Download Monitoring physiological stress in semi-free

Eur J Wildl Res
DOI 10.1007/s10344-014-0842-z
ORIGINAL PAPER
Monitoring physiological stress in semi-free ranging populations
of an endangered Australian marsupial, the Greater Bilby
(Macrotis lagotis)
Edward J. Narayan & Nicole Evans & Jean-Marc Hero
Received: 5 December 2013 / Revised: 10 June 2014 / Accepted: 3 July 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Rapid and reliable physiological evaluation of
stress is necessary for understanding the potential impacts of
environmental changes on managed populations of threatened
mammals. In situ populations of Australia’s iconic marsupial,
the greater bilby (Macrotis lagotis), are nearing extinction due
to the impacts of competition and predation by feral animals
and unpredictable climatic events (summer heat waves). In
this study, we focussed our aim to identify a non-invasive
method to measure adrenal activity in the species and also to
identify potential factors that should be considered when
comparing physiological stress in semi-free ranging populations of the species. We validated an enzyme immunoassay
(EIA) for detecting fecal cortisol metabolites (FCM) from
fresh fecal pellets taken from bilbies within four captive sites
and two semi-free ranging populations around Queensland
and New South Wales, Australia. Our FCM EIA successfully
detected the ‘raise and fall’ pattern of FCM levels within
3 days of exogenous adrenocorticotropic hormone (ACTH)
challenge. Mean FCM levels differed significantly between
the captive sites and between sexes. All male bilbies grouped
outdoor in captivity expressed the highest mean FCM level in
comparison to all captive males that were housed individually
or as groups indoors. Also, semi-free ranging bilbies
expressed higher mean FCM levels than the captive bilbies.
Overall, our study successfully validated a non-invasive tool
for monitoring physiological stress in the greater bilby. In the
future, it will be worthwhile to consider factors such as housing conditions, sex and location when comparing the adrenal
sensitivity to environmental changes, to help evaluate the
Communicated by A. W. Sainsbury
E. J. Narayan (*) : N. Evans : J.<M. Hero
Environmental Futures Research Institute, School of Environment,
Griffith University, Gold Coast campus, Gold Coast QLD 4222,
Australia
e-mail: [email protected]
success of management interventions (such as predator free
enclosures) and support the survival of the species.
Keywords Australian marsupial . Macrotis lagotis .
Semi-wild populations . Predation . Conservation
physiology . Physiological stress
Introduction
Conservation physiology has most recently been defined as ‘an
integrative scientific discipline applying physiological concepts,
tools, and knowledge to characterizing biological diversity and
its ecological implications; understanding and predicting how
organisms, populations, and ecosystems respond to environmental change and stressors; and solving conservation problems
across the broad range of taxa (i.e., including microbes, plants,
and animals) (Cooke et al. 2013, p. 1). Over the past few
decades, there has been an increase in the use of non-invasive
methods for the assessment of glucocorticoids or stress hormones (cortisol) in mammals. The stress endocrine system in
mammals is the hypothalamic–pituitary–adrenal (HPA) axis.
Through intrinsic negative feedback mechanisms between the
brain and the adrenals, the HPA axis regulates the release of
cortisol from the adrenal glands during exposure to environmental stress factors (Young et al. 2004). Cortisol secretion alters
important biochemical processes, such as metabolism, which
then leads to behavioral modifications that enable animals to
cope with acute stimulus, such as predator presence (Wikelski
and Cooke 2006; Narayan 2013; Narayan et al. 2013a). The
process of physiological stress adaptation maintains the internal
homeostatic balance of the animal’s HPA axis with the environment. If the environmental stimuli become prolonged, such as
during extreme hot weather and drought, then the HPA axis
enters into a disrupted state whereby the return of cortisol levels
to baseline levels become difficult (Narayan and Hero 2014a).
Eur J Wildl Res
Consequently, sustained release of corticosteroids from the adrenal glands often also lead to disruptive effects on the reproductive hormones and immune systems, cognition, performance
and behaviour, as shown in mammals (Muller and Wrangham
2004; Sheriff et al. 2009), birds (Ellenberg et al. 2007; Bonier
et al. 2009) and amphibians (Narayan and Hero 2014a, b).
Fecal cortisol metabolite (FCM) analysis is used frequently
in conservation physiology programs for mammals (Dantzer
et al. 2010; Marechal et al. 2011; Fanson et al. 2012),birds
(Palme 2005; Wasser and Hunt 2005),and to a lesser extent,
reptiles (Romero and Wikelski 2001),and fishes (Turner et al.
2003; Ellis et al. 2004). Non-invasive FCM analysis has
contributed new knowledge in relation to wildlife management (Bosacker 2008; Thaker et al. 2010), population density
(Kalz et al. 2006; Park et al. 2011), behaviour and social status
(Muller and Wrangham 2004), reproduction (Hesterman et al.
2008), sociality (Bosacker 2008), territoriality (Barja et al.
2008), animal welfare (Owen et al. 2004), health status
(Chapman et al. 2007), predation threat (Sheriff et al. 2009),
and to predict population declines and mortality (McDonald
et al. 1981; Ellenberg et al. 2007). As with any emerging
quantitative physiological method, non-invasive analysis of
glucocorticoids has pros and cons that need to be considered
for each new study species for which this technique is being
developed (Narayan 2013). Earlier, Sheriff et al. (2011)
reviewed the technical considerations for standardizing the
use of FCM analysis as a biomarker of stress in mammals.
An important validation step includes biological challenge,
using exogenous adrenocorticotrophic hormone (ACTH),
which is used to test whether the assay system is able to
reliably measure the metabolites of target hormone in excreta
of the study species (Narayan et al. 2010, 2013b, c).
Australia is a biodiversity-rich continent; however, it also
has recent high record of mammalian extinctions and species
endangerment, with 21 species already extinct and 31 species
listed as endangered or critically endangered (IUCN 2006;
James and Eldridge 2007). The greater bilby (Macrotis
lagotis) is one of Australia’s most threatened animals and is
currently listed as Vulnerable (C1 ver 3.1) by the IUCN Red
List (IUCN 2006). Once widespread across 70 % of mainland
Australia, this burrowing marsupial has disappeared from
90 % of its historical range and now exists only in fragmented
populations in the driest and least fertile parts of its former
range (Gibson 2001; Smith et al. 2009). This major contraction in range is primarily attributed to predation from introduced species (e.g., red fox [Vulpes vulpes] and feral cats
[Felis catus]), habitat destruction through agriculture and
competition with introduced herbivores (Moseby et al.
2009). Whilst information on the bilby’s reproductive biology,
anatomy and behaviour are available, literature on its stress
physiology is only from single captive population (Narayan
et al. 2012). There is no information available on FCM levels
in semi-wild populations of this marsupial.
The aim of the current study was to develop a non-invasive
tool that could be used to assess adrenal activity in the semifree ranging populations of the bilby. The goal is that the
methods developed could be used in future studies to assess
physiological sensitivity to environmental change in this vulnerable endemic Australian marsupial — aiming to make
suggested in situ management changes — protecting the
species.
Material and methods
Study sites: captivity
Table 1 provides detailed information regarding the four captive study sites. The bilby husbandry and veterinary databases
from each site provided information about age, sex and health
status along with daily activities of individual bilby.
Study sites: semi-free ranging
Currawinya National Park (semi-wild population 1; SW-1),
located in south-western Queensland (28.6817 S, 144.6978 E)
contains a 25-km2 predator-proof enclosure that was built in
the National Park in 2003 to protect the reintroduced population of the greater bilby from ground dwelling predators
(Dunwoody et al. 2009; DEHP 2012). Unfortunately, during
the time of sampling the predator-proof exclusion fence had
been compromised by recurrent rains that caused corrosion
and enabled the infiltration of feral cats (F. catus) into the
enclosure. Consequently, there was a dramatic decline (the
population was reported as critically low) of the bilby population residing within the fence (Williams and Manthey 2012).
Scotia Wildlife Sanctuary (SWS) (Semi-wild population 2;
SW2) (64,653 ha; 141.100 E, 33.100 S) is located 150 km
south of Broken Hill, on the boundary of the arid and semiarid climatic zones of New South Wales. Greater bilbies were
reintroduced within a fenced area (8,000 ha) between 2004
and 2005.A total of 2,103 individual bilbies were residing in
the fenced area at the Sanctuary (Finlayson et al. 2008; AWC
2012). Bilby fecal samples were collected within 210 ha area
of the fenced area, which also contained bridled nail-tail
wallaby (Onychogalea fraenata).
Fecal sampling: captive
At all four captive locations (Table 1), the entire enclosures
were racked in the evening to remove old feces. Fecal samples
were then collected daily early morning (0600 hours) by
husbandry staff over a period of 14 consecutive days during
March and April of 2012. Fresh fecal samples (defecated
within the previous 12 h) were collected from each enclosure
Both individuals were born in
captivity at the Charleville
Breeding Centre.
Fed a diet of dog kibble
and bird seed.
2 male bilbies
Charleville Breeding Centre
(CBC), located in Charleville,
south-western Queensland (C4)
in a single zip-lock bag (labelled with the date and the animal’s
ID) and stored in a −20 °C freezer prior to processing.
Identification of fecal samples in grouped housing: a fecal
marker technique
Each site is coded (C1, C2, C3 or C4) and discussions are made with reference to these codes
None reported.
Fed a diet of seed, dog kibble,
vegetables and various live
insects daily.
2 male bilbies
Currumbin Wildlife Sanctuary
(CWS), Gold Coast,
Queensland (C3)
All individuals were housed separately.
The female’s enclosure consisted of a
10×2.5 m area bedded with coarse
red sand, vegetation and a permanent
artificial burrow site. The male
enclosure consisted of a 3×5 m fenced
area bedded with dry fine sand with
two enclosed sleeping dens
(0.6×0.5 m) bedded with blankets
and towels.
Housed together on permanent display.
3.6×2.5 m area bedded with coarse
red sand. Vegetation provided along
with a permanent artificial burrow site.
Large separate outdoor enclosures
(30×20 m) located away from tourist
activities, large enough to facilitate selfforaging and natural burrowing behaviours.
Ipswich Nature Centre (INC),
Ipswich, Queensland (C2)
2 male and 2 male bilbies
All but two female bilbies
participated in educational
shows.
Male individuals participated
in a school educational visit
on day 10 of sampling
involving transport and
manual handling.
Fed whole grains and carnivore
dental chews supplemented
occasionally with whole insects.
Fed a diet of whole oats, feline
bites, dog kibble and a range
of insects daily.
All individuals were housed separately.
3 male and 4 female bilbies
Dreamworld Theme Park,
Gold Coast, Queensland (C1)
Housing and enclosure description
Animals
Captive study sites
Table 1 Detailed information regarding the four captive study sites
Husbandry regime
Activities
Eur J Wildl Res
A fecal marker was used to identify the feces of bilbies present
in grouped housing at the Ipswich Nature Centre (INC) and
Currumbin Wildlife Sanctuary (CWS). Powdered bakers
green food colouring (Baking Pleasures; ABN: 23 717 428
010; Queensland, Australia) was used as the fecal marker.
Similar products have been used previously to aid fecal identification in a range of mammal species, such as the numbat
(Myrmecobius fasciatus) (Hogan et al. 2013) and domestic
cats (Griffin 2002). At each grouped housing location, one
bilby (Individual A) received a 2-g dose of powdered bakers
food colouring mixed through regular daily food for the
duration of the 14-day sampling period. Individuals were
separated for feeding to ensure that only one bilby was exposed to the food colouring. All feces showing bright green
colouration were identified as belonging to Individual A. All
fecal samples that were absent of any artificial colour were
identified as belonging to Individual B and were analysed
separately. Individuals exposed to the bakers powered food
colouring showed no aversion to taste or consistency of the
powdered food colouring when combined with daily food.
The individuals consumed all food as usual and no adverse
gastrointestinal effects were observed. Visual inspection of
fecal samples confirmed the successful application of bakers’
powdered food colouring for marking an individual’s feces.
Coloured feces (green) were present within 48 h of initial
administration of food colouring and colour intensity was
sufficient to enable the identification of feces containing colour and those that did not. Colour saturation was consistent in
all feces throughout the 14-day sampling period. Serial dilutions of XGreen bakers powdered food colouring did not
show parallelism with serial dilutions of cortisol standard
(R4866) and there was significant difference between bakers
powdered food colouring serial dilution and cortisol standard
serial dilutions (t=−2.12, p=0.040). This indicated that the
XGreen bakers powdered food colouring does not contain a
significant amount of cortisol hormone and as such did not
influence the assay of cortisol metabolites in bilby feces.
Fecal sampling: semi-free ranging
Fecal samples were collected from the semi-free ranging
populations’ using a minimum of ten 250 m×60 cm strip
transects. Transects were positioned at a minimum of 1 km
apart in order to ensure independence of samples. Samples
were collected over 14 days at CNP during March 2012, and
over 7 days at the SWS during July of 2012. Transects were
raked to clear old feces and leaf litter in the evening, and
Eur J Wildl Res
subsequently visited daily for up to 14 days. All fresh (<12 h)
fecal samples found within each transect were collected in ziplock bags (labelled with date, time and transect) and stored on
ice packs until returning from the field site. Fresh feces were
characterised by a layer of mucus, a strong smell, and no signs
of dehydration. Only a single fecal pellet was collected from
each transect to avoid pseudo-replication within and between
transects. Samples were then stored in a −20 °C freezer prior to
assay. Over the 7-day sampling period, a total of 28 fresh bilby
fecal samples were collected from SWS. Due to the extremely
low density of the bilby population at CNP during the time of
sampling, only five fresh fecal samples were collected.
Fecal cortisol metabolite enzyme immunoassay: laboratory
and physiological validations
We analysed bilby fecal extracts for concentrations of FCM
using enzyme immunoassay (EIA) previously described for
other mammals (Wielebnowski et al. 2002; Millspaugh and
Washburn 2004), and recently used for a captive subpopulation of the greater bilby (Narayan et al. 2012; Evans
et al. 2013). Laboratory validation was done using accuracyrecovery checks and parallelism. Recovery of cortisol standard that was added to pooled bilby fecal extracts was y=
0.98x+1.26, r2 =0.997 (n=7), where y is the concentration
observed and x is the concentration expected resulting in an
extraction efficiency of >99 %. Assay sensitivity was calculated as 1.2±0.2 ng/well (n=10). Intra- (within) and inter(between) assay coefficients of variation (CV) were 2.3 % and
6.4 % for the high-binding internal control and 1.9 % and
14.3 % for the low-binding internal control, respectively (n=
15). Serial dilutions of bilby fecal extracts yielded displacement curves parallel to those of the cortisol standard. FCM
concentrations were presented as ng/g FCM net dry feces.
Physiological validation was done using administration of
adrenocorticotropic hormone (ACTH) to four captive individuals (one male and one female from Dreamworld and two
males from CWS). Individuals were removed from their enclosure and manually restrained whilst 250 μl of Synacthen
(Provet Pty Ltd, Australia). Synacthen was available in a glass
ampoule containing 1 ml liquid. Each ampoule contains
250 μg of tetracosactide (as the hexa-acetate salt). Each bilby
received a quarter of the 1-ml dose that was administered as a
single intramuscular injection (Dose = 62.5 μg of
tetracosactide per total body weight of an adult bilby). Daily
fecal samples were collected from 2 to 3 days prior to and 4 to
5 days after injection based on sampling methods described
above. This dose and similar daily sampling design was
previously used for studies on the southern hairy nosed wombat (Lasiorhinus latifrons) and in the roe deer (Capreolus
capreolus) (Dehnhard et al. 2001; Hogan et al. 2011). Each
individual then acted as its own control during a saline challenge that was performed 3 weeks after the ACTH challenge.
The procedure followed the same protocol as for the ACTH
challenge; however, this time, the animals received an injection of 250 μl sterile isotonic saline solution (0.9 % NaCl sol.),
with a 7-day interval between the ACTH and saline injections.
Animals were individually housed during the experiment and
so all samples collected were of known origin. We calculated
the percentage raise in FCM post injection of either ACTH or
saline through time (days). Mean (±SE) FCM plotted for the
ACTH and saline challenge data for visual interpretation of
the results.
FCM concentrations increased to the highest levels within
24 h of administration of the ACTH in male 1 and female 1
(Fig. 1). For male 2, the highest FCM level was noted on day 3
post ACTH challenge (Fig. 1). Male 3 showed a decrease in
FCM level 1 day post ACTH challenge suggesting that the
ACTH dose may have not been sufficient to elicit an adrenocortical response for this individual (Fig. 1). Individual peak
responses were 14.49 ng/g net dry weight (first highest
Female 1), 5.84 ng/g net dry weight (second highest Male 1)
and 4.80 ng/g net dry weight (Male 2) (Fig. 1). Highest
combined sexes mean concentration of FCM was detected at
24 h post ACTH injection at 6.25 ng/g (±2.8) FCM net dry
weight (Fig. 2). Mean FCM concentrations increased by
280.31 % within 24 h of the ACTH injection, began to decline
at day 2 and returned to near pre-treatment FCM levels by day
4 following ACTH injection (Fig. 2). There were no appreciable changes were recorded in FCM concentrations in any of
the four bilbies subjected to the saline challenge with a 5 %
decline in mean FCMs recorded on day 1 following injection
(Fig. 2).
Statistical analysis
Statistical analysis were performed using and SYSTAT
Version 13.0. All data were tested for normality using the
Shapiro–Wilk normality test (applied to the residuals) and
data were log-transformed where necessary to meet the assumptions of equal variances. For all analyses, cortisol was
treated as the response variable and significance was assessed
at the p<0.05 level. A Generalised Linear Model (GLM)
ANOVA was used to compare the mean FCM levels between
the captive groups (study sites), including location and sex as
the factors, and sex×location interaction terms. Post-hoc comparisons between sexes for each study site were analysed
using Tukey's honestly significant difference test.
For the two semi-free ranging populations, FCM data are
presented as individual points; one data point represents one
individual sample per day. The statistical differences in mean
FCM between the semi-free ranging sites and comparisons
with the captive study groups were not analysed because of
the fact that there were limited samples from the semi-free
ranging sites, and due to the potential differences that were
Eur J Wildl Res
Fig. 1 Individual fecal cortisol
metabolite concentrations (ng/g
net dry weight) in bilby subjected
to adrenocorticotropic hormone
(ACTH) challenge. Day of
injection is depicted using vertical
dashed line
unaccounted for such as sampling time of the year, reproductive state and diet.
FCM levels in semi-wild bilby populations
The average FCM concentration for samples from SWS was
higher than that of Currawinya National Park (CNP) (2.78 ng/
g net dry weight cf. 2.26 ng/g FCM net dry weight).
Results
FCM levels in captive bilby populations: comparison
between sites and between sexes
Discussion
There was a significant difference in mean FCM concentrations between the four captive populations (GLM ANOVA;
F=16.53, p<0.001, Fig. 3). Male bilbies grouped in outdoor
enclosure at the Charleville Breeding Centre (CBC) recorded
the highest mean baseline FCM concentration at 0.89 ng/g net
dry weight. Female bilbies at the INC recorded the second
highest mean FCM at 0.74 ng/g net dry weight, followed by
female bilbies at Dreamworld at 0.36 ng/g net dry weight
(Fig. 3). Male bilbies at the INC recorded the third lowest
level of mean FCM at 0.22 ng/g net dry weight. Male bilbies
grouped in indoor enclosure at the CWS recorded the second
lowest mean baseline FCM level at 0.132 ng/g net dry weight.
Male bilbies at Dreamworld recorded the lowest mean baseline FCM level at 0.10 ng/g net dry weight (Fig. 3).
As shown in Fig. 3, there was a significant difference in
FCM levels between male (n=3) and female (n=4) greater
bilbies housed at Dreamworld (t = −0.249, p < 0.001).
Likewise, there was a significant difference in mean FCM
levels between male and female bilbies housed at the
INC (t=−0.481, p<0.001).
We validated a fecal cortisol EIA for evaluating physiological
stress in the Greater Bilby (M. lagotis). ACTH challenge
causes a maximal cortisol response in mammals (Benhaiem
et al. 2012). Our ACTH challenge results verify that the FCMs
excreted after the ACTH injection were related to the stimulation of the HPA axis of the individual bilbies in response to
the ACTH injections. The ‘rise and fall’ pattern showed the
levels of metabolic end-products of free cortisol, denoted as
FCMs. Our results showed that on average FCM levels should
appear in bilby feces in response to acute stressor within 24 h.
This approximation fits well with estimates of 8–12 h for
average FCM expression for the snowshoe hare (Lepus
americanus) (Sheriff et al. 2009), and 12 h in the red-backed
vole (Clethrionoours gapperii) (Harper and Austad 2000).
Actually, this period provides a critical window for obtaining
baseline FCM data from semi-free ranging populations. For
bilbies, if fecal samples can be collected from individuals that
have defecated within less than 24 h after capture then prestimulus baseline FCM level can easily be obtained.
Furthermore, we have demonstrated that fresh (<12–24 h
Eur J Wildl Res
Fig. 2 Mean (±SEM) fecal cortisol metabolite concentrations (ng/g net
dry weight) in bilby subjected to adrenocorticotropic hormone (ACTH)
(n=4) and saline (n=4) challenges on day 0. Fecal samples were collected
2 days prior to and 4 days post administration of ACTH and saline
injections. Day of injection is depicted using vertical dashed line
old) bilby feces are fairly stable to natural weathering conditions up to 13 days; that is, the FCMs in these feces are not
deteriorated (Evans et al. 2013). Therefore, our sampling
methods using transects provide a reliable way of obtaining
fresh bilby fecal samples that will eventually be used for
indicating the physiological sensitivity of semi-free ranging
populations against environmental threats such as stochastic
weather events (storms and droughts) and predation by feral
pests. In some situations, identification of individual feces is
usually problematic in situations such as group housing in
captivity (Fuller et al. 2011). Our data confirms the success of
bakers powdered food colouring (XGreen) as a fecal marker
for the bilby. Administration of approximately 2 g of powdered food colouring once daily produced colour saturated
feces consistently. Food colouring was easily mixed into food
Fig. 3 Fecal cortisol metabolite concentrations (ng/g net dry weight) in
bilby from all captive sampling locations. C1 Dreamworld Theme Park,
C2 Ipswich Nature Centre, C3 Currumbin Wildlife Sanctuary, C4
Charleville Breeding Centre. C3 males were grouped indoor. C4 males
were grouped outdoor. Sample sizes range from 2 to 4 bilbies per site.
Level of significant difference between the sites and sexes are denoted by
asterisks: ***p<0.001
vehicles and was largely recoverable in the feces. Earlier only
one other study has successfully utilised food colourant to
detect feces of individuals within group housed numbat
(Myrmacobius fasciatus) (Hogan et al. 2013).
Both semi-wild populations sampled in this study
(SW1and SW2) were re-introduced within predator-proof
fenced reserves, and play critical roles in the National
Recovery Plan for the bilby (Pavey 2006; Finlayson et al.
2008). Unfortunately, prior to sampling at the SW1, the
predator-proof fence had been compromised by recurrent rains
that caused corrosion to the fencing. Furthermore, a substantial feral cat (F. catus) population was able to establish within
the Currawinya National Park reserve (SW1) and has been
singled out as the main reason for the decimation of this bilby
population (Williams and Manthey 2012). Predation is a fundamental feature of the natural environment and is known to
cause elevations of up to 89 % in glucocorticoid levels in prey
animals (Cavigelli 1999; Sheriff et al. 2009; Thaker et al.
2010). It is known that the bilby is highly sensitive to the risk
of predation (Pavey 2006) and even a slight increase in predator numbers may lead to elevations in FCM concentrations.
In future, fecal hormone monitoring could be used to monitor
the vulnerability of semi-wild bilby populations against feral
prey species in situ. Ground-based predation was not a factor
influencing the average baseline FCM concentrations recorded at the SWS (SW2). Intense management has ensured that
the sanctuary remains free from feral animal species. The only
issue is that the bilby population sampled at SW2 exists at
high density (~1 individual per 3 ha) and co-inhabits the
fenced compound with a particularly high-density population
of bridled nail-tail wallaby (Onychogalea fraenata; ~2
wallabies/ha) (AWC 2012). It is evident that within closed
populations high population densities lead to high levels of
both intra- and inter-specific competition (Rogovin et al.
2003). At the SW2, as in any closed population, the issue of
available space is pertinent and when combined with the
particularly high density of bridled nail-tail wallabies with
which bilbies share the area, the effects of competition are
likely to be significant. Inter-specific competition has been
known to change circadian activity rhythms, habitat preferences, and foraging sites and has been shown to cause significant elevations in FCM levels in mammals Buzzard 2006;
Watts and Holekamp 2008; Forristal et al. 2012). Furthermore,
when space becomes limited then access to food often also
becomes limited (Creel and Creel 1998). The population of
bridled nail-tail wallabies within the breeding compound at
the SW2 has reached to a point where supplementary food is
required (and is provided every other day) (AWC 2012).
Whilst very little is known of the effects of supplementary
feeding on FCM levels in mammals (Krebs 1996), one study
reported that wild supplementary fed elk (Cervus elaphus) had
FCM concentrations 43 % higher than non-supplementary fed
elk during times of food scarcity and also documented strong
Eur J Wildl Res
correlations between FCM levels and animal density. It is
believed that the artificial aggregation around feeding sites
and the associated social interactions may be the primary
cause behind these results (Forristal et al. 2012). Overall, it
is clearly evident from our results that bilbies at both SW1 and
SW2were experiencing stress to some degree due to the
unpredictable environmental stimuli and factors discussed
above. Furthermore, the mean FCM levels were pretty close
to the highest mean level of FCM levels recorded in the bilbies
after the ACTH challenge. Thus, comparatively it is likely that
the HPA axis of bilbies in the semi-wild environment will
express higher circulatory levels of cortisol. It will be interesting to investigate potential modulation of the adrenocortical
response to ACTH in semi-wild bilbies in order to confirm
whether these populations are indeed stressed. We strongly
recommend that the data should be treated with caution when
using them to explain whether the bilbies are facing distress
(pain) or eustress (i.e., good stress). This is because there is no
‘gold standard’ level of bilby FCM level above which the
animal is negatively stressed.
The comparison of average FCM concentrations in captive
populations revealed that individuals housed at the C4 had an
appreciably higher average FCM level than the other three
captive populations sampled. The most apparent difference
between this population and the other captive populations is
that these two individuals were housed in separate large,
unroofed, outdoor enclosures. Bilbies housed at the C1, C2
and C3 were all housed in entirely indoor or roofed enclosures
with access to an indoor area. It is possible that the elevated
FCM levels recorded in the individuals housed at C4are
related to their relative exposure to environmental conditions
and aerial predation from birds of prey. Furthermore, individuals housed at C1, C2 and C3 were provided artificial burrows
and ‘hides’ for resting and are fed within a daily routine,
whereas the two males housed at C3 engage in natural behaviours such as foraging during the night and have to dig their
own burrows for shelter.
The environment in which an animal lives can greatly
influence its biology (Schwarzenberger 2007). Past studies
have also reported significant differences in average baseline
FCM levels between captive and free-ranging populations of
the same species. Some of which found that captive populations had significantly lower averages than their wild conspecifics (e.g., Gilbert’s potoroo (Potorous gilbertii); SteadRichardson et al. 2010) whereas other studies have demonstrated the opposite pattern. (Terio et al. 2004) reported that
concentrations of baseline FCM were significantly higher in
captive cheetahs (Acinonyx jubatus) than in free ranging cheetahs. Based on our findings of difference in mean FCM
between the semi-wild and captive populations, we suggest
that the wide range of unpredictable environment within the
two fenced reserves (SW1 and SW2) sampled for this study
are generally more challenging than the predictable stimuli
(such as adequate security and environmental enrichment)
offered in captivity. Similar results have also been reported
in the Tuco-tuco (Ctenoours sociabilis) (Woodruff et al.
2010), and yellow-bellied marmot (Marmota flaviventris)
(Smith et al. 2012).
In conclusion, by determining average baseline FCM levels
for the bilby in both semi-wild and captive populations, future
research can now focus on investigating the causes of short
term change and/or long-term elevations in FCM levels. The
identification of sources of high FCM in semi-wild populations would provide an opportunity for wildlife managers to
intervene and, for example, change social dynamics, housing
or enrichment or implement feral animal control programs in
order to minimise the effects on the animal’s well-being. Thus
these findings have important implications for future management of threatened mammalian species by providing a simple
quantification tool for monitoring the effects of environmental
change on semi-free ranging populations.
Acknowledgments This project was completed in accordance with
approval from Griffith University’s Animal Ethics Committee (ENV/
17/11/AEC). Field work was conducted under Queensland Department
of Environment and Heritage Protection scientific permit number
WITK10064911. This research was undertaken as an Honours Research
Project by NE that was co-supervised jointly by EJN and J-MH. We
would like to thank the Australian Wildlife Conservancy and Queensland
National Parks and Wildlife Service for providing field equipment, onsite accommodation and other facilities whilst sampling in the field.
Special thanks to numerous volunteers assisted with the fieldwork and
Greg Lollback provided feedback on earlier version of this manuscript.
We also thank the staff of Dreamworld, Ipswich Nature Centre,
Currumbin Wildlife Sanctuary and Charleville Breeding centre for all
their help and cooperation. We also thank the veterinarians Vere Nicolson
and Michael Pyne who assisted with applying the ACTH and saline
treatments. Funding was provided by Save the Bilby Fund and Griffith
University. We are grateful to the editor and two anonymous referees for
helpful reviews and comments.
References
AWC (2012) Scotia Sanctuary. http://www.australianwildlife.org/AWCSanctuaries/Scotia-Sanctuary.aspx. Accessed 3/2/2012
Barja I, Silván G, Illera JC (2008) Relationships between sex and stress
hormone levels in feces and marking behavior in a wild Population of
Iberian wolves (Canis lupus signatus). J Chem Ecol 34(6):697–701
Benhaiem S, Dehnhard M, Bonanni R, Hofer H, Goymann W,
Eulenberger K, East M (2012) Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in
spotted hyenas (Crocuta crocuta). Gen Comp Endocrinol 178:
265–271
Bonier F, Moore IT, Martin PR, Robertson RJ (2009) The relationship
between fitness and baseline glucocorticoids in a passerine bird. Gen
Comp Endocrinol 163(1–2):208–213
Bosacker AL (2008) Stress, evolution, and sociality in wild female
baboons. 3310637. University of Minnesota, Minnesota
Buzzard PJ (2006) Cheek pouch use in relation to interspecific competition and predator risk for three guenon monkeys (Cercopithecus
spp.). Primates 47(4):336–341
Eur J Wildl Res
Cavigelli SA (1999) Behavioural patterns associated with faecal cortisol
levels in free-ranging female ring-tailed femurs, Lemur catta. Anim
Behav 57:935–944
Chapman CA, Saj TL, Snaith TV (2007) Temporal dynamics of nutrition,
parasitism, and stress in Colobus monkeys: implications for population regulation and conservation. Am J Phys Anthropol 134:240–250
Cooke SJ, Sack L, Franklin CE, Farrell AP, Beardall J, Wikelski M,
Chown SL (2013) What is conservation physiology? Perspectives
on an increasingly integrated and essential science. Conserv Physiol
1(1):1–23
Creel S, Creel NM (1998) Six ecological factors that may limit African
wild dogs, Lycaon pictus. Anim Conserv 1(1):1–9
Dantzer B, McAdam AG, Palme R, Fletcher QE, Boutin S, Humphries
MM, Boonstra R (2010) Fecal cortisol metabolite levels in freeranging North American red squirrels: assay validation and the
effects of reproductive condition. Gen Comp Endocrinol 167(2):
279–286
Dehnhard M, Clauss M, Lechner-Doll M, Meyer HHD, Palme R (2001)
Noninvasive monitoring of adrenocortical activity in roe deer
(Capreolus capreolus) by measurement of fecal cortisol metabolites.
Gen Comp Endocrinol 123(1):111–120
DEHP (2012) Currawinya National Park. http://www.derm.qld.gov.au/
parks/currawinya/index.html. Accessed 2/2/2012
Dunwoody E, Liu X, McDougall KA (2009) Spatial analysis of greater
bilby (Macrotis lagotis) habitat in south-west queensland. In:
Ostendorf B, Baldock P, Bruce D, Burdett M, Corcoran P (eds)
Surveying & Spatial Sciences Institute Biennial International
Conference. Spatial Sciences Institute, Adelaide, pp 977–986
Ellenberg U, Setiawan AN, Cree A, Houston DM, Seddon PJ (2007)
Elevated hormonal stress response and reduced reproductive output
in yellow-eyed penguins exposed to unregulated tourism. Gen
Comp Endocrinol 152(1):54–63
Ellis T, James JD, Stewart C, Scott AP (2004) A non-invasive stress assay
based upon measurement of free cortisol released into the water by
rainbow trout. J Fish Biol 65:1233–1252
Evans N, Narayan E, Hero J-M (2013) Effects of natural weathering
conditions on glucocorticoid metabolite measurements in the greater
bilby faeces. Aust J Zool 61:351–356
Fanson KV, Wielebnowski NC, Shenk TM, Lucas JR (2012)
Comparative patterns of adrenal activity in captive and wild
Canada lynx (Lynx canadensis). J Comp Physiol B Biochem Syst
Environ Physiol 182(1):157–165
Finlayson GR, Vieira EM, Priddel D, Wheeler R, Bentley J, Dickman CR
(2008) Multi-scale patterns of habitat use by re-introduced mammals: a case study using medium-sized marsupials. Biol Conserv
141(1):320–331
Forristal VE, Creel S, Taper ML, Scurlock BM, Cross PC (2012) Effects
of supplemental feeding and aggregation on fecal glucocorticoid
metabolite concentrations in elk. J Wildl Manag 76(4):694–702
Fuller G, Margulis SW, Santymire R (2011) The effectiveness of indigestible markers for identifying individual animal feces and their
prevalence of use in North American zoos. Zoo Biol 30(4):379–398
Gibson LA (2001) Seasonal changes in the diet, food availability and
food preference of the greater bilby (Macrotis lagotis) in southwestern Queensland. Wildl Res 28(2):121–134
Griffin B (2002) The use of fecal markers to facilitate sample collection in
group-housed cats. Contemp Top Lab Anim Sci 41(2):51–56
Harper JM, Austad SN (2000) Fecal glucocorticoids: a noninvasive
method of measuring adrenal activity in wild and captive rodents.
Physiol Biochem Zool 73(1):12–22
Hesterman H, Jones SM, Schwarzenberger F (2008) Reproductive endocrinology of the largest dasyurids: characterization of ovarian cycles
by plasma and fecal steroid monitoring: Part I. The Tasmanian devil
(Sarcophilus harrisii). Gen Comp Endocrinol 155(1):234–244
Hogan LA, Johnston SD, Lisle AT, Keeley T, Wong P, Nicolson V,
Horsup AB, Janssen T, Phillips CJC (2011) Behavioural and
physiological responses of captive wombats (Lasiorhinus latifrons)
to regular handling by humans. Appl Anim Behav Sci 134(3):217–
228
Hogan L, Lisle A, Johnston S, Robertson H (2013) Eliminative behaviour
of captive numbats (Myrmecobius fasciatus): pattern and identification of faecal deposits. Zoo Biol, submitted for publication
IUCN (2006) IUCN Red List of Threatened Species.
James AI, Eldridge DJ (2007) Reintroduction of fossorial native mammals and potential impacts on ecosystem processes in an Australian
desert landscape. Biol Conserv 138(3–4):351–359
Kalz B, Jewgenow K, Fickel J (2006) Structure of an otter (Lutra lutra)
population in Germany — results of DNA and hormone analyses
from faecal samples. Mamm Biol 71(6):321–335
Krebs CJ (1996) Population cycles revisited. J Mammal 77(1):8–24. doi:
10.2307/1382705
Marechal L, Semple S, Majolo B, Qarro M, Heistermann M, MacLarnon
A (2011) Impacts of tourism on anxiety and physiological stress
levels in wild male Barbary macaques. Biol Conserv 144(9):2188–
2193
McDonald IR, Lee AK, Bradley AJ, Than KA (1981) Endocrine changes
in dasyurid marsupials with differing mortality patterns. Gen Comp
Endocrinol 44(3):292–301
Millspaugh J, Washburn B (2004) Use of fecal glucocorticold metabolite
measures in conservation biology research: considerations for application and interpretation. Gen Comp Endocrinol 138(3):189–199
Moseby KE, Hill BM, Read JL (2009) Arid recovery — a comparison of
reptile and small mammal populations inside and outside a large
rabbit, cat and fox-proof exclosure in arid South Australia. Aust Ecol
34(2):156–169
Muller MN, Wrangham RW (2004) Dominance, cortisol and stress in
wild chimpanzees (Pan troglodytes schweinfurthii). Behav Ecol
Sociobiol 55(4):332–340
Narayan E (2013) Non-invasive reproductive and stress endocrinology in
amphibian conservation physiology. Conserv Physiol 1:1–16
Narayan E, Hero J-M (2014a) Acute thermal stressor increases glucocorticoid response but minimizes testosterone and locomotor performance in the cane toad (Rhinella marina). PLoS One 9:e92090
Narayan E, Hero J-M (2014b) Repeated thermal stressor causes chronic
elevation of baseline corticosterone and suppresses the physiological endocrine sensitivity to acute stressor in the cane toad (Rhinella
marina). J Therm Biol 41:72–76
Narayan E, Molinia F, Christi K, Morley C, Cockrem J (2010) Urinary
corticosterone metabolite responses to capture and annual patterns
of urinary corticosterone in wild and captive endangered Fijian
ground frogs (Platymantis vitiana). Aust J Zool 58:189–197
Narayan E, Hero JM, Evans N, Nicolson V, Mucci A (2012) Noninvasive evaluation of physiological stress hormone responses in a
captive population of the greater bilby Macrotis lagotis. Endanger
Species Res 18:279–289
Narayan E, Cockrem JF, Hero J-M (2013a) Sight of a predator induces a
corticosterone stress response and generates fear in an amphibian.
PLoS One 8:e73564
Narayan E, Webster K, Nicolson V, Hero J-M (2013b) Non-invasive evaluation of physiological stress in an iconic Australian marsupial: the Koala
(Phascolarctos cinereus). Gen Comp Endocrinol 187:39–47
Narayan E, Clark G, Martin-Vegue P, Parnell T, Mucci A, Hero J-M
(2013c) Faecal cortisol metabolite levels in Bengal (Panthera tigris
tigris) and Sumatran tigers (Panthera tigris sumatrae). Gen Comp
Endocrinol 194:318–325
Owen MA, Swaisgood RR, Czekala NM, Steinman K, Lindburg DG
(2004) Monitoring stress in captive giant pandas (Ailuropoda
melanoleuca): behavioral and hormonal responses to ambient noise.
Zoo Biol 23:147–164
Palme R (2005) Measuring fecal steroids — guidelines for practical
application. In: Bauchinger U, Goymann W, JenniEiermann S
(eds) Bird hormones and bird migrations: analyzing hormones in
Eur J Wildl Res
droppings and egg yolks and assessing adaptations in long-distance
migration. Ann N Y Acad Sci 1046:75–80
Park HC, Han TY, Kim DC, Min MS, Han SY, Kim KS, Lee H (2011)
Individual identification and sex determination of Eurasian otters
(Lutra lutra) in Daegu city based on genetic analysis of otter spraint.
Genes Genomics 33(6):653–657
Pavey C (2006) National recovery plan for the greater bilby, Macrotis
lagotis. Northern Territory Department of Natural Resources,
Environment and the Arts.
Rogovin K, Randall JA, Kolosova I, Moshkin M (2003) Social correlates
of stress in adult males of the great gerbil, Rhombomys opimus, in
years of high and low population densities. Horm Behav 43(1):132–
139
Romero LM, Wikelski M (2001) Corticosterone levels predict survival
probabilities of Galapagos marine iguanas during El Nino events.
Proc Natl Acad Sci U S A 98(13):7366–7370
Schwarzenberger F (2007) The many uses of non-invasive faecal steroid
monitoring in zoo and wildlife species. Int Zool Yearb 41(1):52–74
Sheriff MJ, Krebs CJ, Boonstra R (2009) The sensitive hare: sublethal
effects of predator stress on reproduction in snowshoe hares. J Anim
Ecol 78(6):1249–1258
Sheriff MJ, Dantzer B, Delehanty B, Palme R, Boonstra R (2011)
Measuring stress in wildlife: techniques for quantifying glucocorticoids. Oecologia 166:869–877
Smith S, McRae P, Hughes J (2009) Faecal DNA analysis enables genetic
monitoring of the species recovery program for an arid-dwelling
marsupial. Aust J Zool 57(2):139–148
Smith JE, Monclus R, Wantuck D, Florant GL, Blumstein DT (2012)
Fecal glucocorticoid metabolites in wild yellow-bellied marmots:
experimental validation, individual differences and ecological correlates. Gen Comp Endocrinol 178(2):417–426
Stead-Richardson E, Bradshaw D, Friend T, Fletcher T (2010).
Monitoring reproduction in the critically endangered marsupial,
Gilbert’s potoroo (Potorous gilbertii): Preliminary analysis of faecal
oestradiol-17 beta, cortisol and progestagens. Gen Comp Endocrinol
165(1):155–162
Terio KA, Marker L, Munson L (2004) Evidence for chronic stress in
captive but not free-ranging cheetahs (Acinonyx jubatus) based on
adrenal morphology and function. J Wildl Dis 40:259–266
Thaker M, Vanak AT, Lima SL, Hews DK (2010) Stress and aversive
learning in a wild vertebrate: the role of corticosterone in mediating
escape from a novel stressor. Am Nat 175(1):50–60
Turner JW, Nemeth R, Rogers C (2003) Measurement of fecal glucocorticoids in parrotfishes to assess stress. Gen Comp Endocrinol 133:
341–352
Wasser SK, Hunt KE (2005) Noninvasive measures of reproductive
function and disturbance in the barred owl, great horned owl, and
northern spotted owl. Ann N Y Acad 1046:109–113
Watts HE, Holekamp KE (2008) Interspecific competition influences
reproduction in spotted hyenas. J Zool 276(4):402–410
Wielebnowski NC, Fletchall N, Carlstead K, Busso JM, Brown JL (2002)
Noninvasive assessment of adrenal activity associated with husbandry and behavioral factors in the North American clouded leopard population. Zool Biol 21(1):77–98
Wikelski M, Cooke SJ (2006) Conservation physiology. Trends Ecol
Evol 21(1):38–46
Williams B, Manthey F (2012) Feral cats wreak havoc in raid on
'enclosed' refuge for endangered bilbies. The Courier-Mail, July
19. Online accessible.
Woodruff JA, Lacey EA, Bentley G (2010) Contrasting fecal corticosterone metabolite levels in captive and free-living colonial Tuco-tucos
(Ctenomys sociabilis). J Exp Zool A 313A(8):498–507
Young KM, Walker SL, Lanthier C, Waddell WT, Monfort SL, Brown JL
(2004) Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticold analyses. Gen Comp Endocrinol
137(2):148–165