................. 106
BIOGRAPHICAL SKETCH .......................................................................................... 116
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LIST OF TABLES
Table page 2-1 Treatments for affiliation and territoriality trials ................................................... 34
3-1 Open-Field Trial Correlations.............................................................................. 54
4-1 Summary of lab and field Fecal CORT results.................................................... 82
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LIST OF FIGURES
Figure page 2-1 Preference arena ................................................................................................ 34
2-2 Affiliative behavior .............................................................................................. 35
2-3 Aggressive behavior. .......................................................................................... 36
3-1 Log-logit transformed curves of serially diluted fecal extracts compared with log-logit transformed standard curves. ............................................................... 55
3-2 Mean fecal CORT before and after the swim challenge ..................................... 56
3-3 Individual fecal CORT levels before and after the swim challenge ..................... 57
3-4 Fecal CORT levels vs. immobility. ...................................................................... 58
4-1 Semi-natural enclosure facilities. ........................................................................ 83
4-2 CORT sample-mass effect. ................................................................................ 84
4-3 Open-field trial latency-to-first movement plotted against pre-field fecal CORT . 85
4-4 Home ranges (75% kernel contours). ................................................................. 86
4-5 Boxplots of 75% kernel home range area split by mating tactic ......................... 87
4-6 Boxplots of fecal CORT samples from pre-field behavioral lab trials, split by mating tactic ....................................................................................................... 88
4-7 Boxplots of average field fecal CORT split by mating tactic. .............................. 89
4-8 Anogenital distance split by mating tactic. .......................................................... 90
4-9 Effect of density on 75% kernel home range area, male residents only ............. 91
4-10 Effect of density on number of other males overlapping male resident home-ranges. ............................................................................................................... 92
4-11 Effect of density on number of females overlapping male resident’s home range .................................................................................................................. 93
4-12 Effect of density on average field fecal CORT .................................................... 94
4-13 AGD plotted against final field CORT ................................................................. 95
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4-14 Outlier individual in the three dimensions of pre-field fecal CORT (ng/mg sample), % mate overlap, and home-range area. ............................................... 96
4-15 Density, pre-field CORT, and trial effects on home range area .......................... 97
4-16 Density, pre-field CORT, and trial effects on % mate overlap ............................ 98
4-17 Density, pre-field CORT, and trial effects on % cumulative male overlap ........... 99
4-18 Open-field trial movement index plotted against number of males that overlap a given resident’s home range, broken down by density. ................................. 100
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LIST OF ABBREVIATIONS
AGD Anogenital distance
CORT Corticosterone
HPA AXIS Hypothalamic–pituitary–adrenal axis
RIA Radioimmunoassay
SD Standard deviation
SE Standard error
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
STRESS, CORTICOSTERONE, AND THE SOCIAL BEHAVIOR OF
THE PRAIRIE VOLE MICROTUS OCHROGASTER: AN INTEGRATIVE APPROACH
By
Dimitri Vincent Blondel
August 2013
Chair: Steven Phelps Cochair: Colette St. Mary Major: Zoology
Corticosterone (CORT) is a stress-related hormone found in vertebrates, and is
known to interact with behavior. In the socially monogamous prairie vole Microtus
ochrogaster, acute CORT has been shown to facilitate male social preference for
familiar females, and this has been described as facilitation of the monogamous pair
bond. To test whether this actually instead represents a coping strategy, I checked for
territoriality, a defining component of prairie vole monogamy. Using acute CORT
injections and behavioral trials, I found a facilitation of social preference, but I did not
find increased aggression, suggesting that this is indeed a coping behavior. Consistent
individual differences in stress-reactivity have been found in other taxa to lie along a
bold/shy continuum and to be correlated with CORT. To test whether prairie voles
exhibit the same patterns, I validated a non-invasive fecal CORT assay, and then used
behavioral trials to find that prairie vole behavior is consistent with a bold/shy
continuum. Bolder animals showed higher fecal CORT than shyer animals, which I
interpret as indicative of higher metabolism in bolder animals. I then tested whether
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these patterns are also observed in natural populations, and whether they are affected
by differences in population density. I first evaluated bold/shy behavior in the lab and
then created two different semi-natural enclosure density treatments in the field, high
(240/Ha; 0.1Ha) and low (80/Ha; 0.3Ha). I measured space use via radio telemetry,
and I periodically trapped out animals to measure fecal CORT. High-density males had
lower fecal CORT than low-density males, suggesting more stress in the low-density
condition. My field study confirmed my laboratory result that males exhibit correlated
suites of behaviors consistent with the bold/shy personality paradigm. Fecal CORT
collected prior to enclosure-release was significantly and positively correlated with
several different measures of bold behavior by males in the enclosures, including larger
home ranges and lower percent of home range overlapped by conspecifics. My results
are consistent with the emerging pace-of-life syndrome hypothesis that links higher
metabolism with bolder personality. I suggest that this suite of traits extends to a variety
of social behaviors including space-use in the field.
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CHAPTER 1 INTRODUCTION
Corticosterone (CORT), the main glucocorticoid in rodents, birds, reptiles and
amphibians, has broad effects on social behavior in a variety of taxa, from inhibiting
aggressive behaviors (Wingfield and Silverin, 1986; Yang and Wilczynski, 2003) to
promoting affiliative behaviors (DeVries et al., 1996; Stallings et al., 2001). In its role as
a product of the Hypothylamic-Pituitary-Adrenal (HPA) axis, it performs a variety of
functions including mobilization of glucose reserves, in both the context of normal body
functioning and as part of the vertebrate stress response. Due to the latter, CORT is
also commonly used as an index of stress, and thus can both influence social behaviors
and also be influenced by social behaviors. This dissertation documents a series of
studies on the relationship between CORT and the behavior of the socially
monogamous prairie vole Microtus ochrogaster, with special attention to individual
differences in stress-reactivity and to social behaviors in semi-natural environments.
Prairie voles are small rodents that are native to the grasslands of the central
United States and Canada, and have long been used as a model system for studies on
the evolution and ecology of social monogamy and its underlying mechanisms (Carter
et al., 1995; Getz et al., 1993; Young et al., 2011; Young and Wang, 2004). Monogamy
is a particularly interesting mating system because it is shared, along with humans, by
less than 3% of other mammals (Kleiman, 1977). As such, research on prairie voles
has led to advances in our understanding of monogamy in humans. For example, a
gene originally discovered to be related to pair-bonding in prairie voles (Winslow et al.,
1993; Young et al., 1999) has recently been found to be associated with pair-bonding in
humans (Walum et al., 2008) .
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If allowed to mate, most males and females will form an enduring and exclusive
social (but not mating) preference for each other, termed a pair bond. In prairie voles,
this is correlated with the onset of aggression towards unrelated conspecifics (i.e.
territoriality: (Insel et al., 1995), and shared male-female space use (Reichard, 2003).
DeVries et al. (1996) demonstrated that, in the absence of mating, acute stressors such
as a swim test caused males to form a selective preference for a familiar female. They
then observed the same effect using exogenous acute CORT injections. Based on this,
they argued that CORT facilitates pair bonding. However, in the absence of any other
data, we do not know if this an enduring preference that includes the territoriality found
to be associated with mating-initiated pair bonds in the lab (Insel et al., 1995) and
exclusive space use in the field (Getz et al., 1993; Ophir et al., 2008; Solomon and
Jacquot, 2002). Rather, this social preference could simply be a coping behavior in
response to stress, and physiologically evoked by the CORT produced when
experiencing such stress. In Chapter 2, I test whether territoriality, a critical correlate of
pair-bonds in prairie voles, was also facilitated by acute CORT injections. This
dissertation focused only on male prairie voles because the effects of CORT in prairie
voles are sexually dimorphic (females are LESS likely to establish a social preference
after exogenous acute CORT; DeVries et al., 1996), and studying both males and
females in the context of such different responses to CORT was beyond the scope of a
single dissertation.
The relationship between corticosterone, stress, and prairie vole social behavior
in a natural context is unexplored in the current literature. Lab-documented behaviors
may be influenced by lab artifacts that might not be relevant in a natural environment.
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Further, measuring stress hormone levels in naturally behaving populations provides a
more complete ecological and social picture and a better estimate of such fitness-
associated characteristics as mating opportunities and pairing behavior, while
concurrently being able to measure CORT as an index of stress level and as a metric of
individual differences in stress-reactivity. This would allow us to make inferences
related to both the mechanisms underlying natural variation in social behavior as well as
selective pressures that may have led to the evolution of current behavioral phenotypes.
In order to study these dynamics, CORT levels must be measured. Prairie vole CORT
levels are traditionally obtained via bleeding methods such as retro-orbital bleeding
(DeVries et al., 1995). This has several draw-backs as a means of measuring CORT.
As an invasive procedure, the bleeding is itself a stressor, and if the procedure is not
completed within 2 or 3 minutes, the handling-associated CORT surge will confound the
results (Good et al., 2003). Further, repeated measures would likely affect an
organism’s physiology and behavior; small animals such as voles are simply too small
to undergo repeated measures. Finally, as a point sampling method, the CORT level
measured could represent pulsatile release or stochastic recent events (Harper and
Austad, 2000). For example, there is a circadian rhythm in prairie vole CORT
production, and a point sampling method measuring a difference in CORT between 8
AM and 10 AM might simply reflect that circadian rhythm, rather than any
experimentally interesting variable. Or, a subject may have experienced an
uncontrollable and random event such as time-since-last-food or water, that would not
be experimentally relevant, yet would contribute to the CORT level of a sample. In the
first part of Chapter 3, I validate a novel, non-invasive method of measuring CORT in
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prairie voles: fecal CORT hormone assays. Each CORT measurement using this
method contains an averaged hormone level covering the previous several hours, which
is more representative of an individual’s general hormone status than traditional
bleeding methods.
In many other taxa, there exist correlated and consistent alternative behavioral
and physiological responses to stress (Careau et al., 2008; Koolhaas et al., 1999),
typically called alternative coping behaviors (Koolhaas et al., 1999). These alternative
behaviors are usually defined as two opposing categories of stress-reactivity, proactive
(“bold) and reactive (“shy” or “anxious”), and have been observed in a bimodal
distribution (typically artificially-selected lab strains) as well as a unimodal distribution in
a continuum from one extreme to the other (more typical of wild animals)(Koolhaas et
al., 1999). Stress response behaviors in prairie voles have never been characterized in
this context. Although considered a socially monogamous species, male prairie voles
also exhibit alternative mating tactics. Most males will pair with a female and become a
territorial “resident”. However, as many as 45% of males in a population are non-
territorial “wanderers” that exhibit larger home ranges than residents (Getz et al., 1993;
Ophir et al., 2008; Solomon and Jacquot, 2002). Since proactive and reactive coping
strategies in other taxa differ in their locomotive and exploratory behaviors (rats & mice:
Benus et al., 1987; singing mice: Crino et al., 2010), we hypothesized that male prairie
voles not only exhibit alternative coping strategies, but also that these individual
differences in stress-reactivity may be related to their alternative mating tactics. To first
test for the existence of alternative coping strategies, in the second part of Chapter 3, I
perform open-field behavioral trials on male prairie voles, characterizing their stress-
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response behavior and physiology, including their fecal CORT metabolites. I measure
the CORT using my validated fecal CORT hormone assay method.
Having characterized the basic stress-response behavior and physiology of
prairie voles, we then extend our focus to how these may be related to social behavior
and reproductive tactics in a natural environment. Any field study on prairie voles must
take into account their unusual population dynamics, in which they exhibit wide
fluctuations in population density, from near-population-crashes to as many as 640
individuals per ha (Getz et al., 1993). In other taxa, population density increase is
presumed to act as a stressor, and indeed has been shown to correlate with increased
CORT levels and evidence of increased agonistic interactions (Boonstra and Boag,
1992). However, the effect of population density on prairie vole CORT has never been
examined. Given that prairie voles have not only unusual social behavior and
population density fluctuations but also unusual CORT physiology (10 times the plasma
CORT levels of other rodents, and potentially a glucocorticoid-resistant species;
Hastings et al., 1999; Taymans et al., 1997; Devries, 2002; Ruscio et al., 2007), this
relationship between population density and CORT becomes particularly interesting,
and will not necessarily follow the same pattern observed in other taxa. In Chapter 4, I
manipulate population density in semi-natural enclosure experiments, and estimate the
relative influence of individual differences in stress-reactivity and population density on
male prairie vole space use, reproductive tactic and fecal CORT levels.
In summary, my research explores the relationship between and influences of
corticosterone, stress-reactivity, and population density in the context of the prairie vole
mating system and alternative reproductive tactics. I synthesize both previous lab
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findings and my own lab experiments with the ecologically relevant context of my
fieldwork. I make use of modern technology and validate novel methods while running
experiments that contribute not only to the fields of prairie vole and social monogamy
research, but also to the emerging (Reeder and Kramer, 2005) field of stress biology of
free-ranging mammals.
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CHAPTER 2 CORTICOSTERONE-FACILITATED SOCIAL PREFERENCE IN THE PRAIRIE VOLE:
MONOGAMOUS PAIR BOND OR COPING BEHAVIOR?
Background
One of the more interesting effects of glucocorticoid stress hormones, which
historically were first known for their metabolic and immune functions, is their influence
on an organism’s behavior. Such behavioral responses include freezing behavior,
which increases predator avoidance (rats, De Boer et al., 1990; chickens, Beuving et
al., 1989), and increased sympathy and alertness to infant cries, which facilitates
maternal care (humans, Stallings et al., 2001). Some behavioral responses to
glucocorticoids are considered coping behaviors, which are behaviors that allow
subjects to introduce some control (or even illusion of control) or predictability to their
immediate environment in response to a stressor (Gatchel et al., 1989; Sapolsky, 2002).
Coping behaviors are relatively common (Wechsler, 1995; Weiss, 1968), and if
effective, the coping behavior typically reduces physiological measures of stress and/or
removes the animal from an adverse situation (Wechsler, 1995).
Recent studies on the prairie vole, Microtus ochrogaster, have revealed
glucocorticoid-facilitated social preference behavior (DeVries et al., 1995; DeVries et al.,
1996). In the current study, we ask whether the preference behavior described by
DeVries represents a prairie vole-typical socially monogamous pair bond with its
associated suite of behaviors, or if it is instead restricted to a social affiliation that is a
form of coping behavior. Prairie voles are small rodents that inhabit the grasslands of
much of the North American Midwest, and they have long been used as a model system
for the study of social monogamy (Carter et al., 1995; Getz et al., 1993; Young et al.,
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2011; Young and Wang, 2004), a mating system that is rare in mammals (Kleiman,
1977) .
The defining characteristic of social monogamy is that a particular male and
female recognize each other and form an enduring and exclusive social (but not
necessarily sexual) attachment to each other after mating (Adkins-Regan, 2005; Carter
et al., 1995; Moller, 2003). This is referred to as a pair bond. Such social bonds cannot
be directly measured, but we can use related behavioral processes as an index of a
bond (Carter, 1998). The most commonly used indices of mutual attachment are
maintenance of proximity or voluntary contact with (Carter et al., 1995), and preference
for (Williams et al., 1992; Winslow et al., 1993), a bonded mate, on the part of both
mates. In prairie voles, attachment is associated with resident males and females living
at and defending the same nest against all conspecifics, and with both parents
contributing to the care of the young at the nest (Getz et al., 1993; Hofmann et al., 1984;
Reichard, 2003; Winslow et al., 1993) . The onset of mutual social attachment, parental
care and territorial aggression towards non-mates is referred to as becoming a prairie
vole “resident” (Getz et al., 1987; Insel et al., 1995; Solomon and Jacquot, 2002).
The onset of both social preference and resident aggression requires repeated
bouts of mating within an estrus cycle (Insel et al., 1995). In previous studies,
researchers observed that even in the absence of mating, stress facilitates social
preference formation in male (but not female) prairie voles (DeVries et al., 1996).
Specifically, DeVries observed that acute stressors such as a swim challenge results in
social preference formation (DeVries et al., 1996). Males, exposed to a stressor and
subsequently to a novel female, develop a social preference for that female, even in the
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absence of mating, whereas non-stressed males do not develop such a preference.
Further, they found that exogenous doses of corticosterone (CORT), a glucocorticoid,
also results in preference formation. Thus, CORT seems likely to mediate the influence
of environmental stressors on social preferences.
Although male preference for a familiar female is an important attribute of pair
bonding, a full pair bond requires a mutual social preference that is both selective and
enduring. In prairie voles, if the social preference induced by CORT is pair bonding,
then we would expect it to be accompanied by an increase in territorial aggression.
Thus, the onset of territorial aggression can also serve as a useful proxy for the onset of
an enduring bond in this species (Insel et al., 1995). If males develop territorial
behavior after a combination of CORT exposure and cohabitation with a novel female,
then stress could be considered to facilitate the full monogamous pair-bonding-
associated suite of behaviors.
Preference due to only coping behavior, on the other hand, would consist of a
selective preference but not necessarily an enduring one. More importantly, the
individual would not exhibit the territorial behavior associated with mated prairie voles.
Social affiliation as a coping response to stress is a well-researched phenomenon
(reviewed in House et al., 1988). More specifically, we also know that male prairie voles
socially affiliate with pups as a short-term coping behavior (Bales et al., 2006), which
suggests a similar process may be observed with this CORT-facilitated social
preference behavior. Thus, if social preference induced by CORT in male prairie voles
is due to a coping behavior to reduce the effects of the stressor, then males should not
become territorial after exposure to CORT. This is an important distinction, since a pair
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bond would represent a significant long-term life-history decision, whereas a social
contact coping response alone would likely be a more short-term response to the
stressor. Differentiating between these two hypotheses may help to better understand
the selective pressures that have favored the evolution of this unusual mating system.
We tested whether the effects of CORT on social preferences were sufficient to
induce residency by examining the effects of CORT and mating on the aggressive and
affiliative behaviors of males in the laboratory. We administered exogenous acute
CORT injections to unmated male voles, allowed them to choose between a familiar
(but unmated) female and an unfamiliar female, and then measured their territoriality in
the presence of a novel male intruder. If CORT facilitates the full pair bond and its
associated suite of behaviors, we expect to see both a social preference for the familiar
female and increased territorial behavior relative to control males. If only an affiliative
coping behavior is involved, we expect to see the social preference but no territorial
behavior. Finally, as a complementary series of experiments, we also tested whether
CORT itself inhibits aggression by separately injecting mated territorial males and
measuring their territorial behavior. If CORT inhibits territorial behavior, than we would
not necessarily have been able to detect territoriality in the earlier unmated
experiments. If CORT does not significantly inhibit territorial behavior, then any onset of
territoriality in our unmated experiments should be readily apparent.
Methods
Subject Animals
Study animals were descendants of wild-caught voles from Illinois and
Tennessee. All animals were lab born and raised, and were weaned at 21 days. Upon
weaning, voles were placed in same-sex sibling groups. Housing conditions have been
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known to affect CORT levels of prairie voles, with solo-housed animals exhibiting higher
levels of CORT (Ruscio et al., 2007); therefore, all animals used in this study were
socially housed. Prairie voles reach sexual maturity at 45 days (Mateo et al., 1994).
Only animals 45 days or older were used as subject animals. All procedures were
reviewed and approved by the University of Florida Institutional Animal Care and Use
Committee.
Treatments
We applied six treatments (Table 2-1): Unmated uninjected (N=14), unmated
vehicle (N=12), unmated CORT (N=12), mated uninjected (N=11), mated vehicle
(N=10), and mated CORT (N=10). The mated treatments tested whether CORT itself
has an effect on resident territorial aggression. Since the subjects were socially
housed, we maximized efficiency of animal use by using siblings in the experiment;
however, siblings were separated into different treatments to avoid confounding genetic
and treatment effects. Unmated and mated animals were run separately. The vehicle
was 20% propylene glycol in PBS. The CORT treatment dose was 200 ug.
Procedure
Unmated treatments
“Vehicle” and “CORT” males were first injected intraperitoneally, while
“uninjected” animals were given no injection and no injection-related handling, but were
otherwise exposed to the same procedures. The animals were then placed with 1/2 of
their old bedding in a novel cage with an unfamiliar female for six hours (after DeVries
et al., 1996). Food and water was available ad libitum. These trials were videotaped,
and if any mating occurred, the trial was excluded from our analysis; this occurred in
two trials.
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We constructed a Plexiglas preference arena of dimensions 51 cm high X 42 cm
wide X 51 cm long (Fig. 2-1). Clean bedding was provided. Half of the arena was
divided into two equal sized chambers with an opaque wall. Two females were placed
in the arena: the female from the six-hour cohabitation (“Familiar”) and another
unfamiliar female (“unfamiliar”). Each female was randomly placed into either chamber
B or C. Each female was affixed with a cable-tie collar linked to a chain that was in turn
attached via a ring and swivel to a horizontal rod running across the B and C chambers
at a height of 20 cm. Thus, the female was able to move around within her chamber
without restraint, but was unable to leave the chamber. Female voles attached in this
manner appear to have normal behaviors (grooming, sleeping, physical contact with a
male), and appear comfortable. Female prairie voles exhibit an induced estrus, which
requires two or three days of exposure to pheromones in the male’s urine. Females
used in these experiments had been housed in female-only sibling groups, and
therefore were not in estrus.
The male subject was placed in an opaque PVC tube at the center of Chamber A
and allowed to acclimate for two minutes. After two minutes, the PVC pipe was
removed and the subject male was completely free to move around all three chambers
of the preference arena. Their behavior was video-recorded for three hours, after which
the two females were returned to their prior sib-group housing. Between each trial,
arenas were emptied, cleaned with ethanol and water, and dried.
Following the preference trial, the subject male underwent a resident-intruder
trial. Subject males were placed back alone in their co-habitation cages. They were
presented with a novel intruder male for a period of five minutes. Trials were video-
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recorded; a slanted mirror above the cage allowed for both a top-bottom view and a side
view.
Mated treatments
Mated treatments were only tested for territorial aggression. Vehicle and CORT
males were first injected as in unmated treatments. They were then placed in their
original cages with their mates for 9 hours (in place of the 6 hour cohabitation + 3 hour
preference for unmated treatments), after which the resident-intruder trials were
administered. “Mated uninjected” animals were simply administered a resident-intruder
trial in their home cage (after mate and any pups were removed).
Data Analysis
Video tapes were scored using JWatcher event recorder software
(http://www.jwatcher.ucla.edu/). Multiple researchers scored the videos, each blind to
the treatments. Inter and intra-observer bias was tested during training, and was kept to
a maximum of 3% for the final data.
In preference trials we measured the time each male spent allogrooming
(repetitive manipulation of the fur of another animal, including licking, touching and
patting with paw) and in side-by-side physical contact with each of the females in the
arena. These are the most common affiliative behaviors exhibited by prairie voles. In
resident-intruder trials we measured latency to first aggressive behavior (lunge or
chase) and total frequency of lunges and chases on the part of the resident towards the
intruder. Lunges were defined as when one animal moves suddenly towards other
animal with open mouth. Chases were defined as a first animal following a second
animal closely and rapidly as the second animal moves away from the first. Alpha level
was set at 0.05.
27
Results
In preference trials, unmated CORT males spent significantly more time with the
familiar female than with the unfamiliar female (Fig. 2-2a; Wilcoxon Signed Rank, p =
0.02), but there were no differences in the time spent with familiar and unfamiliar
females for both of the control unmated male treatments (vehicle and uninjected). We
subtracted unfamiliar contact time from familiar contact time to estimate the strength of
preference within a treatment, so as to be able to test for significance across the three
treatments. Unmated CORT males exhibited significantly stronger familiar preferences
than did uninjected and vehicle treated males (Fig. 2-2B; Kruskal-Wallis, p = 0.04).
In resident-intruder trials, we calculated total subject aggression (combined
number of lunges and chases; Fig. 2- 3A). Within each breeding status category, there
was no significant total aggression difference between CORT, vehicle, and uninjected
animals (Kruskal-Wallis); however, there was a non-significant trend within both
breeding statuses in which the uninjected animals exhibited more total aggression than
the vehicle and CORT treatments. We also calculated latency to first lunge or chase
(Fig. 2-3B). Similar to total aggression, within each breeding status category, there was
no significant latency difference between CORT, vehicle, and uninjected animals
(Kruskal-Wallis). Also similar to the total aggression counts, within each breeding
status category, the uninjected animals exhibited greater aggression (in this case,
shorter latency) than the vehicle and CORT treatments of that breeding status, although
this was a non-significant trend. Importantly, in both unmated and mated groups, and
when looking at both total aggression and latency to aggression, CORT males were
neither more nor less aggressive than vehicle males.
28
Mated males were much more aggressive than unmated males (total aggression:
mated = 19.1 mean ± 2.6 standard error; unmated = 6.5 mean ± 1.3 standard error;
Mann-Whitney U, p = 0.0002). We analyzed across breeding statuses using
generalized linear models. For total aggression (Fig. 2-3A), we used a zero-inflated
negative binomial model, with breeding status (mated vs. unmated) and treatment
(uninjected, vehicle and CORT) as covariates. The model showed a significant
breeding status effect (p < 0.001; Z=-3.968) but no treatment effect and no interaction
effect. For latency to first lunge or chase (Fig. 2-3B), we used an exponential
distribution model with censored wait times (Cox and Oakes, 1984). Here we also
found the best model was with a breeding status covariate, also with a significant
breeding status effect (p = 0.027, Z=-2.211). No other effects were significant.
Discussion
Although experimental (unmated, CORT-injected) animals formed a familiar
preference (Fig. 2-2), they did not develop territorial behavior (Fig. 2-3). Total
aggressive behaviors were significantly lower than all of the mated positive controls.
Thus, the combination of a stressor and cohabitation with a female was sufficient to
facilitate social preference formation, but not sufficient to establish the territorial
aggression that typically develops along with the onset of partner preference in prairie
vole pair bonding. This suggests that the social preference formation is a coping
mechanism in response to stress, rather than a full socially monogamous pair bond.
An alternative interpretation of these results is that bonding-related territorial
behavior takes longer to develop than social preference. Aggression and affiliation
have different neuroendocrine pathways (Young and Wang, 2004; reviewed in Nelson
and Trainor, 2007), and may develop at different rates. The CORT/cohabitation
29
combination may have initiated the development of a full pair bond, but it may not have
been sufficiently long for territorial behavior to develop. We consider this an unlikely
alternative, since territoriality develops relatively fast after actual mating, with noticeable
aggression after just one hour of mating (Insel et al., 1995). However, we cannot
completely rule out this alternative.
We also had to consider whether CORT itself might have influenced prairie vole
aggressive behavior. In other taxa, CORT has a varying effect on aggressive behavior
depending on both species and context (Haller et al., 1997), sometimes facilitating
aggression (hamsters, Hayden-Hixson and Ferris, 1991; rats, Haller et al., 1998),
sometimes suppressing aggression (song sparrows, Wingfield and Silverin, 1986; anole
lizards, Yang and Wilczynski, 2003), and sometimes having no effect at all (Haller et al.,
1997). Prior to our study, no one had examined the influence of CORT on prairie vole
aggression. Thus, there was the possibility that the exogenous CORT injections
themselves might have inhibited aggressive behavior that might otherwise have been
observed. To test this, we also examined the effect of CORT on males that had a
mated partner, and thus had an established pair bond and the attendant territorial
behavior. Most importantly, our results showed that these mated, territorial males did
not show any significant difference in aggressive behaviors in response to CORT, when
compared with both a vehicle mated treatment and an uninjected mated treatment
(Figs. 2-3a & b, mated treatments). Although there were differences in experimental
design between mated and unmated subjects, we still found it useful to compare levels
of aggression between the two breeding statuses. The mated treatments showed more
30
aggressive behaviors than the unmated treatments, demonstrating that mated males
exhibited territoriality that was intact and measurable by our methods (Figs. 2-3A & B).
We should also note that for both latency and total aggression data, and for both
unmated and mated animals, the unmanipulated uninjected animals were slightly more
aggressive than the vehicle and CORT injected males. While this was a non-significant
difference, the pattern’s presence across these different aggression measures and
different breeding statuses merits some consideration. It could be that the experience
of the extra handling and the injection resulted in a slight elevation of CORT levels in
the vehicle animals, which was not enough to induce familiar preference formation, but
yet was enough to have a slight influence on aggressive behavior. We can conclude
that while CORT certainly does not facilitate prairie vole aggression in this context, it
might have a slight inhibitory effect that we were not able to detect at a statistically
significant level at our sample size. This is particularly relevant since this is the first
study to look at the effects of acute CORT on aggression in prairie voles. However,
note that even if a slight CORT-related inhibition exists, our experiment should have still
detected pair-bond-associated territoriality: the unmated CORT data would have been
closer to the positive territoriality controls (the mated treatments), even if slightly less
aggressive than the mated unmanipulated treatment, rather than our actual results that
showed no significant difference between unmated CORT and the other unmated
treatments, as well as being significantly less aggressive than all the positive controls,
even those that also were administered CORT (Fig. 2-3A).
Interpreting the social preference formation as a coping mechanism rather than a
fully intact pair bond would be consistent with other prairie vole studies. For example,
31
stressed male prairie voles show significantly more parental care behaviors, such as
increased kyphosis (arched-back huddling) and increased licking and grooming of pups,
and CORT levels are negatively correlated with these behaviors (Bales et al., 2006) .
Taken together, these various data on “familiar” female preference, territoriality, and
parental care suggest that in male prairie voles, social contact may be involved in the
regulation of stress responses. While under acute stress, individuals become
hypervigilant, and glucocorticoids in certain contexts can then act on the brain to
enhance memory formation and storage for emotionally salient events (Cahill and
McGaugh, 1998; McGaugh and Roozendaal, 2002; Meaney, 2001). This could be the
mechanism by which the time spent in social contact with a particular female during the
post-stressor cohabitation, while experiencing elevated CORT levels, leads to the social
preference for that female.
Our results are consistent within the larger literature on the relationship between
stress and affiliation. There is a long-established body of work on social support, and
evidence in many taxa, which has suggested that affiliative social contact can be
causally related to improvements in psychological and physiological functioning
(reviewed in House et al., 1988). Further, affiliative behavior can be closely linked with
glucocorticoids. For example, in rats, degree of maternal licking and grooming behavior
experienced as pups is correlated with basal CORT levels and CORT response to
stressors once those pups reach adulthood (Meaney, 2001). In turn, glucocorticoids
also appear to promote social contact in the context of maternal care: human
postpartum women have higher cortisol levels and also are more sympathetic to cries of
infants (Stallings et al., 2001).
32
To our knowledge, our familiar preference results are the first replication of
DeVries’s 1996 experiments. That is, in the absence of mating, a significant familiar
preference was established in CORT-injected animals, but not in vehicle-injected and
unmanipulated animals (Figs. 2-2a & b). One difference we found from DeVries was
that they used singly housed animals, which did not work for us. Our population was
sensitive to housing conditions to the extent that even unmanipulated singly-housed
individuals were mostly forming familiar preferences (Blondel, unpublished data), and
thus we used socially-housed animals, which when unmanipulated did not form a
significant familiar preference relative to unfamiliars, as expected. A potential sensitivity
to housing-conditions in the context of familiar preference formation is not surprising,
since singly-housed animals have been shown in other studies to exhibit higher levels of
CORT (Ruscio et al., 2007).
Even if the CORT-induced prairie vole social preference is related to coping
behavior rather than representing a full pair bond, it is still a particularly striking
behavioral response considering that prairie voles are thought by some to be a
glucocorticoid-resistant species (DeVries, 2002; Hastings et al., 1999; Ruscio et al.,
2007; Taymans et al., 1997) for various reasons including the fact that they exhibit 10
times the level of circulating CORT relative to other rodents (DeVries, 2002; Ruscio et
al., 2007). Yet, they are still sensitive to circadian cues and social stressors (reviewed
in DeVries, 2002), as well as exhibiting the induced preference response. Further, this
behavioral response to stress raises interesting questions about stress and pairing
behavior in the field, in a natural context. The role of stress on pairing and social
behavior in the wild remains unexplored in the current literature. Are individuals with a
33
higher baseline circulating CORT level more likely to eventually pair (even if this is
initially a coping response) than those with lower CORT levels? What are the fitness
consequences derived from these individual differences? Do individual differences in
stress-reactivity relate to how exclusively paired animals use space? How is pairing
behavior affected by environmental stressors such as increased agonistic interactions
due to increased population density? Our results also suggest directions for future
laboratory research looking more closely at the mechanisms of aggression in prairie
voles. In particular, there is the possibility of a slight inhibition of aggressive behavior in
response to CORT, in the context of a resident facing an unfamiliar intruder. Future
studies should examine this relationship in greater detail, looking at the influence of
acute CORT on prairie vole aggressive behaviors in a variety of contexts. By combining
studies on CORT in the field with genetic paternity analysis, we may also get a better
understanding of the fitness consequences of the influence of CORT and of stress on
behavior, and the role this may have played in the evolution of this mating system.
34
Table 2-1. Treatments for affiliation and territoriality trials
Treatment Purpose
Unmated uninjected Unmanipulated negative control for affiliation Unmated vehicle Vehicle negative control for affiliation Unmated CORT Detection of main treatment effect for both
affiliation and territoriality Mated uninjected Unmanipulated positive control for territoriality Mated vehicle Vehicle positive control for territoriality Mated CORT Test whether CORT inhibits territoriality
Figure 2-1. Preference arena. The wall between chambers B & C is opaque; front walls are clear. Females were restricted to chambers B & C. Males were allowed free movement within the arena.
35
Figure 2-2. Affiliative behavior. A) Time in minutes spent by resident male in affiliative
behavior (grooming and physical side-by-side contact with familiar and with unfamiliar). Trials lasted three hours. Error bars indicate standard error. Sample sizes for each treatment are indicated above each bar pair. Significance of P < 0.05 is indicated by asterisk. B) Box plots of time spent by subject male in affiliative behavior (grooming and side-by-side contact) with familiar minus affiliative behavior with unfamiliar. Non-zero values indicate familiar preference. Sample sizes are indicated above each plot. Significance of P < 0.05 is indicated by asterisk.
36
Figure 2-3. Aggressive behavior. A) Mean number of combined aggressive behaviors
(lunges and chases) during resident-intruder trial. Error bars indicate standard error. Sample sizes indicated below each treatment. There is no significant difference between treatments within each breeding status. B) Latency to first aggression (lunge or chase), during resident-intruder trial. Error bars indicate standard error. Sample sizes indicated below each treatment. There is no significant difference between treatments within each breeding status.
37
CHAPTER 3 STRESS COPING STRATEGIES AND FECAL CORTICOSTERONE ASSAY
VALIDATION IN THE PRARIE VOLE, MICROTUS OCHROGASTER: STRESS-REACTIVE INDIVIDUALS SHOW LESS, NOT MORE, GLUCOCORTICOIDS
Background
Since at least the 5th century BCE, when Hippocrates outlined his four
temperaments (Albores-Gallo et al., 2003), it has been known that there exists a high
degree of variation in the way individuals of a given species respond behaviorally and
physiologically to stress. This individual variation exists across a wide range of taxa
including rats (Cameron et al., 2005), birds (Silverin, 1998), fish (van Raaij et al., 1996),
and even octopi (Mather and Anderson, 1993). These individual differences are often
manifested as correlated suites of behavioral traits; the general patterns of trait
association and their implications have been variously studied as coping styles,
behavioural syndromes, personalities, temperaments, or consistent individual
differences (Biro and Stamps, 2010; Careau et al., 2008; Koolhaas et al., 1999; Sih et
al., 2004). In many taxa, coping styles can be characterized by two alternative styles:
proactive and reactive. Proactive individuals tend to be more active and aggressive,
and have lower circulating levels of stress-related hormones; reactive individuals are
less aggressive and tend to exhibit more freezing behavior and passive avoidance, and
have higher circulating stress-related hormones than proactive individuals (Koolhaas et
al., 1999). One of the goals of this study was to characterize the relationship between
behavior and stress hormone levels of the prairie vole, Microtus ochrogaster, in the
context of these divergent coping styles.
Prairie voles are small rodents that are native to the grasslands of the central
United States and Canada, and have long been used as a model system for studies on
38
the evolution and ecology of social monogamy and its underlying mechanisms (Carter
et al., 1995; Getz et al., 1993; Young et al., 2011; Young and Wang, 2004). Although
prairie voles as a species are considered socially monogamous, this is a facultative
monogamy (Waterman, 2007), and 4% to 45% of males in a given population may
exhibit a non-pairing alternative tactic called “wandering” (Getz et al., 1993; Ophir et al.,
2008; Solomon and Jacquot, 2002). These wandering males are non-territorial and
have significantly larger home ranges than the paired, territorial “resident” males
(Solomon and Jacquot, 2002). Since proactive and reactive coping strategies in other
taxa differ in their locomotive and exploratory behaviors (rats & mice: Benus et al.,
1987; singing mice: Crino et al., 2010), there is a potential that these alternative coping
strategies are related to prairie vole alternative mating tactics. As the first step in
investigating this larger question, herein we first characterize the anxiety-related and
exploratory behavior of male prairie voles, and then correlate these with levels of
corticosterone (CORT).
CORT is a stress-related glucocorticoid produced by the adrenal gland. There
exists between-individual variation in basal circulating levels in a variety of taxa (Bonier
et al., 2009), as well as within-individual variation in CORT on a daily basis in the form
of a circadian CORT pulse (in prairie voles: Taymans et al., 1997) . Such baseline
variation is further modified in the short term in response to acute changes in stressors
or glucose availability (Romero, 2004). In other taxa, including house mice, rats, pigs,
and chickens, proactive individuals tend to have lower CORT levels than reactive
individuals (reviewed in Koolhaas et al., 1999). However, proactive and reactive CORT
39
profiles have never been characterized in prairie voles. Thus, we measured prairie vole
CORT levels in conjunction with standard measures of anxiety-related behavior.
Traditionally in voles and other rodents, CORT has been measured by relatively
invasive methods such as retro-orbital bleeding (DeVries et al., 1995; Taymans et al.,
1997). There are several limitations to measuring CORT in this manner. Most
importantly, the bleeding is itself a stressor, and if the procedure is not completed within
2 or 3 minutes, the handling-associated CORT surge will confound the results (Good et
al., 2003). Further, as an invasive sample collection method, repeated measures would
likely affect an organism’s physiology and behavior. Finally, small animals such as
voles are simply too small to undergo repeated measures.
Therefore, our second goal in this study was to validate a non-invasive CORT
sampling method, and use this method to measure CORT levels that could be
correlated with the coping style behavioral profiles. Within the last 15-20 years, new
techniques have been developed that make it possible to measure glucocorticoids non-
invasively. This includes fecal corticoid assays, which measure glucocorticoid
metabolites, and have been shown to be accurate indicators of both average circulating
CORT and biologically meaningful changes in CORT levels in many other mammalian
taxa (wolves, Wasser et al., 1995; african wild dogs, Monfort et al., 1997; spotted
hyenas, Goymann et al., 1999; ground squirrels, Mateo and Cavigelli, 2005). This
method has also been validated in other small rodents such as house mice (Mus
musculus), deer mice (Peromyscus maniculatus), red-back voles (Clethrionomys
gapperi) (All three Harper and Austad, 2000), old-field mice (Peromyscus polionotus;
Good et al., 2003), and short-tailed singing mice (Scotinomys teguina; Crino et al.,
40
2010). To our knowledge, no one has yet validated this method in prairie voles.
Glucocorticoid metabolism can vary greatly between species (Palme et al., 2005), and
thus a validation must be performed prior to any experimental use of fecal CORT data
for a given species. As an important model system for the study of social monogamy,
validating a non-invasive method of measuring CORT in this taxon will facilitate new
prairie vole research opportunities both in lab and field. We should note that since this
method measures excreted glucocorticoid metabolites, and as such is subject to and
influenced by an organism’s metabolism, the results can sometimes be more
complicated to interpret. However, a validation such as we perform here allows us to
interpret subsequent prairie vole fecal CORT measurements with confidence, and
further, the fecal hormone assay method has considerable advantages over traditional
bleeding methods: beyond its non-invasive nature and more straightforward collection
procedure, each sample also contains an averaged hormone level covering the
previous several hours. This is more representative of an individual’s general hormone
exposure than the point sampling of bleeding methods, which conversely could
represent pulsatile release or stochastic recent events (Harper and Austad, 2000).
Thus, our validation will ultimately allow for a greater range of stress-related research
questions to be investigated in prairie voles, particularly for free-living animals in the
field.
Methods
Subject Animals
Study animals were descendants of wild-caught voles from Illinois and
Tennessee. All animals were lab born and raised, and were weaned at 21 days. Upon
weaning, voles were placed in same-sex sibling groups. Housing conditions have been
41
known to affect CORT levels of prairie voles, with solo-housed animals exhibiting higher
levels of CORT (Ruscio et al., 2007); therefore, all animals used in this study were
socially housed. Prairie voles reach sexual maturity at 45 days (Mateo et al., 1994);
only animals 45 days or older were used as subjects. All procedures were reviewed
and approved by the University of Florida Institutional Animal Care and Use Committee
in accordance with local, state and federal regulations to minimize pain and discomfort.
Fecal Hormone Assay Validation
Test for assay linearity
To biochemically validate our assay, we ran a test for parallelism (Harper and
Austad, 2000; Mateo and Cavigelli, 2005). This checks that assay values vary linearly
with hormone concentration (Heilmann et al., 2011) so that different dilution levels will
provide consistent results, and it is a standard test when validating a fecal hormone
assay for the first time in a new taxon (Beehner and McCann, 2008; Crino et al., 2010;
Harper and Austad, 2000; Heilmann et al., 2011; Mateo and Cavigelli, 2005;
Vasconcellos et al., 2011). We used a pooled sample collected from the fecal CORT
extractions from 16 animals, for a total of 93 different fecal samples that had been
collected from different individuals and at different times of day. Twenty-five microliters
were collected from each sample, resulting in a pooled sample of 2.325 mL. We
performed three separate serial dilutions, each one ranging from 1:2 to 1:2000, using
the steroid diluent provided with the RIA kit (MP Biomedicals, # 07120102 & #
07120103). We quantified CORT at each dilution level for each of the three replicates,
log-logit transformed the antibody binding curves, and used an analysis of covariance to
compare this with slope of the log-logit-transformed antibody binding curves from the
42
standards generated from stock solutions that were supplied with the RIA kits (Harper
and Austad, 2000; Mateo and Cavigelli, 2005).
Swim challenge
To determine whether our fecal assay could detect an acute stressor (Crino et
al., 2010; Harper and Austad, 2000; Mateo and Cavigelli, 2005), we conducted a swim
challenge. Prairie voles swim instinctively when placed in water, and swim challenges
are standard behavioral tests used in this species (Bosch et al., 2009; DeVries et al.,
1996; Grippo et al., 2012). Sixteen animals (eight male, eight female) were used in the
trial. Animals were removed from their sibling-housed cage and placed individually in
new cages with ad libitum food and water, and bedding. They were given 48 hours to
acclimate to their new cages prior to the challenge.
Eight animals (four male, four female) were randomly selected to be control
animals and the other eight (four male, four female) were treatment animals. Each
treatment animal was placed in a ten-gallon aquarium filled with 15 cm of 22°C water
for five minutes. This was repeated every 30 minutes for each treatment animal three
times, for a total of three 5-minute swim challenges spaced over 75 minutes.
After the swim challenge, animals were placed back in their cages. All animals
dried off and quickly resumed their normal behaviors post-swim-challenge. Fecal pellet
samples were periodically collected for the next 20 hours.
Samples were collected from each animal every 2-3 hours, but subjects were
monitored frequently enough that only fresh samples (within 15 minutes) were collected.
We collected as many pellets as the animal produced, up to a maximum of five pellets.
We discarded feces that were contaminated with urine (i.e., visibly wet and/or in a pool
43
of urine; Cavigelli et al., 2005). Samples were stored in polypropylene tubes in a -20°
C freezer. Samples were collected until 20 hours after the beginning of the swim
challenge.
In half of our subjects, we performed a second swim challenge after 48 hours.
The goal of this second challenge was to collect every pellet produced by every animal
so as to detect the beginning of the challenge-induced CORT surge; this provides
information about the gut passage time relative to the stressor and hence when the
CORT surge can first be detected. The challenge was identical to the prior challenge,
but this time four of the previous control animals (two male, two female) became
treatment animals, and four of the previous treatment animals (two male, two female)
became control animals; the other eight animals were not used for this part of the
experiment. Samples were then collected as soon as they were produced, but only
over the next 3.5 hours. The CORT data from these were also combined with the first
challenge to increase our statistical power; when several samples were collected within
the same 2-3 hour time window for a given individual, the CORT values were averaged.
We tested the CORT data for normality (Shapiro-Wilk Normality Test), and then
compared Swim and Control treatments within each time window.
Fecal CORT extraction and radioimmunoassays (RIAs):
Samples were freeze-dried and weighed. To minimize variation in sample weight
prior to extraction and radioimmunoassay, we combined dry pellets collected at the
same time to a weight of 15-20mg per sample. If the entire sample was less than 15
mg, we still used the sample; if there were still remaining pellets after we reached 20
mg, we left those extra pellets out of the analysis and stored them separately. Samples
44
were then placed in individual glass culture tubes with 1 mL 90% methanol solution.
Samples were homogenized with a spatula, and then agitated for 24 hours using a
Thermo Scientific Labquake rotator. Samples were then centrifuged at 2000 rpm and at
4°C for five minutes. We collected ~500 microliters of the supernatant from each
sample, and stored them in glass tubes (test for parallelism) or polypropylene tubes
(swim challenge, open-field trials) at -20°C until assayed. Fecal CORT was then
quantified using a corticosterone 125I-radioimmunoassay kit for mouse and rat serum
(MP Biomedicals, Solon, OH; catalog nos. 07120102 & 07120103), which have a
minimum detectable dose of 7.7 ng CORT/mL. To account for differences in fecal
sample mass, we divided the total CORT (ng/mL) by fecal mass (mg/mL). Our sample
collection, extraction and validation protocols included methods established by Harper
and Austad (2000), Mateo and Cavigelli (2005), and Crino et al. (2010). We achieved
our best results (e.g. consistently readable off the standard curve) using undiluted
extracts for the assays.
Open-Field Test
Open-field tests are a standard approach for measuring anxiety-related and
exploratory behavior in rodents (Crawley, 1985; Prut and Belzung, 2003), and have
successfully been used in previous prairie vole studies (Greenberg et al., 2012;
Olazabal and Young, 2005; Pan et al., 2009). A hard plastic arena was used, with 18
cm high walls that were sloping outward, a 73 cm diameter at the bottom of the walls,
and a 90 cm diameter at the top of the walls. An “inner zone” was marked with raised
pins in a circle 10 cm from the outer wall, such that the “inner zone” had a 63 cm
45
diameter. Bedding was placed on the floor of the arena, such that no floor was visible,
but the tops of the pins were still visible.
Twenty-four male subjects were used. Subject animals were placed in an
opaque chamber in the center of the arena, and given two minutes to acclimate, after
which the chamber was lifted and the animal was free to move about the test arena.
Trials lasted ten minutes, after which subjects were placed in a clean, empty cage, and
fecal samples were collected after the trial in the same manner as the Swim Challenge
documented above. Data from the validation analyses (below) indicate that fecal CORT
data reflect acute stressors after a 2-3hr time lag; thus our fecal CORT measures are
unlikely to have been influenced by the open-field experience. The arena was emptied
of bedding and wiped down with ethanol between each trial.
Video tapes were scored using JWatcher event recorder software
(http://www.jwatcher.ucla.edu/). We measured immobility (latency from the lifting of the
acclimation chamber to the first movement, recorded in seconds), time spent in each
zone, number of crosses from one zone to the other, number of jumps, and number of
posts. We also quantified the levels of CORT metabolites in the fecal samples via the
methods described above.
Results
Fecal Hormone Assay Validation
Test for assay linearity
Our three serial dilutions consisted of 10 samples each for a total of 30 samples.
All 30 samples were run in duplicate in a single radioimmunoassay, for a total of 164
tubes including standards and controls. Sixteen samples had CORT concentrations
that were either too high or too low to be read off the standard curve. The other 14
46
samples were readable. Intra-assay coefficient of variation (CV) for the 82 sets of
duplicate tubes was 4.35%.
Log-logit transformed curves of serially diluted fecal extracts were compared with
log-logit transformed standard curves in a test for parallelism (Fig. 3-1). The three
replicates of the three separate serial dilutions (1:4, 1:10, 1:20, 1:40, 1:100, 1:200) are
shown with replicates staggered for ease of viewing, but they all actually lie directly on
the trendline of the standard curve. The standard curve slope is y = -2.08x + 4.68, r2 =
0.996; the slope of the total combined fecal extracts is: y = -2.10x + 4.71, r2 = 0.993. A
grouped linear regression analysis of covariance between the three serial dilutions and
the standards showed that the common slope is significant ( p < 0.0001; DF = 1; VR =
2907.6) and the difference between slopes is not significant (p = 0.60; DF = 3; VR =
0.6).
Radioimmunoassays
The swim challenge samples and open-field samples came to a total of 169
biological samples, and were run together in duplicate over two radioimmunoassays, for
a total of 356 tubes including standards and controls. Samples were run with no
dilution. Although most samples were run in duplicate, 11 samples had only enough
extract for single RIA tubes. We ran a subset of the samples (n=9) in both assays to
create a correction factor for CORT values so that measurements could be
standardized and compared. Four samples out of the 169 were not used: one for being
too high CORT concentration to read off the standard curve, one for high variance
between duplicate tubes, and the other two as outliers for having abnormally high
CORT levels relative to the other samples (2.5 SD and 10.3 SD above the mean for the
two outliers; the next highest sample measurement after that, out of the 133 samples,
47
was only 1.0 SD above the CORT sample mean), with all of the above indicating
potential error during processing or contamination of samples.
Intra-assay average CV for the 178 sets of duplicate tubes was 1.86%. Inter-
assay average CV for nine overlapped samples that were run in both assays was
22.3%; the CORT measures from each assay for these nine overlapped samples were
significantly correlated (simple linear regression p < 0.0001; R 2 = 0.9931). We used the
resulting slope of y=0.663x+15.28 to convert samples from the second assay to the first
assay, prior to any further analysis.
Swim challenge
The CORT data were not normally distributed (Shapiro-Wilk, p<0.0001,
W=0.89082), so we used non-parametric tests to compare swim treatments with
controls for a given time period (Fig. 3-2). “Time of stressor” indicates the beginning of
the swim challenge for the first swim animal. The combined data from the swim
challenges (Fig. 3-2) showed a CORT surge in the swim treatment relative to the control
animals. This was a significant difference in the second post-stressor collection window
(p=0.005, one-tailed Mann-Whitney U Test). The first and third post-stressor collection
windows approached significance (p=0.070, p = 0.058, respectively; one-tailed Mann-
Whitney U Test). Note that when restricting the data to just the first swim challenge
and leaving out the second challenge data, we still found a significant difference in the
second post-stressor collection window (one-tailed Mann-Whitney U Test, p = 0.019).
Next we plotted the CORT titers of Swim Challenge animals against the exact
time since the beginning of the stressor for that animal (Fig. 3-3). From the beginning
of the swim challenge (beginning of first five-minute swim) to the end of the challenge
(end of third five-minute swim), the duration of the challenge period for each animal
48
ranged from 62 minutes to 77 minutes, with a mean of 68.3 ± 4.0 minutes standard
deviation. The CORT surge had already started by the time the first samples were
produced approximately 100 minutes post-beginning of swim challenge (Figs. 3-2 & 3-
3), and appears to peak approximately 200-300 minutes post-stressor-beginning,
depending on the animal (Fig. 3-3). However, there was individual variation in how
individuals responded to the Swim Challenge, with some individuals showing a more
dramatic CORT surge and other individuals showing a much smaller and gentler
increase over time (Fig. 3-3).
Open-field Trials
Posts, crosses, and jumps were all significantly positively correlated with each
other (Table 3-1; posts & crosses, r=0.580, p=0.0024; posts & jumps, r=0.421,
p=0.0399; cross & jumps, r=0.413, p=0.0440). Crosses and time spent in the inner zone
were also significantly positively correlated (r=0.599, p=0015). Posts and latency-to-
first-movement (“immobility”) were significantly negatively correlated (r=-0.454,
p=0.0283). None of the other variables were significantly correlated.
Only 12 of 24 animals produced fecal samples. One of those animals did not
have an immobility measurement due to video-recording error, so that animal was not
included in the CORT correlation analysis. Among those animals that defecated, CORT
was significantly negatively correlated with immobility (measured as latency to first
movement, Fig. 3-4; p = 0.015 simple linear regression; R² = 0.50). There were no
other significant correlations with other variables. Since only 11 of 23 animals produced
fecal samples and had complete behavioral data, we divided them into “defecators” and
“non-defecators” and compared the open-field behavioral measures (crosses, posts,
jumps, inner duration and immobility) of these two groups. There were no significant
49
differences between the two categories in any of our measures (Mann-Whitney U Test,
α = 0.05, all p > 0.35).
Discussion
Our major objectives in this study were to validate a fecal hormone assay that
could be used to non-invasively measure corticosterone hormones in prairie voles, to
characterize prairie vole behavior in the context of the open-field trial and the
proactive/reactive alternative coping style paradigm, and to use our validated fecal
hormone assay to correlate prairie vole fecal CORT with the open-field behavioral data.
We successfully validated the non-invasive method to measure prairie vole
CORT levels via fecal hormone assays. This was demonstrated by the test for
parallelism (Fig. 3-1) and the stressor-related CORT surge observed in the experimental
animals relative to the control animals (Fig. 3-2), the latter showing that a biologically
meaningful change in CORT levels in response to an acute stressor can be detected in
prairie vole fecal samples. This validation allows other prairie vole researchers to now
use this non-invasive method to measure CORT levels in both lab and field.
Researchers should ideally collect fecal samples relatively soon after removing a
prairie vole from its home cage (in a lab context) or after live-trapping (in a field context),
since fecal samples, while not as sensitive to recent acute stressors as plasma CORT
collection, will nonetheless show a CORT surge within 100 to 300 minutes (Fig. 3-3).
Animals left in traps for several hours may exhibit fecal CORT more indicative of the
trapping experience than of their pre-trapped environment.
The beginning of the CORT surge evident in Figs. 3-2 and 3-3 suggest a gut-
passage time of around 100-300 minutes (~2-3 hours). Gut-passage time has never
been formally measured in prairie voles, but prairie voles exhibit ultradian activity
50
rhythms of 2-4 hours (Calhoun, 1945; Madison, 1985) and digestion studies have
suggested that this is due to a digestive bottleneck limiting food intake, which forces
voles into short bouts of rest and feeding (Zynel and Wunder, 2002).
Of particular interest is the high degree of individual variation in response to the
CORT surges (Fig. 3-3). Some individuals show a dramatic increase in CORT levels,
while others show less of a response; note however that all individuals depicted in this
figure had an identical stressor (i.e. a swim challenge). Other species have been shown
to also have extensive between-individual variation in fecal CORT levels. For example,
house mice (Mus musculus) showed significant correlations between within-individual
fecal CORT levels in samples taken three months apart, in a constant environment,
while also showing extensive between-individual variation (ranging from ~10 to ~220
ng/g CORT); thus, individuals experiencing an identical environment could be
experiencing different levels of stress (Harper and Austad, 2000). Studies in other taxa
known to exhibit alternative coping styles also show similar between-individual variation
in HPA axis reactivity, specifically with proactive individuals tending to show weaker
CORT responses to stressors, and reactive individuals tending to show stronger CORT
responses (reviewed in Koolhaas et al., 1999). Thus, Fig. 3-3 adds further support to
our hypothesis that prairie voles exhibit alternative coping styles.
The behavioral analysis of our open-field trial data revealed correlations between
open-field exploratory behaviors, space use, and temporal characteristics, which
suggests that prairie voles exhibit similar alternative stress coping styles to those
documented in other taxa. Specifically, they are demonstrating correlated suites of
proactive-typical and reactive-typical behaviors. Although correlations among behaviors
51
were strong, the distributions of these behaviors were not bimodal; such continuous,
unimodal distributions have also been characterized in similar stress-reactivity studies in
other taxa (Brockmann, 2008; Koolhaas et al., 1999; Wilson et al., 1994).
The relationship between fecal CORT and immobility (time in seconds to first
movement) was contrary to the typical relationship between serum CORT and open-
field behavior. Freezing behavior and high serum CORT levels are typical of reactive
individuals (Koolhaas et al., 1999), yet, in our results lower fecal CORT levels were
associated with freezing behavior (Figs. 3-4). Since fecal hormone assays are a
relatively recent technological advance, these results may point to a previously
unexplored (e.g. the Koolhaas et al. 1999 extensive review consisted of 100% plasma
CORT studies and no fecal CORT data) aspect of the metabolism of stress and coping,
specifically, that proactive individuals may metabolize CORT more efficiently than
reactive individuals. There are relatively few studies that examine fecal CORT levels in
relation to open-field trials. Our results are consistent with a similar study in another
small mammal, singing mice, that found a significant positive correlation between fecal
CORT and a “proactive” behavioral principal component that was highly positively
loaded with jumps, posts and crosses and highly negatively loaded with latency to first
movement (Crino et al., 2010). Our data suggest a testable prediction that the CORT-
metabolizing enzyme Cyp3A may be more active in proactive individuals relative to
reactive individuals. A similar but broader relationship between metabolism and
coping styles has been recently proposed, but is still in the early stages of an emerging
theoretical framework, and is still relatively unexplored empirically (Biro and Stamps,
52
2010; Careau et al., 2008; Reale et al., 2010). Our data show support for this proposed
proximate link.
While open-field trials have been conducted on prairie voles prior to our study
(Greenberg et al., 2012; Martin, 2011; Olazabal and Young, 2005; Pan et al., 2009;
Yamamoto, 2009), most of these previous studies have been restricted to examining
anxiety-related and exploratory behavior across different treatment groups. These
studies found, for example, that individuals were more likely to exhibit anxious behavior
if they were socially isolated after weaning (Pan et al., 2009), separated from their pups
for longer rather than shorter time periods (Yamamoto, 2009), if they had provided
alloparental care to younger siblings (Greenberg et al., 2012), or if they had been
exposed to serotonin prior to birth (Martin, 2011) . Because these were limited to
comparing anxiety-related behavior across experimental treatment groups, it is not
possible to infer from the studies whether natural behavioral variation exists in an
unmanipulated population. The one exception is Olazabal and Young (2005), which
examined an unmanipulated population in the context of spontaneous maternal vs
infanticidal female behavior. They report on the varying proportions of females that
exhibit these different behaviors, across different labs and experiments (reviewed in
Olazabal and Young, 2005). They also find that maternal females exhibit more
proactive open-field behaviors relative to the more reactive infanticidal females. In
finding correlated anxiety-related behaviors that may be related to stress-coping styles,
their findings are consistent with our own. To our knowledge, ours is the only study that
has correlated prairie vole CORT with open field behavior.
53
Because of the extent of individual variation in physiological responses to stress
(Figs. 3-3 & 3-4), and the counter-intuitive CORT/behavior correlations in our open-field
trials, studies that measure fecal CORT must be designed carefully so as to differentiate
between individual differences in production of CORT in response to a stressor versus
individual differences in metabolism of CORT. Note that even if some individuals do
indeed metabolize CORT faster than others, CORT still remains a valid method of
assessing whether a particular individual has experienced a stressor, as observed in
our swim challenge experiment (Fig. 3-2).
In conclusion, we have shown variation in behavior and CORT in male prairie
voles that is consistent with alternative reactive and proactive coping styles. In future
studies we will examine whether there is any correlation between these alternative
coping styles and the alternative mating tactics of resident and wanderer males.
Further, our data suggest that stress-reactive individuals show lower, not higher, fecal
CORT levels, and this has generated a testable hypothesis that individuals with reactive
coping styles are less efficient at metabolizing CORT. This has important behavioral
and physiological consequences, since long-term presence of circulating CORT can
have negative health impacts that would influence an individual’s evolutionary fitness.
Finally, we have successfully validated a non-invasive method of measuring prairie vole
CORT levels, which will now allow other researchers greater flexibility in field and lab
experimental design, including the ability to take repeated CORT measurements with
fewer negative impacts on the health of the subject and on the integrity of the
experimental data.
54
Table 3-1. Open-Field Trial Correlations.
Correlation Sample Size P-value
Posts, Crosses 0.580 24 0.0024
Posts, InnerDuration 0.203 24 0.3451
Posts, Jumps 0.421 24 0.0399
Posts, Immobile -0.454 23 0.0283
Crosses, InnerDuration 0.599 24 0.0015
Crosses, Jumps 0.413 24 0.0440
Crosses, Immobile -0.223 23 0.3104
InnerDuration, Jumps 0.179 24 0.4075
InnerDuration, Immobile 0.067 23 0.7630
Jumps, Immobile -0.0147 23 0.5083
Behavioral results of open-field trials.
55
Figure 3-1. Log-logit transformed curves of serially diluted fecal extracts compared with
log-logit transformed standard curves. Fecal extracts were run in three replicates of three separate serial dilutions (1:4, 1:10, 1:20, 1:40, 1:100, 1:200) in the same assay. Replicates are staggered for ease of viewing, but lie directly on trendline of standard curve. Standard curve: y = -2.080x + 4.676, r2 = 0.996; Total fecal extracts: y = -2.101x + 4.710, r2 = 0.993.
56
Figure 3-2. Mean fecal CORT before and after the swim challenge. Error bars = ± 1
standard error; * = significance between swim and control, one-tailed Mann-Whitney U Test
57
Figure 3-3. Individual fecal CORT levels before and after the swim challenge, with each
line representing a different animal and each point representing a different sample.
58
Figure 3-4. Fecal CORT levels vs. immobility (time-to-first-movement) from open-field
trials. P = 0.015 (simple linear regression), R² = 0.4987. N = 11.
59
CHAPTER 4 CORTICOSTERONE, STRESS-REACTIVITY AND SOCIAL BEHAVIOR: EFFECTS
OF POPULATION DENSITY AND INDIVIDUAL DIFFERENCES AMONG MALE PRAIRIE VOLES
Background
Glucocorticoids are intimately linked to social behavior. They have been shown
to facilitate various forms of social affiliation such as increased sympathy to crying
babies in humans (Stallings et al., 2001) and establishment of a social preference in
prairie voles (Devries et al., 1996, Blondel et al., Chapter 2). They are also a reliable
index of social stress, such as an increase of glucocorticoids upon separation from a
mate, and a decline back to baseline levels upon reunion, as seen in some mammals
(Mendoza and Mason, 1986) and birds (Remage-Healey et al., 2003). Circulating
glucocorticoid levels can even correspond to social compatibility of pair-bonding, such
as tree shrews that show lower levels of glucocorticoids in pairs that interacted more
harmoniously than other pairs (von Holst, 1998). Individual differences in glucocorticoid
production are also related to alternative coping styles in a variety of species, with more
proactive, “bold” individuals typically exhibiting lower circulating levels, and more
reactive, “shy” individuals exhibiting higher circulating levels (Koolhaas et al., 1999).
While there are many examples of glucocorticoids both influencing and responding to
social contexts, we know little about how environmental stressors and individual
differences interact to shape social behavior in natural settings.
It appears that the socially monogamous prairie vole (Microtus ochrogaster) also
displays alternative coping styles (Blondel et al., Chapter 3), along a continuum from
more proactive individuals to more reactive individuals. This species also has well-
documented alternative reproductive tactics among males. Some males, called
60
“residents,” are territorial and live in a pair with a female, whereas others, called
“wanderers,” remain unpaired and are non-territorial (Getz et al., 1993; Ophir et al.,
2008; Solomon and Jacquot, 2002). We do not currently know whether prairie vole
alternative copying styles are related to their alternative reproductive tactics, nor do we
know what role glucocorticoids play in this social and mating system. In the laboratory,
acute CORT facilitates the formation of specific social preferences (Devries et al., 1996,
Blondel et al. Chapter 2). Although this CORT-induced affiliative behavior has been
previously suggested to reflect pair-bonding (DeVries et al., 1996), we found that this
effect does not extend to the full suite of socially monogamous behaviors that normally
accompany pair-bond formation, such as the onset of territoriality (Blondel et al.,
Chapter 2). How CORT might be correlated with various aspects of affiliation or
reproductive strategy in the field has remained unexplored in the literature.
Prairie voles are also known for their highly fluctuating population densities,
which can range from as low as 11/ha to as high as 640/ha (Getz et al., 1993). High
population density in other rodents has been positively correlated with higher CORT
levels and higher wounding levels (Boonstra and Boag, 1992), which suggests that
higher population density results in increased agonistic interactions and thus acts as a
stressor in those other species. Prairie vole CORT levels, while having been measured
in the lab in a variety of contexts (DeVries et al., 1996; Ruscio et al., 2007), have never
been measured in a natural environment.
To examine the complex interactions of environmental and individual differences,
we examined stress-reactivity in the lab, and patterns of space use and social behavior
61
in semi-natural environments that varied in population density. We asked two main
questions:
Are individual differences in stress-reactivity in prairie voles related to space use and reproductive tactic?
Specifically, are differences in fecal CORT and open-field trial behavior measured in the lab related to subsequent field behavior?
Does high prairie vole population density act as a stressor?
Specifically, will an increase in population density result in an increase in CORT levels, as seen in other taxa?
We used a non-invasive fecal CORT assay (Blondel et al., Chapter 3) to quantify CORT
levels. In order to characterize individual differences in stress-reactivity, we examined
fecal CORT and open-field behavior in the lab. We then randomly assigned individuals
to two replicate semi-natural enclosure field trials, each with high and low-density
treatments. In these treatments, we measured patterns of space use as well as field
fecal CORT from animals. Since our CORT measures are from fecal samples, there is
an added complexity of interpretation caused by the introduction of CORT metabolism
into the estimate of CORT. Thus, any inferences need to carefully consider not only
production of CORT but also individual differences in metabolism of CORT.
Methods
Experimental Design Overview
We measured stress-reactivity of male voles in the lab, via open-field behavioral
trials and lab fecal CORT levels. We then placed the same subject males in semi-
natural enclosures in the field for 19-24 days along with females, and measured their
space use and pairing patterns. We manipulated population density in these
enclosures, creating concurrently a high-density treatment for half of the animals and a
62
low-density treatment for the other half of the animals. At two points in the field trials we
collected field fecal CORT samples: mid-way and at the end. Finally, we took
morphometric measurements at the end of the study.
Subject Animals
Study animals were descendants of wild-caught voles from Illinois and
Tennessee. All animals were lab born and raised, and were weaned at 21 days. Upon
weaning, voles were placed in same-sex sibling groups. Housing conditions have been
known to affect CORT levels of prairie voles, with solo-housed animals exhibiting higher
levels of CORT (Ruscio et al., 2007); therefore, all animals used in this study were
socially housed. Prairie voles reach sexual maturity at 45 days (Mateo et al., 1994);
only animals 45 days or older were used as subjects. All procedures were reviewed
and approved by the University of Florida Institutional Animal Care and Use Committee
in accordance with local, state and federal regulations to minimize pain and discomfort.
Open-Field Test
Prior to the field trials, each male (n=24) was given an open-field trial using the
procedure described in Chapter 3, to measure the stress-reactivity of the animal. In
brief, this involves a ten-minute trial in an open arena during which time the subject is
free to move anywhere within the arena, and after which a fecal sample was collected.
Blondel et al. (Chapter 3) has shown that fecal CORT data reflect acute stressors after
a ~2-3hr time lag; thus, our fecal CORT measures are unlikely to have been influenced
by the open-field experience and should reflect individuals’ typical CORT levels.
63
Fecal CORT Extraction and Measurements
We quantified the levels of CORT metabolites in the fecal samples using the
procedure documented in Chapter 3. In brief, this involves a methanol extraction from
freeze-dried fecal samples, followed by a radioimmunoassay (RIA).
Semi-Natural Enclosures and Density Manipulation
We created the two population density treatments using semi-natural enclosures:
low-density (24 animals in 0.3 hectares = 80 animals/hectare) and high-density (24
animals in 0.1 hectares = 240 animals/hectare). These density treatments fall within the
range of the 11 to 624/hectare reported in wild prairie vole populations (Getz et al.,
1993). Our field site was located in Ava, Illinois, well within the natural geographic
range of prairie voles. The semi-natural enclosures consisted of four abutting
quadrants, with each quadrant measuring 27.4m X 36.6m, and thus ~0.1 hectares each
(Fig. 4-1). Each quadrant had a removable gate, allowing the quadrants to be
combined for up to 0.4 hectares total. Quadrant walls were composed of aluminum
flashing that extended 30cm below-ground (to prevent escape via-tunneling) and 60cm
above-ground. Surrounding the four quadrants was a buffer zone around which was a
second perimeter of flashing. Predation-prevention measures included aviary netting
covering the top and sides of the enclosure, snake-guards on the perimeter, a 2-cable
electrified fence directly above the outer perimeter, and meso-mammal-sized live-traps
placed in the buffer zone. Inside the enclosure were abundant tall grasses and sedges,
the preferred habitat of the prairie vole (Getz, 1985). Clover was planted to supplement
their mostly herbivorous diet (Batzli, 1985) . Although their water requirements are
typically met by water content of vegetation and by dew, we also provided two
agricultural water stations (designed for chickens) in each quadrant, which were kept
64
filled during trials and cleaned between trials. We mowed a 60cm buffer inwards from
the flashing, so as to discourage voles from approaching the flashing. The outer buffer
zone was also kept mowed regularly, and 20 live-traps were maintained in the outer
buffer zone to catch any voles that escaped the enclosed area. After building the
enclosures, they were exhaustively live-trapped to remove any previous wild rodent
residents.
We ran two 19-to-24-day field trials, with each trial consisting of 48 total animals:
24 males and 24 females. Previous studies have shown that adults in the wild usually
exist at a 1:1 sex ratio (Getz and Carter, 1980). Trial 1 was conducted in August
(“Summer” trial) and Trial 2 was conducted in October (“Fall” trial). Animals were
divided equally into the two different density treatments (Fig. 4-1), low-density (80
animals/hectare; 3 quadrants with relevant gates removed to allow travel between them)
and high-density (240 animals/hectare; 1 quadrant with all gates closed). The field trials
were conducted from July 31, 2011 to October 23, 2011. All animals were ear-tagged
and toe-clipped for identification (sometimes ear-tags can be lost), and affixed with
radio-transmitters. In the wild, male space-use is strongly influenced by female
residency (Getz and McGuire, 1993), with males dispersing until they find and join a
female at her nest. Thus, we released females first, and released males after a
minimum of two days, to more closely replicate the natural social environment. This
allowed females to establish territories and shelter prior to being confronted by the
mate-choice decisions of encountering novel males.
The location of animals was noted via radio-tracking from 1-3 times per day
every day, with a minimum of one hour between subsequent fixes for a given individual.
65
Tracking was accomplished using a randomized list of channels that varied with starting
animal and progression forwards or backwards through the list. Animals were tracked
at varying times of the day in order to avoid timetabling issues (Kenward, 2001), and
between one and three night fixes were performed per animal over the course of each
trial. Locations were estimated to within 30 cm. Radio-tracking was performed with a 3-
element yagi antenna via the homing method (Kenward, 2001), by walking a grid of
3mX3m cells. The interior of each cell was never traversed, in order to minimize
disturbance of the vole habitat. A minimum of 30 fixes per animal were collected for
each trial.
On days 8-13 of each trial, animals were live-trapped out and fecal samples were
collected. This was performed between dawn and 10:30 AM so as to minimize effects
of circadian rhythms on CORT. 192 Sherman live-traps (model LFAHD, 3x3.5x9") were
used. These were place in appropriate micro-habitats through-out the grid, and left
locked open and baited daily for a minimum of 5 days prior to any trap-out. Traps were
baited with sunflower seeds and a peanut-butter/oats mixture. During trap-outs, traps
were checked every 15 minutes so that we could calculate time-of-capture to within 15
minutes for use in fecal CORT analysis. Only fecal samples that were collected within
90 minutes of live-trap capture were used in fecal CORT analysis, as otherwise the
CORT levels present in the sample may reflect the trapping event (Blondel et al.,
Chapter 3). On days 19-24 of each trial, all animals were live-trapped out, fecal
samples were collected, and morphological measurements were taken. These included
tail-length, weight, ano-genital distance and foot-length. CORT was extracted and
measured from the fecal samples as described in Chapter 3.
66
Data Analysis
Open-field trials were scored manually for latency-to-first-movement, and using
jwatcher event recorder (http://www.jwatcher.ucla.edu/) for latency-to-first-cross,
number of crosses, and time spent in the inner (central) zone of the arena. Home
ranges from field enclosure space use data were calculated using the Ranges6 program
(http://www.anatrack.com) at the 75% kernel contour, which minimizes the distortion
caused by rare foraging events that would be otherwise apparent at higher percent
contours. Paired males (“residents”) were designated as those males who had a kernel
center within 15 feet of a female, and that female had the male as her closest kernel
center. If the kernel center was longer than this distance but the relative encounter rate
(male encountering a given female and that female encountering that male) was greater
than .50, and neither overlapped any other individuals, then they were still designated a
pair. There was only one instance of this, in a low-density quadrant. All other males
were designated unpaired (“wanderers”). Percent home range overlapped of a male
overlapped by his mate, number of overlaps of a male by other animals of each sex,
and percent of a male’s home range overlapped by animals of each sex were also
calculated using the 75% kernel contour, using the Ranges6 overlap analysis function.
Open-field measures (lab CORT values, open-field behavior), enclosure space-use
measures, field CORT values, and morphometric measurements were all analyzed
using ANOVAs and including effects of density, trial, and factorial interactions for all
predictive and response variables. A male body size index was created by average Z-
scores of hind foot length and tail length. Male anogenital distance (AGD) was
corrected for body size by taking residuals from a regression against body size index.
67
Results
CORT Quantification
The lab-collected open-field trial fecal samples and field-collected fecal samples
came to a total of 138 samples from 46 individuals, and were run together in duplicate
over two radioimmunoassays (RIAs). Each sample consisted of from 1 to 5 fecal pellets
that were produced at the same time by a given individual. We ran a subset of the
samples (n=40) in both assays to create an inter-assay correction factor for CORT
values so that measurements could be standardized and compared. Although most
samples were run in duplicate, five of the samples in this overlapped subset had only
enough extract for single RIA tubes for the second assay. All samples were run with no
dilution.
In total 382 tubes were assayed, including standards, controls, and samples run
in both assays. All samples produced readable CORT values off of the standard curve,
and were used in our analysis. Intra-assay average CV for the 185 sets of duplicate
tubes was 1.48%. Inter-assay average CV for the 40 overlapped samples that were run
in both assays was 22.1%; the CORT measures from each assay for these 40
overlapped samples were significantly correlated (simple linear regression p < 0.0001;
R 2 = 0.93). We used the resulting regression y=1.1258x+36.173 to convert samples
from the second assay to the first assay, prior to any further analysis.
We divided our CORT assay results by the original sample weight to account for
mass. However, even after this calculation, there was a significant effect of sample
mass on CORT (simple linear regression, p<0.0001, R2 = 0.28, Fig. 2A). This is a
common issue observed across taxa when performing methanol-based fecal CORT
extractions on samples less than 0.02g (Millspaugh and Washburn, 2004; Tempel and
68
Gutierrez, 2004). Due to the small size of prairie voles, most of our samples were
below this weight. Although the reason for such a sample mass effect is not known, it
has been suggested that the effect might be caused by a higher concentration of
methanol per unit mass during the extraction process only at those very small sample
masses (Millspaugh and Washburn, 2004). We corrected for this effect of mass by
setting a second order polynomial regression (because the effect was mostly at the
smaller sample weights; p<0.0001, R2 = 0.34, Fig. 4-2A), and using those residuals as
our CORT values, adding each residual to the overall mean of the original uncorrected
CORT values. There was no effect of sample mass on these new mass-corrected
CORT values (simple linear regression, not significant, Fig. 4-2B). All further references
to CORT will imply sample-mass-corrected CORT.
Open-Field Trials
Open-field trial CORT showed a negative correlation to latency-to-first-movement
(Fig. 4-3), which is the same pattern we saw in Chapter 3. This was a non-significant
trend (simple linear regression, 2-tailed p=0.075, R2 = 0.11). Latency-to-first movement
(seconds) was significantly positively correlated with latency-to-first-zone-cross (from
inner to outer arena zone; seconds) (simple linear regression, p<0.0001, R2 = 0.38).
Number of crosses between zones was significantly positively correlated with time spent
in the inner (central) arena zone (seconds) (simple linear regression, p=0.0015, R2 =
0.21). There were no other significant correlations between open-field trial measures.
We created a latency index by averaging the z-scores from latency-to-first-movement
and latency-to-first-cross. We created a movement index by averaging the z-scores
from number of crosses and time spent in inner zone.
69
Semi-Natural Enclosures
For space use in enclosures, we used 75% kernel contours for home ranges
(Fig. 4-4) and analyzed kernel centers and encounter rates to define 27 males as
residents (10 in Summer trial, 17 in Fall trial) and 6 males as wanderers (2 in Summer
trial, 4 in Fall trial). In the combined high density treatments, 83% (15) of males were
residents, and 17% (3) of males were wanderers. In the combined low density
treatments, 80% (12) of males were residents, and 20% (3) of males were wanderers.
We classified 37 females as residents (16 in Summer trial, 21 in Fall trial), and 7
females as wanderers (4 in Summer trial, 3 in Fall trial). In the combined high density
treatments, 94% (15) of females were residents, and 6% (1) of females were
wanderers. In the combined low density, 72% (15) of females were residents and 28%
(6) of females were wanderers. There were 11 male and 8 female mortalities in the
Summer trial, and one male and two female mortalities in the Fall trial. The Summer
trial mortalities are likely due to the record high temperatures in August 2011; there was
zero evidence of predation in any of the mortalities. All mortalities occurred within the
first few days of the beginning of the trial, and were thus unlikely to have had any
significant or confounding impact on the space use data of the surviving animals. As
expected, resident home ranges (mean 126 ±15SE m2) were significantly smaller than
wanderer home ranges (Fig. 4-5, mean 194 ±37SE m2; p= 0.035, one-tailed t-test,
Solomon and Jacquot, 2002; Ophir et al., 2008). Pre-field fecal CORT of residents was
lower than that of wanderers (Fig. 4-6; median resident = 11.3 ±0.7SE ng/mg sample,
median wanderer = 12.5 ±2.4SE ng/mg sample); when considering mating tactic, trial
and density as main effects, there was a significant mating tactic (p=0.04) and trial
effect (p=0.03) but no density effect (ANOVA). There was no significant difference
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between the average field fecal CORT of residents and wanders (Fig. 4-7, median
resident = 9.4 ±0.4SE ng/mg sample, median wanderer = 10.4 ±1.3SE ng/mg sample).
There was no significant difference between residents and wanderers for absolute and
size-corrected AGD (Fig. 4-8). Note that there was a non-significant trend for larger
resident AGD. Due to the relatively low number of wanderers in our dataset, unless
otherwise noted the remainder of the analyses are restricted to the residents.
The population density treatments had several significant effects on the space
use of resident males. High-density home ranges were significantly smaller than low-
density home ranges (Fig. 4-9; p=0.008, ANOVA with density and trial as effect; N=15
high-density (80/hectare), N=12 low-density (240/hectare); mean high area = 92 ±14SE
m2, mean low area = 169 ±23SE m2). The number of males that overlapped a given
resident male’s home range was significantly greater in the high-density treatment than
in the low-density treatment (Fig. 4-10; p=0.01, ANOVA with trial and density as effects);
N=15 high-density, 12 low-density; mean high = 2.5 ±0.3SE overlaps, mean low = 1.6
±0.3SE overlaps). Similarly, the number of females that overlapped a given male
resident’s home range was significantly more in the high-density treatment than in the
low-density treatment (Figure 4-11; p=0.03 with trial and density as effects); N=15 high-
density, 12 low-density; mean high = 3.2 ±0.3SE overlaps, mean low = 2.2 ±0.4SE
overlaps). Population density did not have any significant effect on cumulative percent
overlap by other males (i.e. percent of resident’s home range overlapped by each other
male, summed together), cumulative percent overlap by females excluding the mate,
and percent of a resident male’s home range overlapped by his mate.
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The average field-collected resident fecal CORT levels were significantly lower in
the high-density treatment than in the low-density treatment (Fig. 4-12; p=0.03, ANOVA
using trial and density as effects, N=10 high-density, 10 low-density; mean high = 8.2
±0.5SE ng/mg sample, mean low = 10.1 ±0.6SE ng/mg sample). End-of-trial field-
collected fecal CORT levels were significantly negatively correlated with anogenital
distance of all males (residents and wanderers), even after correction for body size
index (Fig. 4-13; all males; N = 22; absolute AGD, p=0.007, simple linear regression, R2
= 0.31; size-corrected AGD, p=0.007, R2 = 0.31, simple linear regression). Pre-field
CORT levels were not significantly higher than average field-collected fecal CORT
levels, but this trend was close to significance (p=0.051, unpaired t-test, N=18 pre-field,
20 field; mean pre-field = 10.8 ±0.7SE ng/mg sample, mean field = 9.2 ±0.4SE ng/mg
sample)
When regressing pre-field fecal CORT against space use measures, there was
one individual that was consistently an outlier. He is an outlier in at least the three
dimensions of pre-field CORT (2.4 SD below the mean), percent of a male’s home
range overlapped by his mate (0.65 SD below the mean), and home range area (0.61
SD above the mean). When looking at all three dimensions of these variables, this
individual clearly had different patterns than the rest of the resident males (Fig. 4-14).
He was the only resident male to have a low pre-field fecal CORT and a large home
range area (the others exhibited a positive correlation between pre-field fecal CORT
and home range area; Fig. 4-15B), and also the only resident male to have a low pre-
field fecal CORT and a low percent-mate-overlap (the others exhibited a negative
correlation between pre-field fecal CORT and percent mate overlap; Fig. 4-16B). Thus,
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we removed him from all of the pre-field fecal CORT/space use analyses, but still
showed the outlier in Figures 4-15 to 4-17. Pre-field fecal CORT had a significant
positive effect on home range area (Fig. 4-15B; p=0.04, ANOVA with pre-field fecal
CORT, trial and density as effects.) Pre-field fecal CORT also had a significant effect
on percent home range of the male overlapped by his mate, in this case a negative
relationship (Fig. 4-16B; p=0.03, ANOVA with pre-field fecal CORT, trial and density as
effects). When looking at cumulative percent home range overlapped by other males as
a response variable, there was a significant pre-field fecal CORT X density interaction
(Fig. 4-17b, p=0.04, ANOVA using CORT, density and trial as effects). Open-field trial
movement index for resident males was significantly negatively correlated with the
number of other males overlapping the resident’s home range (Fig. 4-18 p=0.04, R2 =
0.16, simple linear regression).
Discussion
We sought to investigate how environmental factors and individual differences in
stress-reactivity contribute to natural variation in social behavior. We manipulated
population density to investigate its consequences on stress, measured by fecal CORT,
and on social behavior, measured by space use. We found a significant effect of
population density on CORT levels, suggesting that low population density
environments are more stressful for prairie voles. To examine the influence of individual
stress-reactivity differences and basal fecal CORT levels, we measured fecal CORT
and stress-reactivity in the lab, and then compared these data to subsequent field social
behaviors inferred from patterns of space use. Pre-field fecal CORT levels predicted
individual differences in male resident space use in the field. Finally, we found that pre-
field CORT was more strongly related to field space use at lower population densities,
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including a density X CORT interaction on measures of exclusive space use. We
discuss our findings in more detail below.
Individual and Population Density Differences in Field Space-Use
At high densities, males had smaller home range size and had higher number of
both male and female overlaps (Figs. 4-9 to 4-11). It is interesting to note that the
percent a male overlapped with his mate did not change between density treatments,
demonstrating a stability of affiliative behavior with a mate even in the face of
dramatically changing population densities. However, high population density appears
to provide more opportunities for extra-pair mating, as the high-density treatment
resulted in nearly twice the average percent of non-mate overlap relative to the low-
density treatment (p=0.14). In the case of male-male interactions, high population
density resulted in a greater number of male overlaps, but the fraction of a male’s home
range overlapped by other males was not significantly different. This suggests that the
same-sex overlaps are at the peripheries of home ranges, which suggests a degree of
exclusive space use, generally considered a proxy for territoriality (Maher and Lott,
1995) and is consistent with what has been reported for most male prairie voles in both
the wild and in semi-natural enclosures (Getz et al., 1993; Ophir et al., 2008; Solomon
and Jacquot, 2002).
Our overall measures of residents and wanderers are consistent with what has
previously been reported in the literature for this prairie vole alternative mating tactic.
Residents had larger home ranges than wanderers, with both tactics exhibiting home
range areas similar to those reported in other studies (Ophir et al., 2008; Solomon and
Jacquot, 2002). We had 18% wanderers (these typically range from 4% to 40%; Getz et
al., 1993; Ophir et al., 2008; Solomon and Jacquot, 2002), and as reported by other
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researchers, this fraction of resident males was not influenced by population density
(Getz et al., 1993). Lastly, we replicated the finding that AGD was larger for residents
than for wanders (Ophir and DelBarco-Trillo, 2007).
Effects of Population Density on Fecal CORT
When interpreting differences between population density treatments, we are
interpreting fecal CORT as in index of stress, since this was an environmental treatment
difference. We found an unexpected effect of population density on fecal CORT levels
in the field. In other rodents, higher population density correlates with higher CORT and
higher wounding and scarring (Boonstra and Boag, 1992), presumably due to increased
agonistic interactions. However, we found that the low population density resident
males had significantly higher CORT than high population density residents.
Prairie voles are a highly social species for whom being housed alone rather than
with siblings is even enough to elevate CORT levels (Ruscio et al., 2007), and is a
species that often exists at high population densities (Getz et al., 1993). In the wild,
reported population densities range from 11/ha to 640/ha, with <100/ha being
considered “low-density” (our low-density treatment was 80/ha), and >100/ha being
considered “high-density” (our high-density treatment was 240/ha)(Getz et al., 1993).
Typically prairie vole populations boom during late-autumn-winter, likely due to
hibernation of their natural predators (Getz et al., 2006; Getz et al., 1990), and crash
during spring-early-autumn; over a period of 6 seasons, the average annual peak late-
autumn-winter density was approximately 270/ha (Getz et al., 1993). Other prairie vole
semi-natural enclosure studies have used population densities of 100/ha (Solomon and
Jacquot, 2002) and 200/ha (Ophir et al., 2008), although neither study measured CORT
levels.
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One explanation for our findings is that prairie voles are adapted socially to high
population densities, and living at lower population densities is experienced as a
stressor. Since low population density has been attributed to predation, it may also be
that low population densities are a cue of predator presence in the environment. Wild
prairie voles living at higher densities were found to have a longer life expectancy than
those at lower densities (Getz et al., 1997), also a surprising result, since the increased
agonistic interactions that likely occur at the higher densities would predict the opposite
pattern. Our findings of elevated CORT at lower densities might contribute to the
shorter life expectancy at lower densities via health issues related to chronic stress.
Note also that theory predicts that it would be more adaptive to pair at lower population
densities (Kokko and Rankin, 2006), and thus, even if the proportion of pairs do not
seem to change between densities, a higher CORT level at lower densities might
promote pair-ponding via an initial social preference (Blondel et al., Chapter 2), which
might have a temporal effect on pairing, i.e. faster time-to-pair. It is interesting that our
density results also suggest an Allee effect (a positive correlation between population
density and fitness; Allee, 1931, Stephens et al., 1999); measuring relative fitness of
high-density and low-density individuals would confirm if we are actually observing such
an effect.
In addition to differences between population densities, we found a significant
decrease in CORT levels between pre-field collected and field-collected samples
(p=0.05). This pattern suggests that despite the success researchers have had with lab
and breeding colonies of this species, prairie voles still experience captivity as a
stressor. Even in a relatively uncontrolled outdoor environment with wide fluctuations in
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temperature and resources, they still exhibited lower CORT levels in their naturally-
adapted environment relative to the consistent and controlled, yet artificial and
movement-constraining, environment of the lab.
Stress-Reactivity in Lab and Field
When considering our lab open-field trial fecal CORT data (all collected prior to
release in enclosures), we are interpreting fecal CORT as a measure of individual
differences in stress-reactivity, rather than as an index of stress in response to some
stressor treatment. This is because all of the individuals were subject to the identical
open-field trial environment, so any differences found in basal fecal CORT would
represent individual differences in stress-reactivity. In our laboratory open-field trials,
we found a negative relationship between latency index and fecal CORT (Fig. 4-3).
Although the trend is not significant (two-tailed p=.075), it is the same pattern we have
observed in other replicates of this experiment in which the pattern was significant
(Blondel et al., Chapter 3); further, this pattern is detectible even given the subtlety of
the measurement and the narrow variation of behaviors measured (mean 2.7 ±0.2SE -
seconds-to-first-movement). Since longer latencies are typically associated with more
reactive, “shy” individuals in the context of stress-reactivity trials (Koolhaas et al., 1999),
higher levels of CORT would be correlated with a more proactive, “bold” individual. A
positive relationship between fecal CORT and proactivity is in contrast to the negative
relationship often reported between plasma CORT levels and proactive behaviors
(Koolhaas et al., 1999). Fecal CORT, however, is dependent on both production and
metabolism (Palme et al., 2005). A similar relationship between proactive individuals
and higher fecal CORT has been observed in singing mice (Crino et al., 2010), which
was suggested to be due to differences in CORT metabolism by the enzyme Cyp3a.
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Indeed, there is emerging evidence in various taxa that personality and overall rates of
metabolism may be linked, with proactive individuals having higher metabolism (Biro
and Stamps, 2010; Careau et al., 2008; Reale et al., 2010).
In the field, those males that eventually became wanderers had higher pre-field
fecal CORT levels, particularly when including density as a main effect (Fig. 4-6). This
suggests that wanderers may tend to be more proactive individuals. Because most
males were residents, we focused our subsequent analysis of individual differences on
males who had pair-bonded. Among residents, higher fecal CORT levels in the lab were
associated with subsequent larger home range size and with less time spent with mate,
suggesting resident males with higher CORT might be more proactive, “bold”
individuals. Increases in home range size and reductions in time with mate would be
predicted to result in more extra-pair female encounters and potential extra-pair mating
opportunities. In contrast, resident males with lower CORT, smaller home range sizes,
and more time spent with mates might be more reactive, “shy” individuals. We would
thus predict more mate-guarding opportunities for these males. Overall, residents seem
to be more reactive than wanderers, and among paired males reactive residents also
seem to be more affiliative; both results are consistent with a “social coping” explanation
for CORT influences on affiliation (Blondel et al., Chapter 2).
When considering the variation in lab (latency-to-move) and field (home range
area, percent mate overlap and percent same-sex overlap) behaviors, we should
consider the potential influence of testosterone. This gonadal steroid hormone is
usually associated in other taxa with aggression and territoriality (Beach et al., 1972;
Edwards, 1969), which in turn is usually associated with the “bold”/proactive end of the
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stress-reactivity continuum (Koolhaas et al., 1999). Thus, it might be proposed that
variation in testosterone is driving the variation in our behavioral measures. However,
some data suggest that aggressive behavior in prairie voles seems to be independent of
testosterone. Adult prairie voles have 10X less circulating testosterone than mice and
rats and 4X less than other voles (Demas et al., 1999; Graham and Desjardins, 1980;
Klein et al., 1997) and castration does not inhibit prairie vole aggression (Demas et al.,
1999; Gaines et al., 1985); some strains of prairie voles do respond to testosterone
implants with increased aggression, although it has been hypothesized that this surge of
testosterone is mimicking a mating-related testosterone surge that initiates the onset of
territoriality as part of monogamous pair-bonding (Gaines et al., 1985). Given these
previous studies, we would predict that circulating levels of testosterone would not be
correlated with our various behavioral measures. Alternatively (but not mutually
exclusive), testosterone may be more important organizationally rather than
activationally in this species, and individual differences during development may
correlate with the other individual differences that we have documented.
The open-field movement index was significantly associated with the number of
other male home ranges that overlapped resident male home ranges in the field. More
active behaviors (number of crosses and time spent in the center of the arena in the
open field test) correlated with fewer males overlapped in the field. These more active
residents may be more aggressively territorial, in which case they would exhibit more
exclusive space use in the field home range patterns, since other males learn quickly to
avoid the more territorial residents. Proactivity is associated with aggressive behavior in
other rodent species (Koolhaas et al., 1999).
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It is important to note that the overall variation observed in both stress-reactivity
and field behavior is not bimodal in distribution, but rather represents a continuous
variation from one extreme to the other. This is not an unusual pattern in alternative
personalities among other taxa (Koolhaas et al., 1999; Wilson et al., 1994).
Interactions Between Population Density and Individual Differences
When pre-field CORT was included as a covariate, we found population density
and stress-reactivity could interact to shape social patterns in the field. In all such
cases (home range area, percent mate overlap, and percent male overlap), the
predictive power of pre-field CORT was always present in the low-density treatment,
and when it was observable in the high-density treatment as well, the correlation was
not as strong. Higher population densities seem to be more constraining on these
various social interaction patterns. In lower population densities, males that are more
proactive may have more flexibility to exhibit larger home ranges.
Anogenital Distance and Field CORT
We observed an interesting relationship between anogenital distance (AGD) and
field-collected CORT. Looking at all males (residents and wanders combined), the
significant negative correlation between field-collected CORT and AGD suggests a
pattern of individual differences, an AGD-related influence of social behavior on CORT,
or some combination of the two. It could simply be that males with larger AGD tend to
have lower CORT in general. It could also reflect a lowering of CORT levels due to
increased affiliative behavior from females, since females exhibit a preference for larger
AGD (Ophir and DelBarco-Trillo, 2007). Such a “calming” effect of affiliative behavior
on CORT has been seen in prairie voles in other contexts, such as the decrease in
CORT after affiliative behavior with pups (Bales et al., 2006) and females (DeVries et
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al., 1995) in the lab, as well as in other taxa when an individual is reunited with a mate
(Mendoza and Mason, 1986). Without further information it is not possible to
differentiate between the two alternative explanations. The morphological variation in
AGD is not likely to be mechanistically caused by differences in CORT, but rather is
more likely related to some combination of genetic influence and intrauterine exposure
to testosterone from neighboring fetuses of differing sexes, which can also result in
differences across a spectrum of physiological, morphological and behavioral traits
(Ryan and Vandenbergh, 2002).
Future Directions
Our results will be enhanced by a paternity analysis run on offspring from our
field trials. This will allow us to measure the relative fitness of individuals along the
bold/shy continuum of stress reactivity and space use that we documented in this study.
We predict a population density effect on fitness differences between bold and shy
individuals; since this variation has been maintained in the species over evolutionary
time, it is unlikely for only one of the extremes to be strongly selected for at all
population densities. We also plan to run quantitative PCR on Cyp3A (a CORT-
metabolizing enzyme), using liver samples from field trials, to measure relative CORT
metabolism in field individuals. This will allow us to test whether the higher fecal CORT
individuals do indeed have a higher metabolism than the lower CORT individuals. It will
also be interesting to examine how aggression fits into this system. We know that all
resident males are territorial; but, are proactive residents with larger home range sizes
and higher pre-field fecal CORT more aggressive than the more reactive residents with
smaller home ranges who spend more time with their mates? Future experiments on
aggression involving resident-intruder experiments in conjunction with fecal CORT and
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open-field trials and semi-natural enclosure experiments will help to clarify this. Finally,
we plan a further analysis of our existing data examining the temporal component of
pairing, by testing whether time-to-pair is similar or different at different population
densities, and whether this is correlated with individual differences in stress-reactivity.
We would predict a faster time-to-pair at lower population densities, given higher CORT
at lower densities, the affiliative effect of CORT on this species, and the adaptive
advantage of pairing at lower densities (Kokko and Rankin, 2006).
Summary
We find both population density and stress-reactivity can have powerful and
interacting influences on social behavior in natural settings. Somewhat surprisingly, low
population densities result in higher fecal CORT and a dramatic shift in many
dimensions of space use. Our data on individual differences indicate that male prairie
voles exhibit not only the alternative tactics of residents and wanderers, with wanderers
displaying larger home range sizes and no mate-guarding (Getz et al., 1993; Ophir et
al., 2008; Solomon and Jacquot, 2002), but also a continuum of variation within a tactic,
ranging from a higher investment in mate-guarding on one end to a higher investment in
exploratory behavior on the other end. As suggested by the pace-of-life syndrome
hypothesis (Reale et al., 2010), boldness, high metabolism, and high dispersal are
reflected in higher fecal CORT and larger HR size; these contrast with shyness, low
metabolism, and greater philopatry represented by residents with lower fecal CORT and
smaller HR size (Table 1). Together these results clarify the complex interactions of
environmental forces and individual differences on the interactions between stress and
complex social behavior. Lastly, this experiment serves to demonstrate the strength of
an integrative approach to the study of behavior.
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Table 4-1. Summary of lab and field Fecal CORT results.
HIGH Baseline Fecal CORT
LOW Baseline Fecal CORT
Lab latency Faster Slower
Personality interpretation Bold/Proactive Shy/Reactive
Metabolism interpretation High Low
Home range size Larger Smaller
Mate Overlap Less More
Male Overlap Less More
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Figure 4-1. Semi-natural enclosure facilities, demonstrating density treatments.
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Figure 4-2. CORT sample-mass effect. A) Effect of sample mass on corticosterone,
showing both simple linear regression (p<0.0001, R2 = 0.28) and 2nd order polynomial (p<0.0001, R2 = 0.34. B) Effect of sample mass on mass-corrected corticosterone, simple linear regression (ns).
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Figure 4-3. Open-field trial latency-to-first movement plotted against pre-field fecal
CORT. p=0.075, R2 = 0.11 (simple linear regression).
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Figure 4-4. Home ranges (75% kernel contours) for the high-density quadrant and for
one low-density quadrant from Trial 2 Dotted lines indicate males, solid lines indicate females. Kernel center (and likely nest site) is indicated by + for males, o for females. Where these would otherwise be overlapping, they have been staggered for visibility. Animals of the same color have been designated pairs (“residents”) and unique colors are unpaired (“wanderers”).
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Figure 4-5. Boxplots of 75% kernel home range area split by mating tactic. R= resident,
W= wanderer. p=.035, one-tailed t-test); N=27 residents, 6 wanderers; median resident = 108 m-squared, median wanderer = 223 m-squared.
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Figure 4-6. Boxplots of fecal CORT samples from pre-field behavioral lab trials, split by
mating tactic. R= resident, W= wanderer. Significant difference when Trial is also included as effect (p=.03, ANOVA); N=18 residents, 6 wanderers; median resident = 11.3 ng/mg sample, median wanderer = 12.5 ng/mg sample.
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Figure 4-7. Boxplots of average field fecal CORT split by mating tactic. R= resident, W=
wanderer. No significant difference (t-test; N=20 residents, 4 wanderers; median resident = 9.4 ng/mg sample, median wanderer = 10.4ng/mg sample.
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Figure 4-8. Anogenital distance split by mating tactic. A) Absolute (mm). B) Size-
corrected, residuals from regression against body size index (positive = masculinized). R= resident, W= wanderer. No significant difference (t-test; N=27 residents, 6 wanderers; median resident = 12.9mm, median wanderer = 11.7mm.
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Figure 4-9. Effect of density on 75% kernel home range area, male residents only. *
indicates significance (p=0.008; ANOVA with density and trial as effects); N=15 high (240/hectare), 12 low (80/hectare); mean high area = 92m-squared, mean low area = 169m-squared. SE indicated.
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Figure 4-10. Effect of density on number of other males overlapping male resident
home-ranges. * indicates significance (p=0.01; ANOVA with trial and density as effects); N=15 high (240/hectare), 12 low (80/hectare); mean high = 2.5 overlaps, mean low = 1.6 overlaps. SE indicated.
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Figure 4-11. Effect of density on number of females overlapping male resident’s home
range. * indicates significance (p=0.03 when Trial is included as effect; ANOVA); N=15 high (240/hectare), 12 low (80/hectare); mean high = 3.2 overlaps, mean low = 2.2 overlaps. SE indicated.
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Figure 4-12. Effect of density on average field fecal CORT. * indicates significance
(p=0.02, t Test); N=10 high (240/hectare), 10 low (80/hectare); mean high = 8.2 ng/mg sample, mean low = 10.1 ng/mg sample. SE indicated.
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Figure 4-13. AGD plotted against final field CORT. A) Absolute AGD (all males; N = 22).
R-squared = 0.31, p=0.007 (simple linear regression). B) AGD corrected for body size by regressing against body size index. R-squared = 0.31, p=0.007 (simple linear regression).
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Figure 4-14. Outlier individual in the three dimensions of pre-field fecal CORT (ng/mg
sample), % mate overlap, and home-range area. Non-parametric density contours (JMP).
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Figure 4-15. Density, pre-field CORT, and trial effects on home range area. A) Effect of
population density on home range area, broken down by trial. B) Pre-field Fecal CORT (ng/mg sample) regressed against home range area. Broken down by density. Pre-field fecal CORT has significant effect, p=0.04, ANOVA with pre-field fecal CORT, trial and density as effects.
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Figure 4-16. Density, pre-field CORT, and trial effects on % mate overlap. A) Effect of
population density on % mate overlap, broken down by trial. B) Pre-field Fecal CORT (ng/mg sample) regressed against % mate overlap. Broken down by density. Pre-field fecal CORT has significant effect, p=0.03, ANOVA with pre-field fecal CORT, trial and density as effects.
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Figure 4-17. Density, pre-field CORT, and trial effects on % cumulative male overlap.
A) Effect of population density on % cumulative male overlap, broken down by trial. B) Pre-field Fecal CORT (ng/mg sample) regressed against % male overlap. Broken down by density. Significant CORT X Density interaction, p=0.04, ANOVA using CORT, density and trial as effects.
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Figure 4-18. Open-field trial movement index plotted against number of males that
overlap a given resident’s home range, broken down by density. Overall correlation p=0.04, R2 = 0.16 (simple linear regression).
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CHAPTER 5 CONCLUSIONS
This dissertation was broadly motivated by previously unexplored questions
regarding role of the stress hormone corticosterone (CORT) and stress itself on the
well-documented social system (Getz et al., 1993) of the prairie vole Microtus
ochrogaster in a natural context. These questions included the influence of individual
stress-reactivity differences on reproductive tactic and space use in the field, whether
high population density acts as a stressor for these free-living populations, and what the
interactions between individual stress-reactivity differences and population density
might be. While prior studies have examined the role of CORT on prairie vole behavior
in the lab (DeVries et al., 1996; Ruscio et al., 2007), and other studies have examined
space use and pair-bonding behavior in the field (Getz et al., 1993; Ophir et al., 2008;
Solomon and Jacquot, 2002), none have focused on the relationship between the two.
To do so required an initial clarification of the effect of acute CORT on the prairie vole
pair-bond in a lab environment, a validation of a non-invasive method of measuring
prairie vole CORT, and finally a semi-natural enclosure experiment that included
population density manipulations, estimates of individual differences in stress-reactivity,
and quantification of CORT levels at various stages of the study.
First, I tested whether territoriality, an important correlate of pair-bonds in prairie
voles (Insel et al., 1995), was facilitated by acute CORT injections along with the
previously documented facilitation of social preference (Chapter 2). Although I was able
to replicate the social preference formation reported by DeVries et al. (1996), I found
that territoriality is not present in males that otherwise had formed a selective
preference. This is not likely due to any CORT-related suppression of aggression, since
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my experiments also showed that the territoriality of mated males is unaffected by
exogenous CORT. This suggests that CORT-injected males, by exhibiting a preference
for the familiar female, are exhibiting a coping behavior rather than a full pair-bond.
There is an analogous affiliative coping behavior in this species, where stressed males
exhibit increased pup grooming relative to unstressed males (Bales et al., 2006).
Alternatively, the mechanisms for development of monogamous territorial behavior
maybe be quite unrelated to social preference and may take longer to develop.
Next, I validated a non-invasive method of measuring CORT in prairie voles:
fecal CORT hormone assays (Chapter 3). I was able to demonstrate that an acute
stressor (in this case a swim challenge) elevates CORT metabolites in the feces, and
thus this method is able to detect biologically meaningful changes in circulating CORT
levels. This will be a useful alternative to traditional bleeding methods, particularly in
prairie vole field studies.
I then characterized the stress-reactivity behavior and physiology of the male
prairie vole in the context of alternative coping styles, using open-field behavior trials
and the newly validated fecal hormone assay (Chapter 3). Several open-field trial
behaviors were significantly correlated with each other. This is consistent with patterns
seen in other taxa consisting of correlated suites of alternative coping styles that vary
along a behavioral continuum from more proactive “bold” individuals to more reactive
“shy” individuals. In the coping style literature, proactive individuals tend to have lower
circulating CORT, and reactive individuals tend to have higher circulating CORT. When
looking at the fecal (not circulating) CORT of our animals, however, we observed the
reverse (yet statistically significant) pattern, with individuals that showed a short latency-
103
to-move (proactive) showing higher CORT levels, not lower. This was not unexpected,
however, as previous research has shown similar fecal CORT patterns in other small
mammal taxa (Crino et al., 2010). One interpretation of these findings is that proactive
individuals metabolize CORT at a higher rate. This is indeed predicted by the newly
emerging pace-of-life literature that links personality to metabolism, in particularly linking
higher metabolism to proactive individuals and lower metabolism to reactive individuals
(Biro and Stamps, 2010; Careau et al., 2008; Reale et al., 2010).
Finally, I applied the validated fecal CORT assay to a semi-natural enclosure
field experiment (Chapter 4). This field experiment asked whether the alternative
copying style behaviors documented in Chapter 3 were in any way related to male
prairie vole space use and reproductive tactic, whether increased population density
acted as a stressor, and whether there was any interaction between these individual
differences and differing social environments. I had predicted that higher population
densities would act as a stressor due to increased agonistic encounters, and that they
would result in higher CORT levels. In fact, the opposite pattern was revealed: the
lower population density animals had a significantly higher CORT level. There are a
few alternative explanations for this that we considered, which are not mutually
exclusive. Prairie vole population density cycles seem to be driven primarily by
predators, with population booms occurring during late autumn and winter months while
predators are hibernating. Since predation seems to drive population crashes (Getz et
al., 1990), a low population density might be an environmental cue to prairie voles that
predators may be prevalent, and CORT levels may be elevated in response. This
could also be evidence of an Allee-type effect in this species; fitness would need to be
104
measured in this case, as an Allee effect would predict higher fitness in the higher
population density trial. Finally, it may be that prairie voles are simply socially adapted
to a high-density environment, and despite most males having mates, low-density
environments are still perceived as stressors.
I also found that individual differences in stress-reactivity, specifically CORT
levels from fecal samples collected in the lab prior to placement in the outdoor
enclosures, had strong predictive power on male space use in the field. Residents had
lower pre-field CORT than wanderers. Within residents, individuals with higher pre-field
CORT levels had larger home ranges in the field, and their mate overlapped their home
range by a smaller percentage. The distribution was not bimodal, so there was a
continuum of resident behavior that trended from smaller home ranges and more mate
overlap, which would be predicted to provide more mate-guarding opportunities, to at
the other extreme larger home ranges with less mate overlap, which would be predicted
to provide more extra-pair female encounter rates. Further, the pre-field CORT showed
a negatively correlated trend with open-field trial latency, which was the same pattern
we observed with different animals in Chapter 3: higher CORT correlated with the
shorter latency in proactive animals. If we interpret the CORT levels as representing
higher metabolism for higher CORT, then these results fit the predictions of the pace-of-
life literature, which suggest positive correlations between high metabolism, high
dispersal, and boldness, and between low metabolism, high philopatry, and shyness
(Reale et al., 2010). When I looked at the interactions between population density and
stress-reactivity, the predictive power of stress-reactivity (pre-field CORT) was strongest
105
in the low population densities, suggesting that at the high population densities space
use is more constrained.
In conclusion, in this series of studies I used CORT as a measure of individual
stress-reactivity and as an index of environmental (social) stress, and was able to show
that individual differences play a large role in a given male’s space use in the field, both
in his potential interactions with his own mate and with extra-pair animals. If CORT
levels do indeed correlate with metabolic rate, this will represent an intriguing new
understanding of how metabolism and behavior are related. Finally, while I found that
acute CORT does not by itself facilitate the full suite of prairie vole monogamous
behaviors, instead facilitating a social preference as a likely coping behavior, it may still
promote pair bonding in the field under certain conditions; for example, in the low
population density treatment, individuals on average had higher CORT, and in such
social environments where potential mates might be more rare and harder to encounter
and predators might be more prevalent, a proclivity to pair might be adaptive. Taken
together, this set of studies demonstrates the strength of an integrative approach to
behavioral ecology. By taking lab data and methods, and applying them to a more
ecologically relevant context, we have been able to make valuable inferences regarding
the mechanisms underlying stress and social behavior in the field, and the potential
fitness implications of these patterns.
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BIOGRAPHICAL SKETCH
Dimitri Vincent Blondel was born in France to Linda and Pierre Blondel. He was
raised in Virginia, and graduated from Charlottesville High School in 1992. His
undergraduate education was at Duke University, Durham, North Carolina.
While at Duke, Dr. Blondel took a primate field biology course at the Duke
Primate Center where he studied the social behavior of free-ranging red-fronted lemurs
(Eulemur fulvus rufus). It was during this experience that he developed a keen interest
in field biology and animal behavior. This interest was further crystallized by a research
project on aggressive behavior among herded ostrich (Struthio camelus) during a
School for Field Studies undergraduate study abroad program at the Center for Wildlife
Management Studies, Kenya. Dr. Blondel graduated with distinction with the Bachelor
of Arts degree in biology and French in 1996.
After graduation, Dr. Blondel gained further experience in both laboratory and
field research while working as a research assistant to Dr. Richard B. Forward, Jr., at
the Duke Marine Laboratory in Beaufort, North Carolina. The research involved the
physiological ecology of the blue crab (Callinectes sapidus). Dr. Blondel subsequently
spent four years working on network management software for Lucent Technologies
(formerly Ascend Communications) in Bohemia, New York. He was able to continue
pursuing his interest in field research during this time by participating in a study on the
ecology and breeding biology of the laughing gull (Larus atricilla), under the supervision
of Dr. Kevin Brown, in the Jamaica Bay Wildlife Refuge, New York.
Dr. Blondel earned his Master of Science degree in the Department of Zoology at
the University of Florida, spending two summers in Panama studying the social system
of the long-tailed singing mouse Scotinomys xerampelinus, under the direction of Dr.
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Steven Phelps and Dr. H. Jane Brockmann. He performed his dissertation work as
described herein in the newly formed Department of Biology at the University of Florida,
co-advised by Dr. Steven Phelps and Dr. Colette St. Mary, and earned his Doctor of
Philosophy degree in 2013. Dr. Blondel is a member of the Animal Behavior Society
and the International Society for Behavioral Ecology.