Faculty of Science
STRESSED SNAKES: INVESTIGATING A POSSIBLE LINK BETWEEN BASELINE LEVELS
OF CORTICOSTERONE AND BODY CONDITION IN THE WESTERN RATTLESNAKE
(CROTALUS OREGANUS)
2019 | COLE RICHARD HOOPER

B.Sc. Honours thesis – Biology

STRESSED SNAKES: INVESTIGATING A POSSIBLE LINK BETWEEN BASELINE
LEVELS OF CORTICOSTERONE AND BODY CONDITION IN THE WESTERN
RATTLESNAKE (CROTALUS OREGANUS).
by
COLE RICHARD HOOPER
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF

BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENT OF BIOLOGICAL SCIENCES
(Animal Biology)

This thesis has been accepted as conforming to the required standards by:
Karl Larsen (Ph.D.), Thesis Supervisor, Dept. Natural Resource Sciences
Mark Rakobowchuk (Ph.D), Co-supervisor, Dept. Biological Sciences
James Sudhoff (DVM) Examining Committee member, Animal Health Technology
Program

Dated this 18th day of June, 2019, in Kamloops, British Columbia, Canada

© Cole Richard Hooper, 2019

ABSTRACT
Western Rattlesnakes (Crotalus oreganus) are vital members of ecological communities in
the arid regions of southern British Columbia. These areas are ecologically unique
compared to the rest of BC, but they are facing higher levels of agricultural and urban
development. Therefore, it is critical for us to understand how exactly the conversion of
natural habitat to a landscape dominated by vineyards, orchards and resorts will
impact wildlife. So far Rattlesnakes have been able to persist in areas with
anthropogenic disturbance, but previous research has shown that there may be hidden
effects on the health of individuals. Rattlesnakes near Osoyoos, BC living in areas with
abundant human disturbance exhibit relatively poor body condition when compared to
those living in undisturbed, natural habitats. Considering the process whereby stress
reduces body condition via tissue catabolism, I developed a prediction. I hypothesized
that habitat disturbance in Osoyoos is leading Western rattlesnakes (Crotalus oreganus)
to exhibit higher levels of baseline stress, causing a reduction in their body condition.
Throughout the summer of 2018, I collected sixty-eight blood samples from rattlesnakes
within the Osoyoos population and analyzed the baseline corticosterone (stress
hormone) content using enzyme-linked immunoassay kits. I found no difference in
baseline corticosterone levels between snakes living in natural habitats and those in
disturbed habitats. In addition I found no overarching relationship between baseline
concentrations of corticosterone and body condition among all the sampled
rattlesnakes. While these results do not agree with our original hypotheses, they still
provide an important insight into the stress ecology of the Western Rattlesnake in BC.
This work will serve as an important baseline for future stress related research on the
Osoyoos Rattlesnake population.
Thesis Co-Supervisor: Professor Dr. Karl Larsen
Thesis Co-Supervisor: Associate Professor Dr. Mark Rakobowchuk
ii

ACKOWLEDGEMENTS
Firstly, I would like to thank my two supervisors, Dr. Karl Larsen and Dr. Mark
Rakobowchuk. It was their encouragement and guidance that made this research such
an enjoyable and valuable learning experience. I am also extremely grateful for the
assistance from Dr. Christine Bishop at Environment and Climate Change Canada. In
addition, this work was partly financed by the UREAP scholarship, so thank you to the
committee for selecting me. This project involved countless hours in the field, so thank
you to everyone in the lab who was able to help, especially Jared Maida, Stephanie
Winton and Dana Eye. Lastly, a special thank you to my parents, whose unwavering
support throughout university is what really made all of this possible.

iii

TABLE OF CONTENTS
TITLE PAGE……………………………………………………………………………………...i
ABSTRACT…………………………………………………………………………………........ii
ACKNOWLEDGEMENTS…………………………………………………………………….iii
TABLE OF CONTENTS………………………………………………………………………..iv
LIST OF FIGURES……………………………………………………………………………….v
INTRODUCTION……………………………………………………………………………….1
METHODS……………………………………………………………………………………...13
Statistical analyses…………………………………………………………………….........15
RESULTS………………………………………………………………………………………..16
DISCUSSION…………………………………………………………………………………...20
LITERATURE CITED………………………………………………………………………….24

iv

LIST OF FIGURES
Figure 1. Map of the Osoyoos Indian Reserve (OIR) study site, with a legend denoting
the different disturbance regimes in the area.………………..……………………………..11
Figure 2. Boxplot comparing the body condition of snakes captured at the three
different sites. No significant difference in body condition was detected between these
groups (F=1.60 df=2 P=0.08)…………………………………………………………………...17
Figure 3. Boxplot of corticosterone concentrations (pg/mL) in blood samples collected
from snakes within the 3 different sites. No difference in corticosterone concentration
was seen between these groups (F=1.98 df=2 P=0.15……………...………………………..18
Figure 4. Linear regression between plasma corticosterone concentration (pg/mL) and
LOG body condition (F=1.11 df=1, P=0.30 R2=0.017). ………………………………………19

v

INTRODUCTION
Twenty-first century wildlife conservation is facing more scrutiny than ever,
driving increased pressure on wildlife researchers to identify the causal mechanisms
behind conservation issues to legislators, courts and land managers. One approach has
been conservation physiology, an emerging discipline (Wikelski and Cooke, 2006) that
uses physiological tools and techniques to look at the causal relationships affecting
organisms as they respond to changing environments and human disturbance.
Environmental disturbances of any kind, natural or anthropogenic, are major selective
forces (Wingfield and Romero 2011). As a result, animals have evolved a range of
responses to these stressors that are defined as adverse stimuli that invoke a stress
response, the cascading physiological and behavioural response by the individual.
The study of stress is one of the largest areas of research within conservation
physiology. Acutely, the stress response is a vital physiological process that enables
organisms to overcome immediate environmental challenges and alterations (Baker et
al. 2013). There are two main physiological branches in the response to stress: (1)
stimulation of the sympathetic nervous system (SNS) releases catecholamines, and (2)
stimulation of the hypothalamic-pituitary-adrenal axis (HPA) works to restore
homeostasis via a process known as allostasis (Sheriff et al. 2011). Through allostasis the
physiological systems of the body revert back to the normal dynamic consistency of
homeostasis (McEwen and Wingfield, 2003). McEwen and Wingfield developed a
model conceptualizing stress, made up of three key concepts: (i) “Allostasis is achieving
stability through change.”, (ii) allostatic loads represent “the result of the daily and
seasonal routines organisms have to obtain food and survive and extra energy needed
to migrate, molt, breed, etc.” and it is tolerated up to a limit, and (iii) allostatic overload,
where the organism can no longer cope with its condition, it is unable to exceed its
energetic demands. Under natural conditions it would be normal for an individual to
1

experience allostatic overload; at that point the stress response would then act to
mobilize energy reserves or change behaviour so that the animal returns to a regular
allostatic load. However, under certain conditions the stress response can become
chronically activated (Baker et al. 2013) and the individual becomes stuck in a state of
allostatic overload. This is an issue as the stress response evolved to solve temporary
issues and is not meant to be activated in the long term. This chronic condition can lead
to additional problems.
The benefits and detriments of stress are directly tied to the physiological
components that the response is derived from, namely the stimulation of the SNS and
HPA axis. The response from the SNS is almost instantaneous after interaction with a
stressor, and induces the secretion of norepinephrine and epinephrine (Reeder and
Kramer, 2005). Together these neurohormones increase heart rate, redirect blood flow
and make energy stores available by stimulating glycogenlolysis and lipolysis.
Stimulation of the HPA axis occurs simultaneously with the SNS, but through a
signalling cascade that eventually signals the production of glucocorticoids (GCs). The
primary GC produced varies depending on the taxa of interest and may be either
cortisol or corticosterone. During acute secretion GCs increase available energy through
gluconeogenesis and decrease glucose use, insulin sensitivity, and protein and fat
metabolism; all these functions help the organism counter a specific stressor. In the
short-term, elevated levels of GCs are critical in aiding an animal’s escape from a lifethreatening situation (Wingfield et al. 1998). However, if the stressor somehow becomes
chronic, prolonging the stress response, deleterious effects start to accumulate; these
include neuronal cell death, hyperglycemia, insulin resistance, muscle and bone
atrophy, hypertension, growth inhibition, and even immune system collapse. This
highlights the need to balance focus, between the positive and negative effects of stress.

2

For example, the implications that elevated levels of stress hormones may have
on reproductive function may be overlooked. While this relationship can be highly
variable between species there is a general pattern. There has been an historic view that
any stress is detrimental towards reproduction but moderately elevated levels of GCs
may be positively associated with reproduction through the mobilization of energy
stores (Moore and Jessop, 2003). While some recent evidence notes the benefits, the
historic point of view is well supported with an abundance of evidence showing a link
between highly elevated levels of GCs and the suppression of reproductive behaviours
and even total inhibition of reproduction. Just as any other life history trait, we must
consider both the costs and benefits associated with the stress response.
Previous research has shown that the intensity and duration of the stress
response is directly linked to the general health of the animal (Boonstra et al. 1998),
making it a valuable metric of study. In quantifying stress, researchers mainly examine
GC concentrations because they persists for minutes to hours (Wingfield and Cooke
2006), unlike the SNS response hormones. An important considerations in study design
is the origin of the sample. It is critical to understand exactly how stress is linked to the
sample type that you have chosen, as the relationship between the material and stress
will vary. Blood plasma (or serum) has been the sample type of choice for most stress
research for some time now. The GC concentration in blood plasma reveals the current
physiological state of the organism, i.e. what the current stress state of the organism is
at that moment in time (Sheriff et al. 2011). It is understood that there are three
components that may influence circulating GC content: (1) endogenous cycles
(circadian rhythms, seasonal variation), (2) prior acute environmental stressors (actual
or perceived; predator encounters, extreme weather, etc) and (3) chronic stressors
(predators, environmental conditions, etc). These factors must be kept in mind when
concentrations of GC are being examined in any analyses. Glucocorticoids in the blood
3

are metabolized in the liver, and then make their way into either the urine through the
kidneys, or the gut through the bile ducts. Afterwards the gluccocorticoid metabolites
(GCMs) do not follow a straightforward path: they may be reabsorbed by the
bloodstream within the gut, and undergo hepatic degradation (Klasing, 2005) or their
structure may be altered by the intestinal microflora (Eriksson and Gustafsson, 1970;
Sadoul and Geffroy, 2019). Both of these processes would then alter the ultimate GC
content of the excreta. While blood plasma provides information about the current
circulating concentration of GCs, it remains important to capture a longer term picture
of the stress the animal is experiencing.
Measuring chronic or long term stress can be accomplished using structures such
as hair or feathers because they have slow growth rates and incorporate GCs into their
structure during development. Thus, GCs accumulate over time in these tissues and
provide information about stress from the chronic perspective. Along with hair and
feathers it is possible to use other components of the integumentary system to
understand chronic stress, such as finger or toe nails (Frugé et al. 2018). In using these
materials, it is important to understand their exact growth rates, so that the time scale of
the GC accumulation can be accurately identified (Sheriff et al. 2011). Currently these
methods are still somewhat exploratory because the exact process by which GCs are
incorporated into these structures is not fully understood. Blood supply during
formation is thought to be the main source of GCs in integumentary structures, but they
could also be absorbed from the GC-containing secretions from a multitude of glands.
Sample selection and collection is only the first step of stress related study.
Following collection of tissues containing GCs, the next challenge becomes the
quantification of GC concentration within these various samples. Immunoassays are the
primary approach to quantifying GCs. The two most common versions utilized for this
purpose are radio-immunoassays (RAI) and enzyme immunoassays (EIA or ELISA).
4

These two approaches are similar in the fact that they are both sensitive, competitive
binding assays, both use an antibody specifically targeted against the GC. Radioimmunoassays use a radioactive isotope that generates a radioactive signal indicative of
the GC concentration. Alternatively, the EIA method uses an enzyme to create a
colorimetric signal that allows for quantification of GC concentration. In order to read
either of these signals, expensive equipment is required, including a scintillation or
gamma reader to register the radioactive signal of the RIA and/or a microplate reader to
read the optical densities of the colorimetric signal of the EIA. An increase in the
commercial availability of these assays in recent years has greatly enhanced the quality
and quantity of stress research on wildlife across the globe.
The vast majority of work on wildlife stress has focused on a small range of taxa.
Typically, researchers target the primary GC produced by their study species, as this is
the most biologically active. In general the primary GC is different between taxa: for
mammals, and fish it is cortisol, for amphibians, reptiles, and birds, corticosterone is
most abundant, but there can be intrataxonomic variation as well. (Sheriff et al. 2011).
There is a vast body of literature regarding cortisol based studies, since cortisol is the
primary GC produced in humans. Researchers have examined a wide range of subtopics relating to cortisol in humans, including: the effects cortisol has on various
components of the metabolism (Brillon et al. 1995; Khani and Tayek, 2001), the link
between cortisol and memory (Newcomer et al. 1999), how cortisol may influence
development (Goodyer et al. 2001) , understanding the heritability of factors controlling
cortisol production (Ising and Holsboer, 1996), even the sociological context of stress
and cortisol production (Taylor, 2012), and this is only to name a few of the major
avenues of study relating to cortisol in humans. While much of the existing research on
stress has been conducted historically with humans in mind, the interest surrounding
stress in animals is growing. We do already have a fairly complex understanding of
5

stress in some laboratory animals, such as muroid rodents and primates, but our
knowledge for most wildlife species is minimal at best (Reeder and Kramer, 2005).
In mammals there has been several key factors identified as determinants in GC
secretion. Coe and Levine (1995) revealed how the activity of the HPA axis and thus the
concentration of GCs varies with circadian and circannual rhythms with respect to
regulating energy balance as it relates to environmental conditions. This kind of
temporal variation is linked to stress in more ways than one, as other work has shown
the impact of seasonal variation of both biotic and abiotic factors greatly alters
adrenocortical activity (Boonstra et al. 2001). The cyclical activity of the HPA axis is
most notable in species at high latitudes, as these areas have the greatest disparity in
environmental conditions and resources between different times of year (Gustafason
and Belt, 1981). While these temporal differences in GC secretion are generally shared
across most animals, there is significant variation between species, and even intraspecific sex differences that should be considered (Reeder and Kramer, 2005). With a
growing understanding of the processes underlying stress and the stress response,
research is moving towards more functional conservation based questions. The
extensive human influence on the planet is behind a lot of these questions, such as how
pollution might be altering the stress response of mammals (Oskam et al. 2005), how
high levels of tourism may increase the stress of local mammals (Zwijacz-Kozica et al.
2013), and the link between habitat disturbance and increased cortisol levels in other
mammalian species (Jaimez et al. 2012). Many of the same results seen for mammalian
species have also been described in the other primary cortisol producing group, the
fishes.
Many researchers have examined cortisol in fishes, likely due to the commercial
value of fisheries. Within this context many of the research questions relate to how
fishery practices or conditions could influence cortisol levels in fish, such as over6

crowding, confinement, and handling (Montero et al. 1999; Pankhurst and Sharples,
1992; Strange and Schreck, 1978), all of which were found to increase the stress levels of
fish, potentially impacting tissue quality. While an abundance of funding from fishing
industries supports a great deal of this direct fishery related research, there is other
work looking at cortisol in natural populations of fish. The conservation theme is
carried over here, especially in the context of anthropogenic factors relating to stress,
including increases in cortisol secretion in response to ship noise (Wysocki et al. 2006),
impaired or reduced cortisol production in fish exposed to environmental pollutants
(Hontella et al. 1992), and how habitat degradation may lead to increased cortisol levels
(Hasler et al. 2015). Similar to mammals, temporal variation has also been noted as a
factor in altering cortisol production, specifically in terms of seasonal and daily timing
(Thorpe et al. 1987), as well as points in the reproductive cycle (Wingfield and Grimm,
1977). Most of the research on stress within the different taxonomic groups follows
similar overarching themes, but the extent of more specific questions varies a great deal.
This variation in specific questions is sometimes linked to the actual GC that is being
examined.
Unlike the mammals and the fishes, corticosterone, not cortisol is the primary
biologically active GC amongst bird species (Sheriff et al. 2011). Although the specific
GC is different, the causes and ultimate effects of stress do not change much despite the
active molecule being different. Just like the other taxa, many avian studies have
examined the link between temporal variation and corticosterone secretion (Romero et
al. 1998; Romero and Remage-Healey, 2000). Other environmental factors that can
activate a stress response have also been identified and these include, but are not
limited to harsh weather (Romero et al. 2000), urbanization (Fokidis et al. 2009), and
even habitat quality (Marra and Holberton, 1998). Extensive work has even shown that
corticosterone may play some role in influencing the life history, as species with higher
7

levels of stress induced corticosterone were found to have higher annual survival rates
(Hau et al. 2010). Two specific studies conducted on birds were part of the foundation
for this Honours thesis. The first found that baseline corticosterone levels rose in
association with a decline in body condition throughout the breeding season in Blacklegged kittiwakes (Rissa tridactyla)(Kitaysky et al. 1999). The other showed that
corticosterone secretion in American redstarts (Setophaga ruticilla) differed between
habitat types, and this effect was compounded in the spring when the habitat types
differed the most in quality (Mara and Holberton, 1998). These studies are especially
relevant since birds and herpetiles both produce the same main GC.
Amphibians and reptiles are the other vertebrate groups that also primarily
secrete corticosterone (Sheriff et al. 2011). As mentioned the foundation for our
understanding of stress in wildlife has been built on the study of endotherms, like
mammals and birds (Claunch et al. 2017). In general, much less research has examined
stress ecology in amphibians and reptiles, possibly as a result of the relatively slow or
variable metabolisms of ectotherms such as amphibians and reptiles. The rates of
physiological processes and reactions are dictated by body temperature, so when body
temperature is variable it becomes difficult to interpret physiology. Some of the work
that has been done has actually identified differences between amphibians and reptiles,
in terms of their corticosterone secretion. Amphibians appear to be more sensitive to
certain geographic variation, their baseline levels of corticosterone are related positively
to latitude, and negatively to elevation (Eikenaar et al. 2012). The reptiles exhibit neither
of these relationships. While some differences are apparent, many of the underlying
factors influencing stress in the other groups are shared, including seasonal and daily
variation (Pancak and Taylor, 1983; Tyrrell and Cree, 1998), environmental conditions
(Cash and Holberton, 2005), and habitat quality (Janin et al. 2011). Recently Lind et al.
(2018) has compiled and listed a number of studies looking at the relationship between
8

body condition and corticosterone in other species of snakes. From studies in that list,
no consistent pattern has been discovered, with results varying between a negative
relationship (Moore et al. 2000: Palacios et al. 2012) and no relationship (Holding et al.
2014; Lind and Beaupre, 2015; Lutterschmidt et al. 2009) between corticosterone and
body condition. Several interesting observations can be made when examining the
differences in the studies on that list; the only other study on a fellow northern species,
the Common garter snake (Thamnonphis sirtalis) found no significant relationship
between body condition and corticosterone (Dayger et al. 2013). In addition the only
studies comparing body condition and corticosterone in the Crotalus genus twice found
that there was no significant relationship between the two variables in Timber
rattlesnakes (Crotalus horridus) (Lind and Beaupre, 2015; Lutterschmidt et al. 2009). A
comparison between body condition and corticosterone is notably absent for Crotalus
oreganus. This leads in to my study species, the northern most population of the
Western rattlesnake (Crotalus oreganus).
The Western Rattlesnake (Crotalus oreganus) in British Columbia is inhabiting the
northern-most limit of its range in North America. As ectotherms their daily and
seasonal activity is dictated by the weather (Gregory, 2007). In Canada the winter
weather is far too cold to permit above ground activity, so snakes hibernate for a large
portion of the year. By limiting the time available for growth, courting and mating, the
Canadian climate has serious effects on the population ecology and life-histories of
these animals. In addition to the natural challenges imposed by life at higher latitudes,
various anthropogenic threats are placing additional pressure on these species.
Lesbarrères et al. (2014) identified the main anthropogenic impacts facing northern
herpetiles as habitat loss and fragmentation, roads, pesticides and other contamination,
infectious diseases and climate change. Among these habitat loss and fragmentation
have been considered the most serious threats to all herpetofauna in northern
9

environments. This is especially prevalent in British Columbia where the Western
Rattlesnake (Crotalus oreganus) occupies arid grasslands and riparian areas, as these
land types are the most desirable for agricultural and urban development (COSEWIC,
2015). As of 2005, approximately 16.1% of all grassland habitat in BC has been lost, but
this effect is even more pronounced in the Okanagan where sub-regions have seen
amounts of land conversion as high as 20-28% percent.
My study population occured at the southernmost tip of the Okanagan valley,
adjacent to the American border near the town of Osoyoos, BC (49°01′56″N
119°28′05″W). The study site itself is located on the east side of Osoyoos Lake, on
Osoyoos Indian band land, an extremely hot and arid area. This site offered a
contrasting environment where I was able to compare snakes from the same population
that were living in either disturbed or undisturbed habitats. The property contained a
section of extensive natural shrub steppe habitat known as the north desert, and an area
of high anthropogenic disturbance in the southern portion. The disturbed resort area
had a variety of different disturbance regimes. The complex included a golf course,
hotel, condominiums, winery, interpretive centre, campground and several roads. In
addition, several key components of the resort were surrounded by a thin gauge wire
fence, built to exclude snakes from these areas. This in itself was an interesting
component of the overall anthropogenic disturbance in the area, since this physical
barrier blocked the normal migration route of snakes in the area (Maida, 2018).

10

Figure 1. Map of the Osoyoos Indian Reserve (OIR) study site, with a legend
denoting the different disturbance regimes in the area.

The site is the subject of a long-running snake research program (>12 yrs) that
provides the background for this thesis. Lomas et al. (2015) examined the effects of
11

anthropogenic disturbance on this population. They used a disturbance ranking system
that separated snakes into disturbance categories based on the straight-line distance to
the nearest disturbance. Western rattlesnakes (Crotalus oreganus) in the higher
disturbance categories (closer distance to disturbance) displayed a lower body
condition than those in the lower disturbance categories. I used a simplified version of
this system where I separated snake captures into three primary areas: (1) the north
desert, where the distance to disturbance is always far (capture location is at the
minimum 250 meters to the nearest disturbance) (2) the snake fence, where the distance
to disturbance is very close (capture distance always less than 3 meters to the fence) (3)
The resort, where the distance to disturbance is short but variable (capture location
between 0-150m to disturbance). Data from small mammal monitoring in the area
found that there was not a significant difference in small mammal numbers or densities
between the disturbed and undisturbed habitats (Maida, 2018). If there is no implicit
difference in food availability, then examining other factors causing this reduction in
body condition remains an important area of research.
I proposed that physiological stress may be playing an underlying role in
influencing body condition in snakes occupying the Osoyoos habitat, similar to that
reported elsewhere. Given the mixed habitat quality of our site, we can draw
comparisons to the Mara and Holberton study (1998) where they found that lower
quality habitats were associated with higher levels of corticosterone. Then considering
the results of Lomas et al. (2015), we can speculate that stress may be the cause based on
the studies that have demonstrated this effect (Kitaysky et al. 1999; Moore et al. 2000;
Palacios et al. 2012). Elevated levels of stress hormones are known to cause tissue
catabolism that ultimately reduces energy reserves (Sheriff et al. 2011). Following the
work by Lomas et al. (2015) I investigated this potential mechanism within the Osoyoos
snake population.
12

I hypothesized that habitat disturbance in Osoyoos is leading Western
rattlesnakes (Crotalus oreganus) to exhibit higher levels of baseline stress, causing a
reduction in their body condition. I predicted that snakes living in the undisturbed
north desert area would exhibit relatively low baseline levels of corticosterone. The
resort and snake fence areas both represent disturbed habitats, but due to the physical
interaction between snakes and the fence I expect them to show the highest levels of
baseline corticosterone. Snakes at the resort do not face any physical barriers, but
instead a variety of indirect forms of disturbance so I expect their baseline
corticosterone secretion to lie between the undisturbed habitat snakes and those living
along the snake fence. I also predict there will be a negative relationship between
baseline corticosterone and body condition across sites, among all sampled individuals.

METHODS
This project was conducted under federal and provincial permits that allowed
capturing and handling of the targeted wildlife species. Protocols for handling were
approved by an animal care committee from Thompson Rivers University. All data
used in this thesis were collected during July and August of 2018, as these were the
months when the Western rattlesnakes have settled into their summer foraging habitat.
All snakes were found through walking surveys, or through visitation of known
congregation sites. I conducted these surveys in the three primary disturbance areas
which I described before: the north desert, the snake fence and the resort. I avoided the
hottest times of day when snakes were hiding, surveying in the morning (06:00-12:00)
and in the late evening (19:00-24:00). As soon as a snake was encountered, a timer was
used to ensure blood samples were obtained within 5 minutes of the initial encounter,
to prevent corticosterone concentration from being influenced by handling (Schuett et
al. 2004). Tongs were used to maneuver the anterior portion of the snake into an acrylic
13

tube. Following immobilization I palpated the snakes to ensure no gravid females were
used for blood samples. At least 0.5 mL of blood was drawn from the caudal vein with a
1mL syringes (Terumo Medical Corporation®, Japan) with 27G x 1/2" gauge needles.
The blood was transferred to Vacutainer™ (Becton Dickinson, United States) tubes
coated in lithium heparin to prevent clotting. These tubes were labelled and stored in a
portable insulated lunch kit with an icepack. Then within 4 hours the samples were
transferred to 1.5mL microcentrifuge tubes (Fisher Scientific, United States), and
centrifuged at 6000 rpm for 10 minutes (VWR Galaxy mini centrifuge, VWR
International, United States). Afterwards the blood plasma supernatant was removed
and transferred to a new microcentrifuge tube (Fisher Scientific, United States) and
frozen. At the end of the field season, blood samples were transported on ice to a -80°C
freezer in the laboratory at Thompson Rivers University, where they remained until
further analysis.
In addition to blood sampling, I also recorded the GPS coordinates (±5m),
disturbance category, snout-to-vent length (SVL), weight, and sex of each captured
animal. The most proximal segment of the rattle was painted light blue to ensure
individuals were not resampled. I also recorded any anecdotal evidence that may have
influenced corticosterone levels such as the snake having been substantially disturbed
prior to capture.
I used corticosterone ELISA kits (ENZO Biochem Inc., United States) to assay
stress levels in the blood samples collected from the snakes. Samples were thawed
before analysis, then duplicate 100µL plasma samples were pipetted into assay wells.
After this 50 µL of both the alkaline phosphatase conjugate and sheep polyclonal
antibody were added to the wells. I prepared the standardized wells by completing
serial dilutions using a solution with a known corticosterone concentration. The plate
was then incubated at room temperature on a shaker (500 rpm) for 2 hours. The wells
14

were subsequently washed by emptying the contents of the wells, and then adding 400
µL of wash solution. This process was completed three times. After the final wash, all
remaining moisture was removed from the wells and 200 µL of pNpp substrate solution
was added to every well, before the last incubation phase at room temperature for 1
hour, without shaking. Following this, 50 µL of the stop solution was added to every
well and the plate was read immediately using a microplate reader at both 406nm and
621nm. To validate samples between kits, I analyzed 5 samples with both kits and
determined the coefficient of variation between the kits. In addition, 5 more samples
had widely varying results within their replicates in the first kit, so they were repeated
on the second to obtain a more consistent result. To derive the concentration of
corticosterone in the samples, a standard curve was generated using the standard
solutions with known concentrations of corticosterone. Using the equation of the line
for these curves and the percentage bound (a function of optical density), I derived the
associated concentration of corticosterone in pg/mL for each sample.
Statistical analyses
Following Lomas et al. (2015) body condition was derived from a regression
between log10-transformed SVL and mass and the residual values were calculated; this
method is considered appropriate for deriving a measurement of body condition in
snakes based on general size (Reist, 1985).
All statistics were performed in Minitab (Version 18.1). All data were first tested
for normality using the Kolmogorov-Smirnov. I compared body condition index
between our 3 primary sites with a one-way ANOVA test. I then repeated this test to
compare the concentration of corticosterone between the sites. Tukey’s honestly
significant difference test was used post-hoc for detecting differences between groups.
Finally I built a linear regression to examine the relationship between LOG BCI and the
concentration of corticosterone (pg/mL).
15

RESULTS
In total 68 blood samples were collected, with 32 from snakes in the resort area,
26 from snakes in the north desert and 10 from snakes located at the snake fence. I
found no significant difference between the mean body condition index values of
snakes captured at the 3 different sites. (F=1.60 df=2 df=65 P=0.08, Figure 2). Similarly
there was no difference in the mean concentration of corticosterone in blood samples
between the 3 different sites (F=1.98 df=2 df=65 P=0.15, Figure 3). The linear regression
between corticosterone concentration and LOG BCI showed a very weak relationship
(F=1.11 df=1, P=0.30 R2=0.017, Figure 4).

16

Figure 2. Boxplot comparing the body condition of snakes captured at the three
different sites. No significant difference in body condition was detected between these
groups (F=1.60 df=2 df=65 P=0.08).

17

Figure 3. Boxplot of corticosterone concentrations (pg/mL) in blood samples collected
from snakes within the 3 different sites. No difference in corticosterone concentration
was seen between these groups (F=1.98 df=2 df=65 P=0.15).

18

R2= 0.017

Figure 4. Linear regression between plasma corticosterone concentration (pg/mL) and
LOG body condition (F=1.11 df=1, P=0.30 R2=0.017).

19

DISCUSSION
Overall there were no trends that matched my predictions. My results comparing
BCI between snakes occupying different portions of the landscape contrasted strongly
with those reported earlier by Lomas et al. (2015) in that there were no apparent
differences between snakes in the different disturbance regimes. There was no
difference in baseline concentrations of corticosterone between the blood samples
collected from snakes at the different sites. It appears that increased habitat disturbance
in Osoyoos is not leading the Western Rattlesnake population (Crotalus oreganus) to
exhibit higher levels of baseline stress, causing a reduction in their body condition. I
attempted (post-hoc) to determine if there was an underlying relationship between
baseline corticosterone and body condition, independent of site, but again there was no
detectable link between them.
While my hypotheses and predictions were not supported, the results are not
unlike those found in the literature. I based my hypothesis upon research that showed a
clear negative relationship between corticosterone concentrations and body condition
(Kitaysky et al. 1999; Moore et al. 2000; Palacios et al. 2012), yet other work has shown
no relationship whatsoever between these metrics (Dayger et al. 2013; Lind and
Beaupre, 2015; Lutterschmidt et al. 2009). The body condition index reflects the amount
of stored energy an animal has available, or in other words, it is the physiological
energy balance of the individual and the allocation of that energy. This is a critical
internal process that must result in a favourable distribution of energy to life-history
processes such as foraging, reproduction, and predator avoidance or else there may be
significant fitness losses (Dayger et al. 2013). Since one of the primary functions of GCs
is to mobilize energy stores, a negative relationship often exists between circulating GC
concentrations and body condition (Kitaysky et al. 1999; Moore et al. 2000; Palacios et al.
2012). However in our case, and in others no relationship between the two variables
20

was found (Dayger et al. 2013; Lind and Beaupre, 2015; Lutterschmidt et al. 2009). This
suggests the presence of this relationship is contextual, and varies depending on the
environmental conditions and life-history. Previous research on the Common Garter
Snake (Thamnophis sirtalis) has come to similarly-conflicting conclusions about the
relationship between corticosterone and body condition. A study of the species in
Oregon (Lat 45°N) found a negative correlation between body condition and
concentrations of corticosterone (Moore et al. 2000). Conversely, a parallel study in
Manitoba at a higher latitude (51°) found no significant relationship (Dayger et al. 2013).
Although more comparative data are needed, there may be life-history elements that
northern populations specifically balance off energetically, resulting in little
relationship between body condition and corticosterone.
The mean baseline corticosterone levels I recorded in this study (=57405 pg/mL)
were approximately five times higher than those detected in a similar study of timber
rattlers (C. horridus) in Pennsylvania (Lutterschmidt et al. 2009). This further suggests
that underlying differences in baseline GC levels may exist between populations and
species. It also adds credibility to the argument that northern populations of snakes
may be less likely to exhibit a relationship between corticosterone and body condition.
Since the season for breeding and reproduction is condensed by the climate (Gregory,
2007) there may be higher levels of corticosterone secretion by all individuals
independent of site. This higher level of corticosterone secretion could facilitate more
rapid reproductive behaviour and the mobilization of the necessary energy stores to
accomplish this. In order to validate this claim a much broader data set would be
required to make such a wide comparison between northern and southern populations.
Although it could be a plausible explanation, there are other components that should be
considered when interpreting these results.

21

The discrepancy in my results compared to those of other studies may be due to
the fact I was unable to control for all the factors that determine GC content. There are 3
main elements influencing circulating GC content: (1) Endogenous cycles (Circadian
rhythms, seasonal variation), (2) Prior acute environmental stressors (actual or
perceived; predator encounters, extreme weather, etc.) and (3) Chronic stressors
(predators, environmental conditions, etc) (Sheriff et al. 2011). My hypothesis and
predictions were based on the third element, in that I suggested that long-term poor
environmental conditions due to human disturbance would elevate baseline
corticosterone concentrations. However, controlling the 1st and 2nd elements is not
always possible. It is difficult to know the individual experiences of these animals and
as a result other acute environmental stressors (unknown to us) may be influencing the
results. In addition other endogenous factors and cycles, including sex (Honman et al.
2003), age (Reichert et al. 2012), reproductive stage (Rubenstein and Wikelski, 2005), and
time of year or day (Pancak and Taylor, 1983; Tyrrell and Cree, 1998) also may be
affecting GC levels.
Endogenous cycles in particular may have had a profound effect on my results.
Western Rattlesnakes are cryptic and difficult to locate, even when relatively abundant.
Thus, a blood sample was taken every time an individual was encountered, provided
they met the base criteria. This meant that blood samples were taken from individuals
at different times of year, varying times during the day, and under different weather
conditions and then all incorporated into the same model. One such factor that I did not
account for was the reproductive stage any particular individual was in. Given that
among other species, peak corticosterone secretion generally occurs during the mating
season (Dayger et al. 2013), levels in Western Rattlesnakes should peak during late July
or early August (COSEWIC, 2015). This window in time coincided with the latter part of
my blood sample collection period. This implies all sampled rattlesnakes captured
22

during this period may have been exhibiting higher than normal levels of baseline
corticosterone. In turn, the similarity between mean baseline corticosterone
concentrations I detected may be due to the coincidental reproductive stage all
individuals were experiencing. Moreover, corticosterone secretion must be relatively
similar in all individuals at certain points of the breeding season because there will be a
specific range of GC concentrations that allows mating to occur (Dayger et al. 2018).
While it is important to consider these endogenous factors, there are ways to enhance
the complexity of my methods to avoid these issues to some degree.
A more parsimonious explanation for my results is that baseline stress alone is
not the most revealing metric and it is unsuitable to address questions around
comparing stress between habitats. It is possible that the snakes in anthropogenically
disturbed habitats are facing greater environmental stressors, without exhibiting higher
levels of baseline corticosterone due to a difference in the sensitivity of the HPA axis
(Dayger et al. 2013).

In addition, the work of Sapolsky et al. (1984), found that

sensitivity to stress could be altered not only within the HPA axis, but at the cellular
level as well. Sustained elevated levels of circulating corticosterone will result in a
reduction in the number of cytosolic corticosterone receptors within the targeted cells.
Both of these physiological mechanisms work to mitigate the negative effects of chronic
stress, cells dampen their receptivity to corticosterone to buffer the higher
concentrations of corticosterone; effectively reducing the link between corticosterone
and any physical effects such as reduced body condition. Rich and Romero (2005) have
detailed the downstream effects of acute stress response suppression: European
starlings exposed to chronic stress were found to downregulate corticosterone secretion
upon stimulation from acute stressors. While this effect may be present in the Western
rattlesnake, it cannot be examined if the data consists of baseline concentrations of
corticosterone alone.
23

I have identified a number of issues with my protocols and these could be
addressed in future work on stress in the Osoyoos Rattlesnakes if more elaborate
methods are used, ones that can decouple the effects of chronic and acute stress. One
approach could be a capture stress protocol (Pakkala et al. 2013), a strategy developed
to detail the magnitude of the acute stress response. Another option could be to utilize
more experimental techniques such as using clipped scutes or entire sheds (Berkens et
al. 2013) as measures of chronic stress. Lastly, it should become a focus to maximize the
potential sample size. My sample (N=68) was relatively small compared to those in the
Lomas et al (2015) study, where data was collected over multiple years (N=623).
While I did not detect differences in corticosterone levels between snakes
occupying different habitats, my work still serves as an important baseline for future
study. Conservation physiology strives to uncover the underlying mechanisms behind
conservation issues, in part so that law and policy makers can be adequately informed
so that proper protections can be implemented. While I was unable to support my
hypothesis, I have discovered several interesting components about the stress
physiology and ecology of the Western rattlesnake in southern British Columbia. This
information could lead to more directed questions about the real implications of human
disturbance on the stress response of the Western rattlesnake and the downstream
effects. Stress ecology is an important component of conservation biology that must be
understood in order to effectively protect species from the negative effects of human
development.

24

LITERATURE CITED
Baker MR, Gobush KS, Vynne CH. 2013. Review of factors influencing stress hormones
in fish and wildlife. Journal for Nature Conservation; 21:309–318.
Berkvens CN, Hyatt C, Gilman C, Pearl DL, Barker IK, Mastromonaco GF. 2013.
Validation of a shed skin corticosterone enzyme immunoassay in the African House
Snake (Lamprophis fuliginosus) and its evaluation in the Eastern Massasauga Rattlesnake
(Sistrurus catenatus catenatus). General and Comparative Endocrinology; 194:1–9.
Boonstra R, Hik D, Singleton GR, Tinnikov A. 1998. The impact of predator induced
stress on the snowshoe hare cycle. Ecological Monographs; 68(3):371–394.

Boonstra R, Hubbs AH, Lacey EA, McColl CJ. 2001. Seasonal changes in glucocorticoid
and testosterone concentrations in free-living arctic ground squirrels from the boreal
forest of the Yukon. Canadian Journal of Zoology; 79(1):49–58.

Brillon DJ, Zheng B, Campbell RG, Matthews DE. 1995. Effect of cortisol on energy
expenditure and amino acid metabolism in humans. American Journal of PhysiologyEndocrinology and Metabolism; 268(3):E501–E513.

Cash WB, Holberton RL. 2005. Endocrine and behavioral response to a decline in
habitat quality: Effects of pond drying on the slider turtle, Trachemys scripta. Journal of
Experimental Zoology Part A: Comparative Experimental Biology; 303A(10):872–879.

Claunch NM, Frazier JA, Escallón C, Vernasco BJ, Moore IT, Taylor EN. 2017.
Physiological and behavioral effects of exogenous corticosterone in a free-ranging
ectotherm. General and Comparative Endocrinology; 248:87–96.

Coe CL, Levine S. 1995. Diurnal and annual variation of adrenocortical activity in the
squirrel monkey. American Journal of Primatology; 35(4):283–292.

25

Committee on the Status of Endangered Wildlife in Canada (COSEWIC). 2015.
COSEWIC Assessment and Status Report on the Western Rattlesnake (Crotalus oreganus)
in Canada.

Dayger CA, Cease AJ, Lutterschmidt DI. 2013. Responses to capture stress and
exogenous corticosterone vary with body condition in female red-sided garter snakes
(Thamnophis sirtalis parietalis). Hormones and Behavior; 64(4):748–754.

Dayger CA, LeMaster MP, Lutterschmidt DI. 2018. Physiological correlates of
reproductive decisions: Relationships among body condition, reproductive status, and
the hypothalamus-pituitary-adrenal axis in a reptile. Hormones and Behavior; 100:1–11.

Eikenaar C, Husak J, Escallón C, Moore IT. 2012. Variation in testosterone and
corticosterone in amphibians and reptiles: Relationships with latitude, elevation, and
breeding season length. The American Naturalist; 180(5):642–654.

Eriksson H, Gustafsson J-Å. 1970. Steroids in germfree and conventional rats. European
Journal of Biochemistry; 15(1):132–139.

Fokidis HB, Orchinik M, Deviche P. 2009. Corticosterone and corticosteroid binding
globulin in birds: Relation to urbanization in a desert city. General and Comparative
Endocrinology; 160(3):259–270.

Frugé AD, Cases MG, Howell CR, Tsuruta Y, Smith-Johnston K, Moellering DR,
Demark-Wahnefried W. 2018. Fingernail and toenail clippings as a non-invasive
measure of chronic cortisol levels in adult cancer survivors. Cancer Causes & Control;
29(1):185–191.

Goodyer IM, Park RJ, Netherton CM, Herbert J. 2001. Possible role of cortisol and
dehydroepiandrosterone in human development and psychopathology. British Journal
of Psychiatry; 179(3):243–249.

26

Gustafson AW, Belt WD. 1981. The adrenal cortex during activity and hibernation in the
male little brown bat, Myotis lucifugus lucifugus: Annual rhythm of plasma cortisol
levels. General and Comparative Endocrinology; 44(3):269–278.

Gregory PT. 2007. Biology and conservation of a cold-climate snake fauna.
Herpetological Conservation; 2: 41-56.

Hau M, Ricklefs RE., Wikelski M, Lee KA., Brawn JD. 2010. Corticosterone, testosterone
and life-history strategies of birds. Proceedings of the Royal Society B: Biological
Sciences; 277(1697):3203–3212.

Hasler CT, Suski CD, Chapman JM, Cooke SJ, Jeffrey JD. 2015. Linking landscape-scale
disturbances to stress and condition of fish: Implications for restoration and
conservation. Integrative and Comparative Biology; 55(4):618–630.

Holding ML, Frazier JA, Dorr SW, Pollock NB, Muelleman PJ, Branske A, Henningsen
SN, Eikenaar C, Escallón C, Montgomery CE, et al. 2014. Wet- and dry-season steroid
hormone profiles and stress reactivity of an insular dwarf snake, the Hog Island Boa
(Boa constrictor imperator). Physiological and Biochemical Zoology; 87(3):363–373.

Homan RN, Reed JM, Romero LM. 2003. Corticosterone concentrations in free-living
spotted salamanders (Ambystoma maculatum). General and Comparative Endocrinology;
130(2):165–171.

Hontela A, Rasmussen JB, Audet C, Chevalier G. 1992. Impaired cortisol stress response
in fish from environments polluted by PAHs, PCBs, and mercury. Archives of
Environmental Contamination and Toxicology; 22(3):278–283.

Ising M, Holsboer F. 2006. Genetics of stress response and stress-related disorders.
Dialogues in clinical neuroscience; 8(4):433–444.

27

Jaimez NA, Bribiescas RG, Aronsen GP, Anestis SA, Watts DP. 2012. Urinary cortisol
levels of gray-cheeked mangabeys are higher in disturbed compared to undsiturbed
forest areas in Kibale National Park, Uganda. Animal Conservation; 15:242–247.

Janin A, Léna J-P, Joly P. 2011. Beyond occurrence: Body condition and stress hormone
as integrative indicators of habitat availability and fragmentation in the common toad.
The New Conservation Debate: Beyond Parks vs. People; 144(3):1008–1016.

Katz FH, Shannon IL. 1964. Identification and significance of parotid fluid
corticosteroids. Acta Endocrinologica; 46(3):393–404.

Khani S, Tayek JA. 2001. Cortisol increases gluconeogenesis in humans: its role in the
metabolic syndrome. Clinical Science; 101(6):739.

Kitaysky AS, Wingfield JC, Piatt JF. 1999. Dynamics of food availability, body condition
and physiological stress response in breeding Black-legged Kittiwakes. Functional
Ecology; 13(5):577–584.

Klasing KC. 2005. Potential impact of nutritional strategy on noninvasive measurements
of hormones in birds. Annals of the New York Academy of Sciences; 1046(1):5–16.

Lesbarrères D, Ashpole SL, Bishop CA, Blouin-Demers G, Brooks RJ, Echaubard P,
Govindarajulu P, Green DM, Hecnar SJ, Herman T, et al. 2014. Conservation of
herpetofauna in northern landscapes: Threats and challenges from a Canadian
perspective. Biological Conservation; 170:48–55.

Lind CM, Beaupre SJ. 2015. Male snakes allocate time and energy according to
individual energetic status: Body condition, steroid hormones, and reproductive
behavior in Timber Rattlesnakes, Crotalus horridus. Physiological and Biochemical
Zoology; 88(6):624–633.

28

Lind CM, Moore IT, Vernasco BJ, Farrell TM. 2018. Seasonal testosterone and
corticosterone patterns in relation to body condition and reproduction in a subtropical
pitviper, Sistrurus miliarius. General and Comparative Endocrinology; 267:51–58.

Lomas E, Larsen KW, Bishop CA. 2015. Persistence of Northern Pacific Rattlesnakes
masks the impact of human disturbance on weight and body condition. Animal
Conservation; 18:548–556.

Lutterschmidt WI, Lutterschmidt DI, Mason RT, Reinert HK. 2009. Seasonal variation in
hormonal responses of timber rattlesnakes (Crotalus horridus) to reproductive and
environmental stressors. Journal of Comparative Physiology B; 179(6):747–757.

Maida JR. 2018. Impacts of fencing and development on Western Rattlesnake (Crotalus
oreganus) spring movements in British Columbia (Masters Thesis). Thompson Rivers
University.

Marra PP, Holberton RL. 1998. Corticosterone levels as indicators of habitat quality:
effects of habitat segregation in a migratory bird during the non-breeding season.
Oecologia; 116(1):284–292.

McEwen BS, Wingfield JC. 2003. The concept of allostasis in biology and biomedicine.
Hormones and Behavior; 43(1):2–15.

Montero D, Izquierdo MS, Tort L, Robaina L, Vergara JM. 1999. High stocking density
produces crowding stress altering some physiological and biochemical parameters in
gilthead seabream, Sparus aurata, juveniles. Fish Physiology and Biochemistry;20(1):53–
60.

Moore IT, Jessop TS. 2003. Stress, reproduction, and adrenocortical modulation in
amphibians and reptiles. Hormones and Behavior; 43(1):39–47.

29

Moore IT, Lerner JP, Lerner DT, Mason RT. 2000. Relationships between annual cycles
of testosterone, corticosterone, and body condition in male Red‐Spotted Garter Snakes,
Thamnophis sirtalis concinnus. Physiological and Biochemical Zoology; 73(3):307–312.

Newcomer JW, Selke G, Melson AK, Hershey T, Craft S, Richards K, Alderson AL. 1999.
Decreased memory performance in healthy humans induced by stress-level cortisol
treatment. Archives of General Psychiatry; 56(6):527–533.

Oskam I, Ropstad E, Lie E, Derocher A, Wiig Ø, Dahl E, Larsen S, Skaare JU. 2004.
Organochlorines affect the steroid hormone cortisol in free-ranging Polar Bears (Ursus
maritimus) at Svalbard, Norway. Journal of Toxicology and Environmental Health, Part
A; 67(12):959–977.

Pakkala JJ, Norris DR, Newman AEM. 2013. An experimental test of the capturerestraint protocol for estimating the acute stress response. Physiological and
Biochemical Zoology; 86(2):279–284.

Palacios MG, Sparkman AM, Bronikowski AM. 2012. Corticosterone and pace of life in
two life-history ecotypes of the Garter Snake Thamnophis elegans. General and
Comparative Endocrinology; 175(3):443–448.

Pancak MK, Taylor DH. 1983. Seasonal and daily plasma corticosterone rhythms in
American Toads, Bufo americanus. General and Comparative Endocrinology; 50(3):490–
497.

Pankhurst N, Sharples D. 1992. Effects of capture and confinement on plasma cortisol
concentrations in the Snapper, Pagrus auratus. Marine and Freshwater Research;
43(2):345–355.

Reeder DM, Kramer KM. 2005. Stress in free-ranging mammals: Integrating physiology,
ecology, and natural history. Journal of Mammalogy; 86(2):225.

30

Riechert J, Chastel O, Becker PH. 2012. Why do experienced birds reproduce better?
Possible endocrine mechanisms in a long-lived seabird, the Common Tern. General and
Comparative Endocrinology; 178(2):391–399.

Reist JD. 1985. An empirical evaluation of several univariate methods that adjust for
size variation in morphometric data. Canadian Journal of Zoology; 63(6):1429–1439.

Rich EL, Romero LM. 2005. Exposure to chronic stress downregulates corticosterone
responses to acute stressors. Comparative and Evolutionary Physiology; 288(6):16281636.

Romero LM, Reed JM, Wingfield JC. 2000. Effects of weather on corticosterone
responses in wild free-living Passerine birds. General and Comparative Endocrinology;
118(1):113–122.

Romero, LM, and Remage-Healey, L. 2000. Daily and seasonal variation in response to
stress in captive Starlings (Sturnus Vulgaris): Corticosterone. General and Comparative
Endocrinology; 119(1): 52–59.

Romero LM, Soma KK, Wingfield JC. 1998. Hypothalamic-pituitary-adrenal axis
changes allow seasonal modulation of corticosterone in a bird. American Journal of
Physiology-Regulatory, Integrative and Comparative Physiology; 274(5):R1338–R1344.

Rubenstein DR, Wikelski M. 2005. Steroid hormones and aggression in female
Galápagos marine iguanas. Hormones and Behavior; 48(3):329–341.

Sadoul B, Geffroy B. 2019. Measuring cortisol, the major stress hormone in fishes.
Journal of Fish Biology; 94(4):540-555.

Sapolsky RM, Krey LC, McEwen BS. 1984. Stress down-regulates corticosterone
receptors in a site-specific manner in the brain. Endocrinology; 114(1):287–292.
31

Schuett GW, Taylor EN, Van Kirk EA, Murdoch WJ. 2004. Handling stress and plasma
corticosterone levels in captive male Western Diamond-backed Rattlesnakes (Crotalus
atrox). Herpetological Review; 35(3):229–233.

Sheriff MJ, Dantzer B, Delehanty B, Palme R, Boonstra R. 2011. Measuring stress in
wildlife: techniques for quantifying glucocorticoids. Oecologia; 166(4):869.
Strange RJ, Schreck CB. 1978. Anesthetic and handling stress on survival and cortisol
concentration in yearling Chinook Salmon (Oncorhynchus tshawytscha). Journal of the
Fisheries Research Board of Canada; 35(3):345–349.

Taylor CJ. 2012. A sociological overview of cortisol as a biomarker of response to the
social environment. Sociology Compass; 6(5):434–444.

Thorpe JE, McConway MG, Miles MS, Muir JS. 1987. Diel and seasonal changes in
resting plasma cortisol levels in juvenile Atlantic Salmon, Salmo salar L. General and
Comparative Endocrinology; 65(1):19–22.

Tyrrell CL, Cree A. 1998. Relationships between corticosterone concentration and
season, time of day and confinement in a wild reptile (Tuatara Sphenodon punctatus).
General and Comparative Endocrinology; 110(2):97–108.

Waye HL, Mason RT. 2008. A combination of body condition measurements is more
informative than conventional condition indices: Temporal variation in body condition
and corticosterone in brown tree snakes (Boiga irregularis). General and Comparative
Endocrinology; 155(3):607–612.

Wikelski M, Cooke SJ. 2006. Conservation physiology. Trends in Ecology & Evolution;
21:38–46.

32

Wingfield JC, Grimm AS. 1977. Seasonal changes in plasma cortisol, testosterone and
oestradiol-17β in the Plaice, Pleuronectes platessa L. General and Comparative
Endocrinology; 31(1):1–11.

Wingfield JC, Maney DL, Breuner CW, Jacobs JD, Lynn S, Ramenofsky M, Richardson
RD. 1998. Ecological bases of hormone-behavior interactions: The “emergency life
history stage.” American Zoologist; 38(1):191.
Wingfield JC, Romero LM. 2011. Adrenocortical responses to stress and their
modulation in free-living vertebrates. In: Comprehensive Physiology. American Cancer
Society; p. 211–234.
Wood P. 2009. Salivary steroid assays – research or routine? Annals of Clinical
Biochemistry; 46(3):183–196.
Wysocki LE, Dittami JP, Ladich F. 2006. Ship noise and cortisol secretion in European
freshwater fishes. Biological Conservation; 128(4):501–508.
Zwijacz-Kozica T, Selva N, Barja I, Silván G, Martínez-Fernández L, Illera JC, Jodłowski
M. 2013. Concentration of fecal cortisol metabolites in chamois in relation to tourist
pressure in Tatra National Park (South Poland). Acta Theriologica; 58(2):215–222.

33