Pahuja - Physiological Stress Response of Koala Joeys To Visitors

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1 Physiological stress response of koala joeys to visitors.
2 Harsh K. Pahuja1* Izzy Bee2, Ali Bee2 and Edward J. Narayan 1,3
3 1
School of Agriculture and Food Sciences, Faculty of Science, The University of Queensland, St Lucia, Brisbane,
4 Queensland 4072, Australia.
5 2
Magnetic Island Koala Hospital, Horseshoe Bay, Magnetic Island, Queensland 4819, Australia
6 3
Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Brisbane,
7 Queensland 4072, Australia
8 * Correspondence: uqhpahu1@uq.edu.au
9 Abstract
10 Koala (Phascolarctos cinereus) joey rescues are increasing over the years, and rehabilitation of a joey requires
11 extensive care, close proximity and handling by humans. These novel environments are likely to present a suite of
12 biotic and abiotic stressors during rehabilitation. In this study, we longitudinally monitored the faecal cortisol
13 metabolites (FCMs) of three koala joeys within the context of potential stressors at the Magnetic Island Koala
14 Hospital, Queensland, Australia. A total of 92 faecal samples were analysed for FCMs using a polyclonal R4866
15 cortisol enzyme-immunoassay which has been previously validated in koalas. The iterative baseline method was
16 used to establish FCM profiles of all individuals, and to identify significant peaks in FCM concentrations. Visitor
17 events were identified and confirmed as an acute stressor based on the FCM profiles of the koala joeys. All three
18 koala joeys elicited a significant rise in FCM concentrations after each visitor encounter. To our knowledge, this is
19 the first study to report on the acute stress response of koala joeys to visitors. We recommend that visitor encounters
20 be kept to a minimum, and perhaps avoided altogether especially for joeys that are being rehabilitated to be released
21 back into the wild.
22 Keywords: cortisol; joey; koala; stress response; visitors
23
24
bioRxiv preprint doi: https://doi.org/10.1101/2023.10.09.561560; this version posted October 12, 2023. The copyright holder for this preprint
25 Introduction
26 The Koala (Phascolarctos cinereus) is an iconic marsupial endemic to Australia (Woinarski & Burbidge,
27 2020). Retrospective studies assessing the trends of koala admissions to wildlife hospitals highlight that the clinical
28 outcome for the majority of these koalas is that they either naturally succumb to their injuries and/or diseases, or
29 they need to be humanely euthanised due to their critical condition (Burton & Tribe, 2016; Gonzalez-Astudillo et al.,
30 2017; Griffith et al., 2013; Taylor-Brown et al., 2019). Increased mortality of adult female koalas is likely to cause
31 more rescues of young orphans because during the early stages of their life, joeys are sustained by their mothers. In
32 the study by Taylor-Brown et al. (2019), koala joeys formed the second largest group (after possums) that were
33 rescued as orphans. Furthermore, as highlighted by Pahuja and Narayan (2023a), koala joey rescues are also
34 increasing slightly over the years. Koala joeys are born in an altricial state, which is followed by a ~6-month period
35 of pouch occupancy (Gipp, 2004). During this extensive dependant period, maternal milk, eucalyptus and ‘pap’
36 constitute the diet of joeys (Barker et al. 2013; Morris et al. 2016). When orphaned, of course, this process needs to
37 be replicated by humans and requires extensive care, close proximity and handling. Being raised by a human in
38 captivity is likely to serve as a ‘stressor’ for the koala joey because of the novel environment.
39 A stressor is commonly defined as any noxious stimuli that could potentially cause an imbalance in the
40 animal’s homeostatic system (Moberg & Mench, 2000). When encountered with a stressor, the body elicits an array
41 of responses; one of which is the neuroendocrine activation of the hypothalamo-pituitary-adrenal (HPA) axis
42 (Sheriff et al., 2011). This results in the secretion of glucocorticoids (GCs) which have a range of effects (e.g.
43 energy mobilisation for fight-or-flight response) that allow the animal to achieve homeostasis i.e. cope with the
44 stressor (Moberg & Mench, 2000). As a result, GCs are often referred to as the stress hormone (Cope et al., 2022),
45 and short-term elevation of GCs are indicative of acute stress (Hogan et al., 2012). Prolonged GC elevation can have
46 deleterious impact on an animal’s health given the diverse biological effects of GCs on various body systems, and is
47 thus indicative of chronic stress (Hogan et al., 2012; Narayan et al., 2012). GCs (or its metabolites) can be measured
48 in a range of sample types, such as blood, faeces and fur. However, given the ease of collection and minimal
49 handling associated with collecting scat samples, monitoring faecal glucocorticoid metabolites (FGM)s is the most
50 commonly employed method in studies on Australian marsupials (Hing et al., 2014). Cortisol is the principal GC
51 produced by koalas (Narayan et al., 2013) and is increasingly acknowledged as a practical endocrine indicator of
52 stress (Hing et al., 2014; Narayan & Williams, 2016). Whilst there have been a number of studies investigating the
53 physiological stress response of marsupials (Cope et al., 2022; Hing et al., 2017; Hogan et al., 2011; Hogan et al.,
54 2012; Narayan et al., 2012), including koalas (Charalambous et al., 2021; Pahuja & Narayan, 2023b; Webster et al.,
55 2017), there is a paucity of literature on the capacity of koalas to respond to the stressors of captivity. In order to
56 improve the rehabilitation outcomes and ensure the welfare of koalas in captivity, it is imperative to identify the
57 potential stressors of captivity and understand how these animals respond to it.
58 In the present study, we monitored the faecal cortisol metabolites (FCM)s of three orphaned koala joeys
59 undergoing rehabilitation over a period of one-month before release. During this period, we also monitored potential
60 stressors faced by the joeys during rehabilitation. Of particular interest then was to examine whether koala joeys
61 elicit a physiological stress response to these stressors. We hypothesised that stressors are likely to cause a
62 physiological change in the joeys which will be reflected and capture as elevated FCMs. The primary aim of this
63 study was to expand the current knowledge on koala stress biology by monitoring FCMs within the context of
64 potential stressors. To our knowledge, this is the first study to report on the physiological stress response of koala
65 joeys to potential stressors.
66 Material and Methods
67 Animal Ethics
68 All procedures in this research were performed in accordance with the relevant guidelines and regulations
69 set by The University of Queensland Animal Ethics Committee: Native or Exotic Wildlife and Marine Animals
70 (NEWMA) (approval number 2022/AE000035).
71 Study Site and Animals
72 This research study was conducted in collaboration with the Magnetic Island Koala Hospital. All koala
73 joeys were housed at 3 Corica Crescent, Horseshoe Bay, QLD, Australia 4819 (GPS coordinates: 19°7′26.06″S,
74 146°51′34.06″E). A total of 3 orphaned koala joeys [1 male (M1) and 2 females (F1 and F2)] were studied based on
75 whichever was currently in care during the period of data collection (2 nd April 2022 to 2nd May 2022). All koala
76 joeys were housed individually in a 6 x 6 x 3.5 (L x W x H) meter enclosure donated by the Glencore Australia,
77 Copper Refineries Pty. Ltd. The enclosure was designed with a cement slab floor, insulated roof, snake proof mesh
78 and trim deck metal, and furnished with logs, branches, ropes and polyvinyl pipes of varying dimensions. The joeys
79 were provided with ad libitum water and feed. The feed consisted of a mix of various species of Eucalyptus leaves
80 freshly cut and sourced from a local Eucalyptus plantation. The Eucalyptus leaves were changed daily and mainly
81 consisted of (but not limited to) river red gum (E. camaldulensis), swamp mahogany (E. robusta), red mahogany (E.
82 resinifera), mountain mahogany (E. notabilis) and tallow (E. microcorys) The age for all koala joeys during data
83 collection was estimated by the veterinarian to be between 7-9 months based on tooth wear and body weight. No
84 joeys had any history of disease, and remained clinically healthy throughout the study period as reported by the
85 veterinarian.
86 Data collection (Faecal sample collection and recording stressors)
87 In order to evaluate daily changes in FCM concentrations, fresh faeces were collected routinely from all
88 three koala joeys daily, over a one-month study period. One faecal sample (comprising on average of 3-5 faecal
89 pellets) per day per animal was collected during routine cleaning of the enclosure (0900h to 1100 h), with minimal
90 disturbance to the animal. Samples were collected by hand, using a new pair of gloves, and immediately transferred
91 into a resealable polyethylene ZipLock© bag and labelled with the animal’s name, date and the time of sample
92 collection. All faeces were removed from each enclosure at the end of each day so that we could ensure fresh faecal
93 sample collection on the following day. The samples were then stored at −20°C until they were sent to the laboratory
94 on ice via overnight freight. The faecal samples were transited on ice via overnight freight and upon delivery, they
95 were immediately refrozen at −20°C until processing.
96 The staff at the Magnetic Island Koala Hospital also observed and recorded any potential stressors
97 encountered by the joeys. A range of stressors have been identified for marsupials in captivity [see Hing et al.
98 (2014), Hogan et al. (2012), Charalambous et al. (2021)], including (but not limited to) maintenance activities in and
99 around the enclosure, moving animals to a different enclosure, visitor encounters, loud noises, extreme
100 environmental events, handling for routine health checks and so on. This non-exhaustive list of potential stressors
101 was used as a reference to observe and record potential stressors that could be affecting the koala joeys.
102 Faecal sample processing and extraction

103 Faecal sample processing and FCM extraction followed the procedures previously described for koalas
104 (both, adults and joeys) (Charalambous et al., 2021; Narayan et al., 2013; Pahuja & Narayan, 2023b) Briefly, frozen
105 faecal samples were weighed and then dried in an oven at 65°C for a 24 h period, or until completely dried. Once
106 completely dry, the samples were ground into a fine powder using a mortar and pestle. Analytical grade ethanol
107 (10% v/v) was used to clean the mortar and pestle between each individual sample processing to avoid cross-
108 contamination. The ground-up powder was sifted through a fine mesh strainer to remove all coarse particles. A 0.2 ±
109 0.001 g sample of sifted product was weighed out into a labelled Eppendorf tube and then stored in a −20°C freezer
110 until use.
111 For hormone extraction, samples were removed from the −20°C freezer and 2 mL of 90% (v/v) analytical
112 grade ethanol solution was added to the Eppendorf tube. Tubes were vortexed at medium-high speed (5 min, 5000g)
113 on an Eppendorf mini-spin centrifuge for a minimum of 30 s to thoroughly mix the solution. Tubes were then placed
114 into a water bath (>60°C) for 20 min to allow hormones to dissolve in the solution. While in the bath, tubes were
115 gently manually shaken to ensure faeces remained submerged in ethanol and did not spill over the top of the tube.
116 After 30 min, the supernatant was transferred into an Eppendorf tube, closed and then centrifuged at 3000g for 5 min
117 to separate any solid faecal residue. If the sample looked turbid or had suspended particles, tubes were centrifuged
118 again for 5 min and then pipetted into a new and clean labelled Eppendorf tube. Following this, for the polyclonal
119 cortisol antibody assay (R4866; Late C. J. Munro, University of California, Davis), 0.6 mL of the aqueous solution
120 was aliquoted into a new and clean labelled Eppendorf tube. The tubes were kept in a laminar flow chamber for 24 h
121 to allow enough time for the ethanol to completely evaporate. Once ethanol was completely evaporated, 1 mL of
122 assay buffer (39 mM NaH2PO4·H2O, 61 mM NaHPO4, 15 mM NaCl and 0.1% bovine serum albumin, pH 7.0) was
123 added to each tube. The tubes were vortexed at medium-high speed (5 min, 3000g) in an Eppendorf mini-spin
124 centrifuge for 30 s, and then centrifuged at 10000g for 10 min. Following this, 850 µL of supernatant was pipetted
125 into a new and clean labelled Eppendorf tube, ensuring any solid remnants in the solution were avoided.
126 Enzyme-immunoassay (EIA)
127 All consumables for the EIA were obtained from Sigma-Aldrich, The R4866 EIA has previously been
128 validated in koalas using physiological (ACTH challenge), biological and laboratory validation techniques, and is
129 routinely used for monitoring physiological stress in koalas (Charalambous et al., 2021; Narayan, 2019; Narayan &
130 Vanderneut, 2019; Narayan et al., 2013; Webster et al., 2017). In addition, this assay has also been validated in the
131 laboratory for monitoring FCMs in koala joeys (Pahuja & Narayan, 2023b). Glucocorticoid concentrations for faecal
132 extract using the R4866 assay were determined using a polyclonal anti-cortisol antiserum diluted to 1:15 000,
133 horseradish peroxidase (HRP) conjugated cortisol 1:80 000 and cortisol standards (1.56–400 pg/well). Cross
134 reactivity of the R4866 anti-cortisol antiserum is reported as 100% with cortisol and less than 10% with other
135 steroids (with prednisolone 9.9%, prednisone 6.3%, cortisone 5% and < 1% with corticosterone) tested (Munro &
136 Stabenfeldt, 1985). Sample extracts were assayed in duplicate on Nunc Maxisorp™ plates (96 wells) (Sigma
137 Aldrich, Sydney, Australia). Plates were coated with 50 µL cortisol antibody diluted to the appropriate concentration
138 in a coating buffer (50 mmol/L bicarbonate buffer, pH 9.6), and left to stand and incubate for a minimum 12 h in a
139 fridge at 4°C. The plates were washed the next day using an automated plate washer (ELx50, BioTek™, Sursee,
140 Switzerland) with phosphate-buffered saline containing 0.5 mL/L Tween 20 to rinse away any unbound antibody.
141 Stocks of standards, faecal extracts and horseradish peroxidase labels were diluted to the appropriate concentration
142 in assay buffer. The extraction dilution rates (1:2) were based on the concentrations of pooled samples that resulted
143 in 50% binding in a previous study monitoring FCMs in orphaned koala joeys (Pahuja & Narayan, 2023b). For each
144 assay, 50 µL of cortisol standard and diluted faecal extract was added to each well based on the plate map.
145 Immediately following this, 50 µL of HRP label was added to the wells. Plates were covered and incubated at room
146 temperature for 2 h. Following a wash, 50 µL of substrate buffer (0.01% tetramethylbenzidine and 0.004% H 2O2 in
147 0.1 M acetate citrate acid buffer, pH 6.0) was then added to generate a colour change. Colour reaction was halted
148 after 15 min using 50 µL of stop solution (50 μL of 0.5 mol/L H 2SO4), and the plates were read primarily at 450 nm
149 and secondarily at 630 nm on an ELx800 (BioTek) microplate reader. The sensitivity of the faecal cortisol FCM was
150 calculated as the value 2 standard deviations from the mean response of the blank samples. The sensitivity of the
151 faecal cortisol metabolite EIA was 1.2 ± 0.2 pg well-1 (n = 3). Intra- and inter-assay coefficients of variation (CV)
152 were determined by calculating the mean of CV within a plate (for intra-assay) and between the plates (for inter-
153 assay) assayed during analysis. The intra- and inter-assay coefficients of variation were 4.26 ± 1.10 % (n = 3) and
154 6.42 ± 3.71 % (n = 3) respectively.
155 Data processing and analysis

156 All FCM concentrations were expressed as (ng/g) on the basis of net dry faeces weight. Analysis of
157 monthly profiles of FCM secretion involved establishing a baseline level in order for significant peaks to be
158 identified. FCM concentrations from individual koala joeys were analysed using an iterative baseline method
159 (Brown et al., 1994). In this method, the baseline is calculated through an iterative process, excluding concentrations
160 greater than ‘mean + 2 SD’. In successive iterations, all concentrations greater than ‘mean + 2 SD’ were excluded
161 until no points fall above this threshold. The mean iterative baseline was the mean of the remaining concentrations at
162 the completion of the iterative process. Concentrations that fall above the threshold (mean + 2 SD) of the final
163 iteration were classified as significant peaks. The ‘mean + 2 SD’ of the final iteration represents the ‘cut-off’ (i.e.
164 baseline threshold) value and all concentrations above this value were considered to be an indicative of a significant
165 acute stress response. Several measures were calculated to summarize faecal hormone profiles for each individual
166 koala joey : (1) overall mean of all samples for the collection period, (2) baseline mean that excluded all values
167 greater than ‘mean + 2 SD’ of the final iteration, and (3) peak mean that only included all values > 2 SD above the
168 baseline mean. These values are highlighted in Table 1. The iterative baseline calculation can be performed using
169 the R package hormLong derived by Fanson and Fanson (2015) for large datasets and/or when monitoring multiple
170 hormonal profiles. However, given the nature and scope of the current study, these calculations were performed in
171 Microsoft Excel 2021©.
172 Statistical analysis
173 Statistical analysis was performed in GraphPad Prism version 9.3.1. The parametric assumptions of
174 normality and homoscedasticity were tested and a single factor ANOVA, followed by Tukey’s multiple comparison
175 test, was run to determine whether overall mean, baseline mean, baseline threshold and peak mean FCM
176 concentrations differed (P < 0.05) among individuals.
177 Results
178 Identification of stressors
179 The staff at the Magnetic Island Koala Hospital identified visitors as the only potential stressor for the three
180 koala joeys undergoing rehabilitation. Visitors mainly included potential donors and their associated members
181 and/or media, in an attempt to raise funding for improving the conservation of koalas. A visitor event could be
182 described as photographing the joeys from a safe distance from inside the enclosure and/or media interviews to raise
183 awareness. Visitors were always accompanied by hospital staff and followed a strict no-contact policy. Visitors were
184 confirmed as a stressor based on the FCM profiles of all three koala joeys. These results are presented in detail
185 below.
186 FCM profiles and acute stress response of koala joeys to visitors
187 The daily FCM concentrations of the three koala joeys (Fig. 1) ranged from 10.41 to 33.27 ng/g dry faeces
188 for individual F1, 14.42 to 33.65 ng/g dry faeces for individual M1 and 15.41 to 59.82 ng/g dry faeces for individual
189 F2. There were significant differences in the overall mean (F2,88 = 7.689, p < 0.05), baseline mean (F2,75 = 13.21, p <
190 0.05), baseline threshold (F2,9 = 1.104, p < 0.05) and peak mean (F2,9 = 0.503, p < 0.05) FCM concentrations between
191 the three koala joeys over the one-month sampling period (Table 1). The overall mean FCM concentrations of F1
192 (19.36 ± 6.22 ng/g dry faeces) and M1 (22.81 ± 4.76 ng/g dry faeces) were significantly lower (p < 0.05 for both
193 comparisons) relative to F2 (34.02 ± 11.12 ng/g dry faeces), but did not differ significantly amongst each other (p =
194 0.24) (Table 1). The iterative baseline mean FCM concentrations of all three koala joeys were significantly different
195 from each other (p < 0.05 for all comparisons) (Table 1). The baseline threshold FCM concentrations of F1 (21.48 ±
196 4.67 ng/g dry faeces) and M1 (29.07 ± 1.40 ng/g dry faeces) were significantly lower (p < 0.05 for both
197 comparisons) relative to F2 (49.6 ± 2.86 ng/g dry faeces), but did not differ significantly amongst each other (p =
198 0.09) (Table 1). The peak mean FCM concentrations of F1 (29.59 ± 4.16 ng/g dry faeces) and M1 (32.19 ± 1.33 ng/g
199 dry faeces) were significantly lower (p < 0.05 for both comparisons) relative to F2 (55.52 ± 4.03 ng/g dry faeces),
200 but did not differ significantly amongst each other (p = 0.78) (Table 1). There were no consistent patterns in FCM
201 profiles of koala joeys in relation to their age and/or sex (Fig. 1).
202 Table 1. Iterative baseline method representing the overall mean, baseline mean, baseline threshold and peak mean
203 for each individual koala joey involved in the study.
Baseline
Approximate Overall mean Baseline mean Peak mean
Individual threshold
age (in months) (ng/g) (ng/g) (ng/g)
(ng/g)
F1 9 19.36 ± 6.22 a 16.51 ± 2.48 a 21.48 ± 4.67 a 29.59 ± 4.16 a
M1 9 22.81 ± 4.76 a 21.61 ± 3.72 b 29.07 ± 1.40 a 32.19 ± 1.33 a
F2 7 34.02 ± 11.12 b 31.44 ± 9.07 c 49.6 ± 2.86 b 55.52 ± 4.03 b

204 (Note: For each comparison, different superscripts (a, b, c) within the same column indicate significant mean
205 differences.)
206 All koala joeys displayed a significant increase (above baseline threshold) in FCM concentrations within 1-
207 3 days after each of visitor event (Fig. 1). After the 1st visitor event (Days 1-2), all koala joeys exhibited a significant
208 peak in FCM concentrations (Day 4) followed by an immediate return to baseline concentrations. The FCM
209 concentrations of all koala joeys remained within the baseline threshold limit (Days 5-14) until the next visitor event
210 (Days 15-16). Following the 2 nd visitor event, all koala joeys exhibited a significant peak in FCM concentrations
211 (F1: Day 18; M1: Day 19; F2: Day 19) (Fig. 1). The FCM concentrations of M1 and F2 immediately returned within
212 the baseline threshold concentrations until after the next visitor event. It is important to note that for F1, the FCM
213 concentrations decreased consistently over the following days, but remained above the baseline threshold. Following
214 the 3rd visitor event (Days 22-23), all koala joeys exhibited a significant peak in FCM concentrations (Day 24). The
215 FCM concentrations of M1 and F2 immediately returned within baseline threshold, however, the return to baseline
216 was slightly delayed for F1 (Fig. 1). The FCM concentrations of all joeys remained within the baseline threshold
217 until they were released on Day 31.

VISITOR
VISITOR
VISITOR
© Magnetic Island Koala Hospital

218
219 Fig. 1 FCM profiles of koala joeys undergoing rehabilitation at the Magnetic Island Koala Hospital. All open circles
220 illustrate baseline FCM concentrations, whereas closed circles illustrate significant peaks in FCM concentrations as
221 identified by the iterative baseline method. Vertical boxes delineate visitor events as identified by the staff at
222 Magnetic Island Koala Hospital. Solid horizontal line represents the baseline threshold value. Dashed horizontal line
223 represents the mean iterative baseline.
224 Discussion
225 The primary aim of this study was to monitor FCMs in koala joeys within the context of potential stressors.
226 As hypothesised, potentially stressful stimuli (i.e. visitors) resulted in an increase in FCM concentrations on all three
227 encounters, across all koala joeys. These findings confirm that visitors elicit an acute stress response in koala joeys.
228 To our knowledge, this is the first study to report on the acute stress response of koala joeys to visitors.
229 Individual differences in the FCM profiles of koala joeys was not an unexpected finding. These results are
230 consistent with previous research on rescued koala joeys highlighting inter-individual differences (Pahuja &
231 Narayan, 2023b). In the current study, the natural variability in FCM profiles is reflected in the overall mean,
232 baseline mean, peak mean, baseline threshold and daily variation in FCM concentrations of the koala joeys. GCs are
233 known to reflect inter- and intra- individual variation which can be mediated by several factors such as age, sex,
234 body condition, genetics, season, habituation, temperament, personality and so on (Cockrem, 2013; Guindre-Parker,
235 2020). When considering baseline FCM concentrations, F2 had significantly higher baseline FCM concentrations
236 relative to F1 and M1. As highlighted by Guindre-Parker (2020), baseline GCs are likely to be strongly correlated
237 with energetic balance of the animal. Since F2 is the youngest animal, it is possible that the elevated baseline FCM
238 concentrations of F2 are a result of its high energetic demands. However, it is also possible that since F2 had spent
239 the least amount of time under rehabilitation, it was habituated to a lesser degree to the captive environment
240 resulting in elevated baseline FCM concentrations [see (Hogan et al., 2012)]. Furthermore, as noted by the
241 veterinarian, F2 was rejected by the mum while in care, which could also contribute to the high baseline FCM
242 concentrations [see (Romero et al., 2009)]. Therefore, it is impossible to isolate a single factor that causes variability
243 among FCM profiles of individuals, because it is most likely the result of synergistic interaction between numerous
244 factors.
245 All three visitor events evoked a significant acute stress response in all joeys. It has been documented
246 previously that visitors can evoke a stress response from adult koalas subjected to photography regimes (Webster et
247 al., 2017). Although, in our study all three visitor events evoked a significant acute stress response in all joeys, the
248 magnitude of stress response and the time taken to return to baseline concentrations differed for each individual and
249 for each visitor event. Incorporating these findings into the reactive scope model (RSM) [as proposed by (Romero et
250 al., 2009)] highlights new avenues about the physiological coping mechanisms of koalas. The RSM proposes that
251 the physiological mediators associated with the stress response can be divided into four distinct ranges – (i)
252 predictive homeostasis (ii) reactive homeostasis (iii) homeostatic failure and (iv) homeostatic overload. The
253 predictive range encompasses physiological mediator levels that vary in response to predictable environmental
254 change i.e. tidal, seasonal and circadian rhythm. The reactive range encompasses physiological mediator levels that
255 vary in response to unpredictable changes i.e. response to stressors. The predictive and reactive range together form
256 the normal reactive scope of an animal, and is essential for the day-to-day functioning of the animal. The
257 homeostatic overload range encompasses physiological mediator levels above the normal reactive scope i.e. chronic
258 stress. The homeostatic failure range encompasses physiological mediator levels below the normal reactive scope
259 i.e. insufficiency to respond to stressors. The homeostatic overload and failure serve as two extremes of the RSM
260 and are associated with detrimental pathological effects (Romero et al., 2009).
261 In our study, the baseline concentrations (represented by open circles in Fig. 1) can be presumed to be an
262 indicative of the predictive homeostasis range, representing the natural rhythm in FCMs. After the 1st visitor event,
263 the FCM concentrations of all individuals increased significantly, indicating a classic activation of the HPA axis,
264 followed by an immediate return to baseline concentrations indicating the deactivation of the HPA axis (via the
265 negative feedback loop) once the stressor had passed (Sheriff et al., 2011). The significant increases in FCM
266 concentrations (represented by closed circles in Fig. 1) can be presumed to be an indicative of the reactive
267 homeostasis range wherein the transient rise in FCM concentration enables the animal to cope with the stressor and
268 re-establish homeostasis (Wingfield, 2013). Following the 2nd visitor event, the FCM concentrations of F1 were
269 elevated for a longer duration, relative to M2 and F2 which followed a similar pattern of immediately returning to
270 baseline concentrations. Two scenarios are possible in this case for F1 – (i) presence of a prolonged stressor i.e.
271 visitors were present on successive days (ii) delayed termination of stress response. Both of these scenarios make the
272 animal susceptible to enter the homeostatic overload range that can have deleterious impact on the animal (Romero
273 et al., 2009). Since the staff at the Magnetic Island Koala Hospital confirmed the absence of visitors on successive
274 days, the presence of prolonged stressor can be ruled out. Therefore, it could be presumed that the termination of the
275 stress response was delayed due to weaker efficacy of the negative feedback loop. Delayed termination of stress
276 response and its impact on the animal has been discussed thoroughly by Romero (2012) by highlighting the example
277 of Galápagos marine iguanas (Amblyrhynchus cristatus). Essentially, a weaker negative feedback extends the stress
278 response, and mimics a prolonged and sustained stressor such that the FCM concentrations are elevated into the
279 reactive homeostasis range for a much longer period. As a result there the threshold between the reactive
280 homeostasis range and homeostatic overload range progressively shrinks (Wright et al., 2023). For F1, this could
281 imply that an additional stressor is likely to drive this animal into the homeostatic overload range and the
282 pathological consequences associated with it. This obviously depends on duration and magnitude of the additional
283 stressor (Romero et al., 2009). In comparison to the first two events, the stress response was elicited more
284 immediately during the 3rd visitor event across all joeys. It is possible that this could be due to the substantially
285 shorter recuperation time between the 2nd and 3rd visitor event. Frequent repeated stressors do not allow enough time
286 for the recovery of threshold between the reactive homeostasis range and homeostatic overload range (Wright et al.,
287 2023). This implies that visitors that elicit an acute stress response from the koala joeys, if repeated frequently over
288 several days (without recovery time) could drive the animal into the homeostatic overload range leading to chronic
289 stress (unless the joeys get acclimatised to visitors). This again obviously depends on the rate of decline of the
290 threshold between the homeostatic overload and the reactive homeostasis and whether or not joeys are acclimatised
291 to visitors (Wright et al., 2023). We would like to reiterate that the discussion about the aforementioned scenarios
292 are based on presumptions, and there is no direct evidence to confirm this. However, there are real-life examples
293 [see (DuRant et al., 2016; Romero, 2012)] that incorporate the reactive scope model to justify (and predict) the
294 physiological response to stressors with empirical evidence. Future research on koalas involving a variety of acute
295 stressors may help get insights about the reactive scope model and the physiological coping mechanisms of koalas.
296 The concentration of FCMs in all koala joeys decreased within 1-3 days following visitor events, and
297 remained within baseline until the next visitor event, indicating that the joeys were not chronically stressed. Instead,
298 these results strongly suggest that the koala joeys are physiologically responding to environmental cues (visitors in
299 this case) to maintain homeostasis. It is important not to overstate the impact of visitors observed in this study. It
300 was evident that visitors were aversive enough to activate the HPA axis, but there was no direct evidence that this
301 translated into a detrimental effect on the welfare of the joeys. In this study, all visitor encounters were acute,
302 resulting in only transient elevations of FCM concentrations and there was no evidence of chronic stress in any
303 animal.
304 Conclusion
305 In summary, this study has provided a better understanding of the koala joey stress physiology and opened
306 up new avenues for incorporating stress physiology models to comprehend the stress response of koalas. FCM
307 profiles of koala joeys varied significantly and can be attributed to the natural variation between individuals. Daily
308 profiling of FCM secretion revealed that visitors serve as an acute stressor for rehabilitating koala joeys and evoke a
309 significant stress response. Whether or not koala joeys would elicit a similar response when encountered by familiar
310 people (i.e. husbandry staff) remains to be investigated. The findings of this study have established a basis for the
311 future monitoring of wellbeing in koala joeys and provide insight into how existing stress management strategies
312 can be enhanced. It is important that visitor encounters be kept to a minimum and perhaps avoided altogether
313 especially for young rehabilitating joeys. However, at the same time we also acknowledge that visitors, donors and
314 the media, all contribute substantially to the overall broader goal of improving the conservation of koalas.
315 Abbreviations
316 FCM: Faecal cortisol metabolite; EIA: Enzyme-immunoassay; GC: Glucocorticoid; HPA: Hypothalamo-pituitary
317 adrenal; HRP: Horseradish peroxidase; ACTH: Adrenocorticotropic hormone; RSM: Reactive Scope Model.
318 Declarations: Not applicable
319 Ethics approval and consent to participate: The study was conducted according to the guidelines of the Native or
320 Exotic Wildlife and Marine Animals (NEWMA) Animal Ethics Committee and approved by the Institutional
321 Review Board (or Ethics Committee) of The University of Queensland (approval number: AE000035).
322 Consent for publication: Not applicable
323 Availability of data and material: All data are contained within the article.
324 Competing interests: The authors have no competing interests to declare that are relevant to the content of this
325 article.
326 Funding: This research received funding from the Australian Koala Foundation (AKF). H.P. also received funding
327 through The University of Queensland Research Training Program (RTP) Scholarship.
328 Authors' contributions: Conceptualization, E.N. and H.P.; methodology, E.N. and H.P.; formal analysis, E.N. and
329 H.P.; investigation, E.N. and H.P.; resources, E.N. and H.P.; sample collection, A.B. and I.B. data curation, E.N. and
330 H.P.; writing—original draft preparation, H.P.; writing—review and editing, E.N.; H.P.; A.B. and I.B.; supervision,
331 E.N.; project administration, E.N. All authors have read and agreed to the published version of the manuscript.
332 Acknowledgements: We thank the Magnetic Island Koala Hospital for their collaboration. We immensely
333 appreciate the time, effort and the support offered by the staff in providing us with the samples and the veterinarians
334 for providing identifying potential stressors encountered by the joeys.
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411 Figure Legends
412 Fig. 1 FCM profiles of koala joeys undergoing rehabilitation at the Magnetic Island Koala Hospital. All open circles
413 illustrate baseline FCM concentrations, whereas closed circles illustrate significant peaks in FCM concentrations as
414 identified by the iterative baseline method. Vertical boxes delineate visitor events as identified by the staff at
415 Magnetic Island Koala Hospital. Solid horizontal line represents the baseline threshold value. Dashed horizontal line
416 represents the mean iterative baseline.
417 Table Legends
418 Table 1. Iterative baseline method representing the overall mean, baseline mean, baseline threshold and peak mean
419 for each individual koala joey involved in the study.

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bioRxiv preprint doi: https://doi.org/10.1101/2023.10.09.561560; this version posted October 12, 2023.

The copyright holder for this preprint

1 Physiological stress response of koala joeys to visitors.

4 Queensland 4072, Australia.

7 Queensland 4072, Australia

21 back into the wild.

22 Keywords: cortisol; joey; koala; stress response; visitors

65 joeys to potential stressors.

66 Material and Methods

70 (NEWMA) (approval number 2022/AE000035).

71 Study Site and Animals

86 Data collection (Faecal sample collection and recording stressors)

95 were immediately refrozen at −20°C until processing.

102 Faecal sample processing and extraction

110 until use.

126 Enzyme-immunoassay (EIA)

154 6.42 ± 3.71 % (n = 3) respectively.

155 Data processing and analysis

171 Microsoft Excel 2021©.

172 Statistical analysis

176 concentrations differed (P < 0.05) among individuals.

178 Identification of stressors

203 for each individual koala joey involved in the study.

M1 9 22.81 ± 4.76 a 21.61 ± 3.72 b 29.07 ± 1.40 a 32.19 ± 1.33 a

F2 7 34.02 ± 11.12 b 31.44 ± 9.07 c 49.6 ± 2.86 b 55.52 ± 4.03 b

217 until they were released on Day 31.

© Magnetic Island Koala Hospital

© Magnetic Island Koala Hospital

223 represents the mean iterative baseline.

318 Declarations: Not applicable

322 Consent for publication: Not applicable

334 for providing identifying potential stressors encountered by the joeys.

339 Queensland. Animals, 6(9), 56.

343 Endocrinology, 181, 45-58.

345 C. A. (2022). Validation of an enzyme immunoassay to measure faecal glucocorticoid metabolites in

347 12(13), 1627.

351 endocrinology studies (2167-9843).

357 Reports, 7(1), 1-11.

361 Integrative and Comparative Biology, 60(1), 79-88. https://doi.org/10.1093/icb/icaa003

363 Australian marsupials. Conservation Physiology, 2(1).

366 endangered woylies (Bettongia penicillata). EcoHealth, 14, 128-138.

372 and Comparative Endocrinology, 179(3), 376-383.

374 welfare. CABI Pub.

378 environmental trauma and disease. Scientific Reports, 9(1), 1-9.

381 Research, 18(3), 279-289.

386 Comparative Endocrinology, 187, 39-47.

390 and mortality in rescued koala joeys. Wildlife Research.

392 undergoing rehabilitation. Australian Mammalogy.

398 for quantifying glucocorticoids. Oecologia, 166(4), 869-887.

407 e.T16892A166496779. Retrieved 12 August 2021, from https://dx.doi.org/10.2305/IUCN.UK.2020-

411 Figure Legends

416 represents the mean iterative baseline.

417 Table Legends

419 for each individual koala joey involved in the study.

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