Study on Reduced Stress Levels in Koalas Provided with Water Supplementation

Koalas (Phascolarctos cinereus) are a defining faunal symbol of Australia (Cork et al., 2000). The Koala is an endemic, herbivorous marsupial species native to Australia (Adams-Hosking et al., 2011). However, the last few decades have been characterised by significant declines of between 50-80% of northern Koala populations (McAlpine et al., 2015; Rhodes et al., 2011; Seabrook et al., 2011). As such, since 2012 Koala populations in New South Wales (NSW) and Queensland (QLD) have been listed as vulnerable to extinction by the Environmental Protection Biodiversity Conservation Act 1999 (EPBC Act) and ICUN red list; emphasising that Koala’s face an ever-growing number of threatening processes (Beyer et al., 2018). These include predation from wild canines, habitat loss and fragmentation, vehicle collisions, the pressure of diseases such as Chlamydia and Koala retrovirus and Australia’s changing climate (Dique et al., 2003; Lunney et al., 2007; Melzer et al., 2000; Polkinghorne et al., 2013; Rhodes et al., 2011).

Australia’s changing climate has resulted in increasing ambient temperatures, which combined with the specificity of Koalas habitat and diet contributes to their increased vulnerability to climate change (DeGabriel et al., 2010; Ellis et al., 2010). Inland areas of QLD and NSW have experienced increased ambient temperatures, and occurrence of droughts and reduced humidity and rainfall, which may potentially, directly and indirectly, affect koala health and survival (Collins, 2000; Hughes, 2003; Plummer et al., 1999). As arboreal folivores, follicular moisture supplies the majority of the Koalas nutritional requirements (Ellis et al., 1995; Moore et al., 2005).

Increasing temperatures and diminished rainfall reduces the nutritional and moisture content of species which are critical components of Koala’s diet (Cork et al., 2000; Davies et al., 2014). This reduced water availability results in physiological stress in a number of species including koalas (Davies et al., 2014; Fredriksson et al., 2007; Van Horne et al., 1998). This may result in altered behaviour, metabolism, energy allocation and reproductive cycles and a reduced immune system, increasing Koalas vulnerability to current threatening processes, or chronic stress levels may cause death (McEwen & Wingfield, 2010; Romero, 2004; Sapolsky, 2002).

Therefore, the aim of this investigation is to examine whether providing koalas access to water through supplementary drinking stations will aid in improving long-term population health. Physiological stress from a lack of water availability may be the cause of koalas increased susceptibility to other threating processes thus, reducing the stress associated with a lack of water may result in improving koala long-term population health and improved resilience to other key threatening processes and aid in the conservation of this species.

Hypothesis: Koalas provided with water supplementation will have reduced stressed levels

Study Objectives:

• Do koalas supplied with water supplementation have reduced stress levels?
• Do the stress levels of koalas change seasonally?
• If water supplementation used, does the use change seasonally?
• Is there a seasonal difference in leaf moisture in Wonderful plains?

Experimental Design

Ethics and research permits will be acquired for all activities related to studying the koalas including sampling and experimental procedures according to the animal ethics committee, as well as a scientific license from the NSW National Parks and Wildlife Service.

A pilot study will be conducted in order to understand the Wonderful plains koala population number, composition and distribution. This will involve placing telemetry collars fitted with GPS trackers on at least 60 koalas. They will be set to record the Koalas position six times over 24 hours, observing movements, social groups and resource use for six months (Davies et al., 2013b). In addition to field-based observations such as the presence of food tree species and koala scats. The analysis of these results will be used to confirm the study design for the main study.

The main study will be conducted over two years to examine the effect of water supplementation. Based on the home ranges and social groups determined in the pilot study, ten groups of least six adult koalas will be identified and organised into treatment groups (Figure 1). Five of these groups will be the controls (no water supplementation) and five treatment groups (provided with water supplementation). The treatments will be randomly allocated to the ten groups of koalas. Each group will be at least 500 m away from each other to ensure independence (Davies et al., 2013b). Two water stations will be placed in each of the five water treatment groups, in the fork of trees (between 1. 5 and 2m high) which are heavily used to ensure the water station is found and to avoid the stations being used by other animals. The water stations will consist of an automatically refilling water bowl, that is connected to a rainwater tank to provide water ad libitum. The use of the water stations will be monitored by motion trigger cameras placed 1m above the drinking stations. The footage will be analysed in J-watcher program, using an ethogram to quantify the animal activity budget in relation to the use of the water stations (e. g. the number of visits and time at the stations). Temperature will be monitored through the use of Thermochron iButtons placed 1. 5m up the southern side of the trees with water stations on them. Maximum daily temperature, daily humidity, wind speed and average rainfall quantity and frequency will be obtained from the local weather station.

Once a month at least fresh two scat samples will be collected from the base of trees from each of the ten treatment locations and faecal cortisol analysis will be conducted following the protocol described by Davies et al. (2013a) and Johnston et al. (2013). Briefly, faecal pellets will be refined to remove debris such as leaf particles, hormones will be extracted, and the faecal cortisol metabolite (FCM) concentration will be determined using cortisol enzyme immunoassay (EIA). At the same time, sites will be managed, including, cleaning water bowls to prevent disease transmission, refilling water tanks, and checking equipment. Once a season (four times a year) leaves will be collected from trees known to be used by the koalas in each of the 10 treatment locations. The leaves moisture content will be analysed following the protocol described by Mella et al. (2019). Briefly, this includes weighing the fresh leaves then drying them in an incubator/oven, weighing their dry weight and calculating the water content as a percentage of the fresh weight.

A power calculation was used to identify the sample size that could distinguish a discernible difference in the population health between the control and treatment groups. At least 28 koalas are required in each treatment (control and water supplementation) for a 95% confidence interval (Ausvet). Therefore at least 60 Koalas are required to ensure statistically significant results. R studio will be used to test assumptions and conduct the statistical analysis with significance set at p ≤ 0. 05. Individual two-way ANOVAs will be used to analyse the effect of treatments, water usage and seasonal attributes on FCM, the effect of season, seasonal attributes, leaf moisture and FCM on water usage and the season and seasonal attributes on leaf moisture.

A generalised linear mixed model (GLMM) will compare the faecal cortisol metabolites with treatment (control and water supplementation), year and season as a fixed factor and site, rainfall and temperature as a random effect. Water usage will also be assessed using a GLMM with FCM, leaf moisture content and site as random effects and season as a fixed factor. Leaf moisture will also be analysed using a GLMM with tree site, species, temperature and rainfall as random effect and season and year as fixed effects.