Study design

Twelve wild-caught koalas were captured from mature manna gum (E. viminalis) forest over a 3.5 km2 area at Cape Otway (38° 50′ 8.07“ S, 143° 31’ 7.00” E) and brought into captivity at the Conservation Ecology Centre, Cape Otway, Victoria (Additional file 2: Figure S2; Additional file 3: Table S1). Koalas were captured in two cohorts of six koalas (3 male and 3 female). The first cohort was held between October and December 2017 and the second was kept from January to March 2018. See Additional file 2: Figure S3 for an outline of the study design.

The koalas were allowed to habituate to their enclosures for a minimum of four days before being administered with 400 mg per kg (of body weight) of Cobalt ethylenediamine tetraacetic acid (Co-EDTA) and 800 mg per kg of Chromium-mordanted plant fibre particles (500–1000 μm). Faeces sampled were collected for 14 subsequent days, to determine the retention time of these inert digestion markers by koalas (manuscript in preparation). During this time the koalas were exclusively fed manna gum foliage.

After the completion of the retention time study, messmate foliage was introduced to the koalas and all koalas were offered both messmate and manna gum foliage for a period of three nights in the case of cohort 1 and eight nights for cohort 2. This difference between the cohorts was solely for unavoidable logistical reasons. Nonetheless, the GI microbiomes of both cohorts were sampled three days after the introduction of messmate (see below).

The koalas were then assigned to either the control or treatment group (3 koalas per group in each cohort; 3 females and 3 males per group across the two cohorts) and administered a daily dose of faecal inoculum for a period of 9 d. A duration of 9 d was selected due to the unusually long passage rate of solute digesta markers in koalas (which is thought to be indicative of microbial retention times). Cork and Warner [59] measured mean retention time of solute associated Cr-EDTA in captive koalas to be approximately 9 d, while Krockenberger and Hume [32] found mean solute retention time to be 4.5 d in wild koalas, a difference attributed to the wild vs captive contrast. As the koalas in this study were housed in captivity, it was conservatively anticipated that 9 d of inoculation would be required to ensure the koalas’ entire intestinal fill was inoculated.

For the treatment koalas, the inoculum was sourced from wild, radio-collared koalas feeding on messmate foliage (see donor koalas section below). Control koalas were inoculated with mixed faecal material from themselves and the other control koalas in that cohort. The control replicated the effect of dosing and the potential disruption to the established GI microbiome from exposure to exogenous microbes.

The koalas were retained in captivity for a further 18 d post-inoculation, providing for a 9 day ‘wash-out’ period during which microbes that failed to establish would be eliminated from the koalas’ gastro-intestinal tracts and 9 subsequent days to assess koala feeding behaviour and GI microbiomes post-establishment. Throughout these 18 days, only messmate foliage was made available to koalas between 20:00 and 08:00 when most koala feeding occurs [60]. Both messmate and manna gum foliage were made available during the day to ensure that those koalas unwilling or unable to eat messmate could meet their nutritional needs. A second digesta marker dose was administered 4 d after the last inoculation for the retention time study.

The composition of the koalas’ GI microbiomes was tracked over the course of the experiment. Faecal samples were collected for microbiome analysis at: 1) capture; 2) immediately prior to the first introduction of messmate; 3) three days after the introduction of messmate; and 4) immediately; 5) nine; and 6) 18 d after the final inoculations. The feeding preferences of the koalas were tracked throughout the study by measuring the koalas’ daily diurnal and nocturnal intakes of messmate and manna gum (see below). The koalas were released at the point of capture after the completion of the experiment, with the exception of one control koala that was released 9 d after the completion of the inoculations due to an unacceptable level of weight loss (> 10% of capture weight). All other koalas generally maintained weight over the coarse of the experiment after an initial drop during the first week in captivity (Additional file 2: Figure S4).

Captive koalas

Koalas were captured using a standard noose and flag technique [61]. The koalas were anesthetised after capture and restrained with isoflurane (Isoflurane, Delvet Pty, Ltd.) in order to assess their health, age, sex and to establish if they possessed pouch young. None of the animals used in the study had pouch young or had any observable illness or injury. The majority of females on the Cape Otway Peninsula had previously been fitted with contraceptive implants (Levonorgestrel; Elorn Projects Pty Ltd., Southport, Queensland, Australia) in an effort by the Victorian Government to control koala overabundance at this site. We preferentially selected contracepted female koalas to minimise the chance of including pregnant koalas in the study. All selected koalas were in good condition (condition score ≥ 7) [62] and exhibited tooth wear from classes 3 to 5, corresponding to ages from 4 to 12 years [63].

Husbandry

The koalas were housed individually in 2 × 2.5 m yards. Each yard was equipped with two resting forks and two feeding stations, with a 2 × 1.5 m area beneath the resting forks covered in artificial turf. The remaining floor area consisted of grass. Part of each yard was sheltered from the sun and rain with shade cloth. All Eucalyptus foliage provided to the koalas was cut as large branches from manna gum and messmate trees on Cape Otway. The branches were kept in water at all times to reduce desiccation. The koalas were provided water ad libitum. Faecal pellets were cleared from the artificial turf and branches daily.

Donor koalas

Healthy adult koalas occupying patches of messmate trees (E. obliqua) on Cape Otway (Additional file 2: Figure S2) were used as faecal donors for the captive treatment koalas (Additional file 3: Table S1). At the beginning of the study, these koalas were captured, fitted with VHF radiotransmitter collars and released at the point of capture. The koalas were then radio-tracked over a period of at least 2 weeks prior to the faecal inoculations, as well as for 2 to 6 months during and after the faecal inoculations. The koalas’ daytime locations and resting tree species were recorded during this period on between 26 and 78 occasions.

Five koalas each were used as donors for cohort 1 and cohort 2. Two females and three male koalas were used as donors during cohort 1. Two of these koalas were observed in messmate stringybark (E. obliqua) trees on at least 78% of occasions, while a further two koalas were found in messmate on 50 and 61% of occasions. When these koalas were not observed in messmate they were generally found in manna gum, although they were occasionally found in non-food trees (0–14% of occasions), such as blackwood (Acacia melanoxylon). The fifth koala was often found in non-food trees and on the ground (18% of occasions) and was found in messmate on 63% of occasions, however, this increased to 76% of occasions when ground/non-food tree locations were excluded. Three of the same koalas were used as donors during cohort 2. However, the two koalas observed in messmate trees the least were replaced with two male koalas such that the donors for the second cohort consisted of one female and four males. The two alternative males were observed in messmate trees on 82 and 94% of occasions.

Several studies have concluded that changes in the faecal microbiome community structure are minimal within the first 24 h post production [64,65,66,67]. Therefore, faecal collections for the preparation of the treatment inoculum were made by locating the donor koalas in the late afternoon (typically after 17:00 h) on each day prior to an inoculation day. The donor koalas’ faecal pellets were collected overnight by placing a shade cloth sheet on the ground beneath the koala. The pellets were then collected from the drop-sheet early the following morning and using gloves, placed into a plastic zip-lock bag, with fresh inoculum processing commencing within 1 h of collection. Subsamples of the donor koalas’ pellets were retained for microbiome analysis.

At the conclusion of the faecal collection periods the koalas were captured and their collars were removed before release at point of capture.

Faecal inoculations

Captive koalas in both cohort 1 and 2 were inoculated with two full capsules containing fresh, processed faecal material each day of the 9-day inoculation period. In addition to this, koalas in cohort MG2 received 1–2 capsules containing dried ground faecal material per day (produced a week prior to the inoculations). Koala faecal microbial communities are known to differ from those higher in the gastrointestinal tract [17]. However, faecal material was the only practical/ethical source of GI microbes for this study and has been successfully used in other studies [8, 30].

The capsules were administered to the koalas using a ‘pill popper’ while they were restrained in a hessian sack by an investigator. Administration of the capsules took less than 5–10 min per koala (including capture time). Antibiotics were not administered to the koalas as antibiotics do not necessarily improve the establishment of inoculated microbes and can result in a lasting reduction in microbial diversity [31].

To prepare the fresh capsules for administration, fresh pellets from up to five messmate donor koalas were pooled (Additional file 3: Table S2). Fresh pellets collected from the captive control koalas were also pooled separately. The faecal inoculum was then prepared according to a modified protocol from Hirsch et al. [30]. The faecal pellets were mixed with ¼ Ringer’s solution and worked into a slurry, first by manual manipulation and then using a vortex mixer. The slurry was then centrifuged for 15 min and the supernatant removed by pipetting. Three layers of pellet typically formed: a lower pellet of large fibrous fragments; a fine particle layer and a white layer that was assumed to be bacterial (Additional file 2: Figure S1). The upper fine particle and bacterial layers were collected, pooled and centrifuged in a Clements (GS 150) centrifuge at 3 700 rpm for 60 min. The supernatant was then removed and the resulting pellet mixed for use as inoculant. The inoculant was dispensed into size 0 acid-resistant hypromellose capsules (DRCaps, Capsugel®). The capsules were then banded with a shellac solution (37% w/v shellac, 61.5% v/v ethanol and 1.5% v/v Tween 20) and subsequently placed within a size 00 acid-resistant hypromellose capsule, which was then thinly coated in the shellac solution. The capsules were left to dry (for approximately 15 min) and then administered within an hour to the koalas.

Prior to the faecal inoculation study, we tested the performance of these capsules in vitro and showed that bacteria can survive within the capsules in the presence of acid (pH 1.7–1.9) for greater than 10 h (exceeding the transit time of digesta in a koala’s stomach; [15]) and that the capsules degrade when placed in a neutral solution similar to the environment of the mid and hindgut (Additional file 1). Samples from each of the pellet layers as well as the final inoculant were collected for microbiome assessment.

To prepare the dry capsules for administration, faeces from the control and treatment donors was air dried at ambient temperature in a brown paper bag for 24–48 h. The pellets were then ground into a fine powder using a domestic coffee grinder. The powder was then packed into Size 00 acid-resistant hypromellose capsules and thinly coated with shellac solution. The dried faecal material was administered to the koalas within 2 weeks of production.

GI microbiome assessment

A total of 98 faecal samples were collected for microbiome assessment. This included 70 samples from the captive koalas over the course of the study, ten samples from donor koalas, two from other koalas found in messmate forest, six from other koalas located in manna gum forest (including one that become a captive koala in MG2), three treatment inoculant samples and seven samples from layers formed during centrifugation of the donor faecal samples (three from a single donor koala and four from a mixed sample). The full set of six samples were collected from ten of the captive koalas. We were unable to collect the final sample from the control koala released early (ID:B) and the capture sample from a treatment koala (ID = G) was lost during transport.

For each faecal sample, total genomic DNA was extracted from approximately 50-70 mg of material taken from the centre of a faecal pellet. The material was beaten for 5 min at 2,000 rpm using the MoBio PowerLyzer24 in a MoBio bead tube containing 0.1 mm dia. Zirconian/silica beads and 750ul of TLA buffer (Promega). The samples were centrifuged at 10,000 g for 30 s. DNA was then extracted from 150 ul of the supernatant using the Maxwell 16 robotic system and corresponding Tissue DNA kit (Promega) following the manufacturer’s instructions. Negative controls were included for each extraction kit.

A 589 bp section of the 16 s rRNA gene (V5 – V8 region) was amplified using 803F and 1392R primers [68] from the DNA extracts following the workflow outlined by Illumina (#15044223 Rev.B) except that Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) was used. PCR products were indexed with unique 8 bp barcodes using the Illumina Nextera XT 384 sample Index Kit A-D (Illumina FC-131-1002). Indexed amplicons were isolated using Qiagen QIAquick Gel Extraction Kit, as per manufacturer’s instructions. Paired-end sequencing was performed at the Australian Centre of Ecogenomics, on the Illumina Miseq using the version 3 reagent kit for 300 cycles.

Forward reads were processed and assigned taxonomic designations by QIIME 2 (v. 2017.10; [69]) using the SILVA 128 database [70, 71]. The resulting microbial feature by sample table was rarefied to 10 000 reads per sample using the vegan package [72] in R (version 3.5.0; [73]). All community composition analyses were performed on the rarefied dataset. Microbiome richness was estimated by a count of unique features recovered per sample after rarefaction and by calculating the Chao Index of alpha diversity using the package fossil in R [74]. Microbiome diversity within koalas was estimated using the Shannon diversity index as calculated using the vegan package. Weighted Unifrac distances between samples were calculated in QIIME 2. Variation in the relative abundance of the microbial groups was described by principal coordinate analysis (PCoA) generated from the weighted Unifrac distances using the vegan package. Significant differences between groups in microbial composition were determined by PERMANOVA from the weighted Unifrac distances using the vegan package. Other differences were tested using linear regression models, t-tests and the Wilcoxon rank sum test, where the residuals did not conform to normality.

Indicator species analysis was conducted using the indicspecies package in R [75] to determine which microbial features were significantly associated with a messmate diet when compared to a manna gum diet. All koalas found in manna gum forest (excluding the koala that later become a captive koala; n = 5) and the captive individuals at capture (with the exception of two captive koalas with GI microbiomes that did not resemble that of typical manna gum koalas; n = 9; see results: success of the faecal inoculations), were included in the manna gum group for the analysis. A single sample from each treatment donor (with the exception of the koala that was only found in messmate on 50% of occasions; n = 6) and two other koalas found in messmate forest were included in the messmate group. This analysis returned two statistics for each identified feature (indicator species): A = the probability that a sample came from a messmate koala given the feature was found; and B = the probability of finding that feature in a messmate koala. We used the suggested parameters in the indicspecies package documentation and considered microbial features for which A exceeded 80% and B exceeded 35% to be true indicator species.

Diet assessment

We measured dry matter intake (DMI) of messmate and manna gum foliage by weighing branches immediately before they were offered to koalas, and immediately after they were removed to give a gross fresh matter intake. Gross wet matter intake was corrected for the change in mass of control branches that were kept outside koala pens. A sample of foliage from each control branch (20–60 g) was dried for 24 h at 80 °C to calculate the dry matter (DM) content of foliage and this was used to calculate the gross DMI of koalas. Koalas also regularly dropped leaves onto the ground while they were feeding. These leaves were collected each time branches were removed from the pens, identified as messmate or manna gum, dried and weighed. The dropped leaves’ dry weight value was subtracted from the gross DMI for each tree species to give the actual DMI.

We calculated average daily intake (ADI) for each koala prior to the introduction of messmate, when the koalas were feeding exclusively on manna gum. This provided a measure of the individual differences in maintenance energy requirements between the koalas due to differences in body size as well as digestive and metabolic efficiency. We also calculated the night time DMI of messmate for each koala prior to the faecal inoculations. This provided a measure of each koala’s individual willingness to feed on messmate prior to experimental manipulation. We then determined the night-time DMI of messmate for each koala during and after the faecal inoculations. For all of these calculations, feeding periods (12 h windows) in which the control branches increased in weight by more than 10% or decreased by more than 5% were excluded because changes beyond these limits introduced increased error into our estimations of DMI. Branches generally increased in weight when water collected on the leaves during rain and this was seen to be a consistent change across branches. Branches typically lost weight if the leaves lost moisture when exposed to sun and wind. These changes were observed to be somewhat idiosyncratic between branches and so a more stringent limit was imposed for weight loss than gain. These limits meant that a large proportion of day time intake measurements were excluded such that total intake and manna gum intake (that was fed only during the day during most of the experiment) could only be reliably assessed on a subset (52%) of days. However, our estimates of night-time intake, when messmate was primarily consumed were reliable on the vast majority of days.

Total dry matter intake

We used linear mixed models to assess the factors affecting total daily dry matter intake over the experiment. The models were constructed in R using the package lme4 [76] with the significance of the fixed effects calculated using the package lmerTest [77]. Koala ID was included as a random effect in all analyses to account for the repeated measures study design. The date was also included as a random factor. The phase of the experiment, treatment group and the change in the koalas’ GI microbiomes over the course of the experiment were considered as explanatory variable in separate models. To allow for the possibility that intake may have changed over time in the treatment group and in koalas that showed a change in their GI microbiomes, we also included the number of days after the first faecal inoculation was administered as an interaction term with the experimental group (treatment/control) and GI microbiome change explanatory variables. Non-significant variables were removed by backward elimination and all reported p values were taken from the final model.

Intake of messmate prior to the inoculations

We also used linear mixed models to assess the factors affecting night-time messmate intake over the first three days after its introduction and prior to the inoculations. We determined if intake of messmate differed between koalas, over time or if it could be explained by the composition of the koalas’ GI microbiomes. The koalas’ GI microbiomes at capture and immediately prior to the introduction of messmate were fitted as predictors in separate models. The Bacteroidetes to Firmicutes (B:F) ratio and the position of the koalas’ GI microbiomes on the first axis of the PCoA based on the weighted UniFrac distance matrix were used as measures of the koalas’ GI microbiome compositions. Manna gum ADI and cohort (1 or 2) were included as fixed co-variates in the analyses. The date was also included as a random factor to account for variation in environmental conditions, such as weather, that may have influenced feeding. Non-significant variables were removed by backward elimination.

Intake of messmate during and after the inoculations

Linear mixed effects models were also used to assess how messmate intake was influenced by the faecal inoculations and whether it was associated with the koalas’ GI microbiomes. Night-time messmate DMI was considered the best response variable due to its reliable estimation (see above), however, a subset of analyses were also performed using the proportion of total daily intake that was messmate as the explanatory variable to confirm our findings. Manna gum ADI, pre-inoculation intake of messmate and cohort were included as fixed co-variates in the night-time messmate DMI analyses. Koala ID was included as a random effect in all analyses to account for the repeated measures study design. The date was also included as a random factor.

A series of potential explanatory variables were assessed in separate models: 1. whether an koala belonged to the treatment or control group; 2. the koalas’ GI microbiomes at capture (B:F ratio and PC1 score); 3. the koalas’ GI microbiomes immediately prior to a phase; 4. the koalas’ GI microbiomes immediately after a phase; and 5. the overall change in the koalas’ GI microbiomes over the course of the experiment. The Akaike information criterion (AIC) was used to assess whether the GI microbiome prior to or after a phase was a better predictor of messmate intake when both variables were significant. To allow for the possibility that messmate intake may have changed over time in the treatment group and in koalas that showed a change in their GI microbiomes, we also included the number of days after the first faecal inoculation was administered as an interaction term with the experimental group (treatment/control) and GI microbiome change explanatory variables. We modelled the entire period during and post-inoculation combined as well as the different phases separately (i.e. during, washout and post-establishment). Non-significant variables were removed by backward elimination and all reported p values were taken from the final model.