Warmer ambient temperatures reduce protein intake by a mammalian folivore.

The interplay between ambient temperature and nutrition in wild herbivores is frequently overlooked, despite the fundamental importance of food. We tested whether different ambient temperatures (10°C, 18°C and 26°C) influenced the intake of protein by a marsupial herbivore, the common brushtail possum (Trichosurus vulpecula). At each temperature, possums were offered a choice of two foods containing different amounts of protein (57% versus 8%) for one week. Animals mixed a diet with a lower proportion of protein to non-protein (P : NP, 0.20) when held at 26°C compared to that at both 10°C and 18°C (0.22). Since detoxification of plant secondary metabolites imposes a protein cost on animals, we then studied whether addition of the monoterpene 1,8-cineole to the food changed the effect of ambient temperature (10°C and 26°C) on food choice. Cineole reduced food intake but also removed the effect of temperature on P : NP ratio and instead animals opted for a diet with higher P : NP (0.19 with cineole versus 0.15 without cineole). These experiments show the proportion of P : NP chosen by animals is influenced by ambient temperature and by plant secondary metabolites. Protein is critical for reproductive success in this species and reduced protein intake caused by high ambient temperatures may limit the viability of some populations in the future. This article is part of the theme issue 'Food processing and nutritional assimilation in animals'.

Health Assessments of Koalas after Wildfire: A Temporal Comparison of Rehabilitated and Non-Rescued Resident Individuals.

Many koalas (Phascolarctos cinereus) required rehabilitation after the 2019/20 Australian megafires. Little is known about how the post-release health of rehabilitated koalas compares to non-rescued resident koalas. We evaluated health parameters in rehabilitated koalas and resident koalas in burnt and unburnt habitat in southern New South Wales, Australia. Health checks were undertaken within six weeks of fire (rehabilitated group), 5-9 months post-fire and 12-16 months post-fire. Body condition improved significantly over time in rehabilitated koalas, with similar condition between all groups at 12-16 months. Rehabilitated koalas therefore gained body condition at similar rates to koalas who remained and survived in the wild. The prevalence of Chlamydia pecorum was also similar between groups and timepoints, suggesting wildfire and rehabilitation did not exacerbate disease in this population. While there was some variation in measured serum biochemistry and haematology parameters between groups and timepoints, most were within normal reference ranges. Our findings show that koalas were generally healthy at the time of release and when recaptured nine months later. Landscapes in the Monaro region exhibiting a mosaic of burn severity can support koalas, and rehabilitated koala health is not compromised by returning them to burnt habitats 4-6 months post-fire.

Fundamental dietary specialisation explains differential use of resources within a koala population.

The diets of individual animals within populations can differ, but few studies determine whether this is due to fundamental differences in preferences or capacities to eat specific foods, or to external influences such as dominance hierarchies or spatial variation in food availability. The distinction is important because different drivers of dietary specialisation are likely to have different impacts on the way in which animal populations respond to, for example, habitat modification. We used a captive feeding study to investigate the mechanisms driving individual dietary specialisation in a population of wild koalas (Phascolarctos cinereus) in which individuals predominantly ate either Eucalyptus viminalis or Eucalyptus obliqua foliage. All six koalas that primarily ate E. viminalis in the wild avoided eating E. obliqua for more than 1 month in captivity. In contrast, all seven koalas that primarily ate E. obliqua could be maintained exclusively on this species in captivity, although they ate less from individual trees with higher foliar concentrations of unsubstituted B-ring flavanones (UBFs). Our results show that fundamental differences between individual animals allow some to exploit food resources that are less suitable for others. This could reduce competition for food, increase habitat carrying capacity, and is also likely to buffer the population against extinction in the face of habitat modification. The occurrence of fundamental individual specialisation within animal populations could also affect the perceived conservation value of different habitats, translocation or reintroduction success, and population dynamics. It should therefore be further investigated in other mammalian herbivore species.

Food intake: an overlooked driver of climate change casualties?

Reduced voluntary food intake is a common response of endotherms to warmer temperatures. However, the implications of this are rarely considered for wild animals exposed to higher temperatures caused by climate change. We provide a conceptual model to demonstrate the potential consequences of elevated temperatures on food intake and survival.

The distribution and abundance of an unusual resource for koalas (Phascolarctos cinereus) in a sodium-poor environment.

Environmentally available sodium tends to decrease with increasing elevation, and sodium resources in these sodium-poor environments are critical for the survival of herbivores. Eucalypt leaves in the subalpine Monaro region of NSW, Australia contain much less sodium than eucalypt leaves at lower elevations, and subalpine koalas obtain this much needed resource by eating the bark from some Eucalyptus mannifera trees. To better understand the availability of salty-barked trees, we searched for evidence of koala bark chewing at 100 randomly generated locations in the region. We found 318 E. mannifera trees with koala chew marks. We also analysed sodium concentrations in the bark of three unchewed E. mannifera trees from each site to determine whether there were trees with high bark sodium content that had not yet been utilized by koalas. Although 90% of unchewed trees had sodium concentrations less than 225.4 mg.kg-1 DM, some unchewed trees contained high sodium concentrations (up to 1213.1 mg.kg-1 DM). From the random survey, we can extrapolate that 11% of trees in this area have bark sodium above 300 mg.kg-1 DM, which is based on the concentration of bark sodium observed in at least moderately chewed trees. We would expect to find 0.24 of these trees per 200 m2, or 720,000 salty-barked trees in the 30 km by 20 km study area. Bark chewing by koalas is widespread in the area, and trees with salty bark are more common than initially thought. We discuss correlations with the occurrence of salty-barked trees and other landscape attributes; however, questions remain about why some E. mannifera trees have much more bark sodium than others. Studies such as this one should be expanded to identify sodium resources and their availability for other herbivorous species, since many are predicted to move to higher elevations in response to climate change.

New approaches to tannin analysis of leaves can be used to explain in vitro biological activities associated with herbivore defence.

Although tannins have been an important focus of studies of plant-animal interactions, traditional tannin analyses cannot differentiate between the diversity of structures present in plants. This has limited our understanding of how different mixtures of these widespread secondary metabolites contribute to variation in biological activity. We used UPLC-MS/MS to determine the concentration and broad composition of tannins and polyphenols in 628 eucalypt (Eucalyptus, Corymbia and Angophora) samples, and related these to three in vitro functional measures believed to influence herbivore defence: protein precipitation capacity, oxidative activity at high pH and capacity to reduce in vitro nitrogen (N) digestibility. Protein precipitation capacity was most strongly correlated with concentrations of procyanidin subunits in proanthocyanidins (PAs), and late-eluting ellagitannins. Capacity to reduce in vitro N digestibility was affected most by the subunit composition and mean degree of polymerisation (mDP) of PAs. Finally, concentrations of ellagitannins and prodelphinidin subunits of PAs were the strongest determinants of oxidative activity. The results illustrate why measures of total tannins rarely correlate with animal feeding responses. However, they also confirm that the analytical techniques utilised here could allow researchers to understand how variation in tannins influence the ecology of individuals and populations of herbivores, and, ultimately, other ecosystem processes.

Occurrence and distribution of unsubstituted B-ring flavanones in Eucalyptus foliage.

A group of plant specialised metabolites (PSMs) collectively known as unsubstituted B-ring flavanones (UBFs) have previously been found in the foliage of some species from the genus Eucalyptus L'Hér. (Myrtaceae), specifically from the subgenus Eucalyptus (monocalypts). Captive feeding studies using artificial diets suggest that these compounds may potentially influence the feeding preferences of marsupial folivores, such as koalas. Understanding natural variation in the composition and concentration of UBFs in eucalypt foliage is a first step to deciding whether, through their effects on herbivory, they might have broader effects on ecosystem dynamics. We used ESI-LCMS/MS and HPLC to characterise and quantify UBFs in 351 individual trees from 25 monocalypt species. We found large variation in the total UBF concentration both between and within species. For example, the mean concentration of UBFs in Eucalyptus muelleriana was 0.2 mg g-1 dry wt, whereas it was 105.7 mg g-1 dry wt, with a range of 78.2-141.3 mg g-1 dry wt, in Eucalyptus mediocris. Different eucalypt species contained different subsets of ten UBFs, and three species showed potential chemotypic variation between individuals within species. Our results suggest that UBFs naturally vary between monocalypt species and individuals at concentrations that could realistically be expected to affect the feeding dynamics of marsupial eucalypt folivores. UBFs could be measured relatively rapidly and cheaply in future studies using near-infrared reflectance (NIR) spectroscopy, as we were able to successfully predict the total UBF concentration of samples from their NIR spectra, with an r2 value of 0.98 and a standard error of prediction (SEP) of 6.07. This work further solidifies NIR spectroscopy as a powerful tool enabling ecologists to analyse the chemical composition of large numbers of samples.

Faecal inoculations alter the gastrointestinal microbiome and allow dietary expansion in a wild specialist herbivore, the koala.

BACKGROUND: Differences between individuals in their gastrointestinal microbiomes can lead to variation in their ability to persist on particular diets. Koalas are dietary specialists, feeding almost exclusively on Eucalyptus foliage but many individuals will not feed on particular Eucalyptus species that are adequate food for other individuals, even when facing starvation. We undertook a faecal inoculation experiment to test whether a koala's gastrointestinal (GI) microbiome influences their diet. Wild-caught koalas that initially fed on the preferred manna gum (Eucalyptus viminalis) were brought into captivity and orally inoculated with encapsulated material derived from faeces from koalas feeding on either the less preferred messmate (E. obliqua; treatment) or manna gum (control). RESULTS: The gastrointestinal microbiomes of wild koalas feeding primarily on manna gum were distinct from those feeding primarily on messmate. We found that the gastrointestinal microbiomes of koalas were unresponsive to dietary changes because the control koalas' GI microbiomes did not change even when the nocturnal koalas were fed exclusively on messmate overnight. We showed that faecal inoculations can assist the GI microbiomes of koalas to change as the treatment koalas' GI microbiomes became more similar to those of wild koalas feeding on messmate. There was no overall difference between the control and treatment koalas in the quantity of messmate they consumed. However, the greater the change in the koalas' GI microbiomes, the more messmate they consumed after the inoculations had established. CONCLUSIONS: The results suggest that dietary changes can only lead to changes in the GI microbiomes of koalas if the appropriate microbial species are present, and/or that the koala gastrointestinal microbiome influences diet selection.

A hot lunch for herbivores: physiological effects of elevated temperatures on mammalian feeding ecology.

Mammals maintain specific body temperatures (Tb ) across a broad range of ambient temperatures. The energy required for thermoregulation ultimately comes from the diet, and so what animals eat is inextricably linked to thermoregulation. Endothermic herbivores must balance energy requirements and expenditure with complicated thermoregulatory challenges from changing thermal, nutritional and toxicological environments. In this review we provide evidence that plant-based diets can influence thermoregulation beyond the control of herbivores, and that this can render them susceptible to heat stress. Notably, herbivorous diets often require specialised digestive systems, are imbalanced, and contain plant secondary metabolites (PSMs). PSMs in particular are able to interfere with the physiological processes responsible for thermoregulation, for example by uncoupling mitochondrial oxidative phosphorylation, binding to thermoreceptors, or because the pathways required to detoxify PSMs are thermogenic. It is likely, therefore, that increased ambient temperatures due to climate change may have greater and more-specific impacts on herbivores than on other mammals, and that managing internal and external heat loads under these conditions could drive changes in feeding ecology.

Intraspecific Variation in Nutritional Composition Affects the Leaf Age Preferences of a Mammalian Herbivore.

Ecologists have long been interested in how the nutritional composition of leaves changes as they age, and whether this affects herbivore feeding preferences. As a consequence, the literature abounds with reports that younger leaves contain higher concentrations of nitrogen and plant secondary metabolites (PSMs) than do older leaves. Most of these studies, however, base their conclusions on average values that often mean little to herbivores. We examined this issue in the well-studied marsupial-eucalypt system, using Eucalyptus melliodora and captive common brushtail possums (Trichosurus vulpecula) offered branches from individual trees containing both young and mature leaves. Like many plants, the concentrations of N and PSMs differed among individual E. melliodora. Although young leaves were, on average, "better defended" by the PSM sideroxylonal than were mature leaves, some trees produced leaves that were relatively undefended at both ages. In response, possums chose different proportions of young and mature leaves depending on the chemistry of the individual tree. Possums did not always prefer leaves with lower concentrations of sideroxylonal (mature leaves) or those with higher concentrations of available N (young leaves). Instead, the sideroxylonal concentration of young leaves dictated their choice: possums preferred young leaves with low sideroxylonal concentrations, but not with high concentrations. By skewing their feeding toward trees producing young leaves with low concentrations of PSMs, possums may influence plant fitness. Researchers will detect these potentially important interactions only if they are aware that measuring variation among plants discloses more information than do average relationships.

Correction to: Oxidizable Phenolic Concentrations Do Not Affect Development and Survival of Paropsis Atomaria Larvae Eating Eucalyptus Foliage.

Ian Wallis was inadvertently omitted as an author in this study. Ian Wallis assisted with the collection of the leaf samples that were used in this study, and built the chambers that the insects were housed in.

Genus-wide variation in foliar polyphenolics in eucalypts.

Many studies quantify total phenolics or total tannins, but understanding the ecological role of polyphenolic secondary metabolites requires at least an understanding of the diversity of phenolic groups present. We used UPLC-MS/MS to measure concentrations of different polyphenol groups - including the four most common tannin groups, the three most common flavonoid groups, and quinic acid derivatives - in foliage from 628 eucalypts from the genera Eucalyptus, Angophora and Corymbia. We also tested for phylogenetic signal in each of the phenolic groups. Many eucalypts contained high concentrations of polyphenols, particularly ellagitannins, which have been relatively poorly studied, but may possess strong oxidative activity. Because the biosynthetic pathways of many phenolic compounds share either precursors or enzymes, we found negative correlations between the concentrations of several of the constituents that we measured, including proanthocyanidins (PAs) and hydrolysable tannins (HTs), HTs and flavonol derivatives, and HTs and quinic acid derivatives. We observed moderate phylogenetic signal in all polyphenol constituents, apart from the concentration of the prodelphinidin subunit of PAs and the mean degree of polymerisation of PAs. These two traits, which have previously been shown to be important in determining plants' protein precipitation capacity, may have evolved under selection, perhaps in response to climate or herbivore pressure. Hence, the signature of evolutionary history appears to have been erased for these traits. This study is an important step in moving away from analysing "totals" to a better understanding of how phylogenetic effects influence phenolic composition, and how this in turn influences ecological processes.

Oxidizable Phenolic Concentrations Do Not Affect Development and Survival of Paropsis Atomaria Larvae Eating Eucalyptus Foliage.

Insect folivores can cause extensive damage to plants. However, different plant species, and even individuals within species, can differ in their susceptibility to insect attack. Polyphenols that readily oxidize have recently gained attention as potential defenses against insect folivores. We tested the hypothesis that variation in oxidizable phenolic concentrations in Eucalyptus foliage influences feeding and survival of Paropsis atomaria (Eucalyptus leaf beetle) larvae. First we demonstrated that oxidizable phenolic concentrations vary both within and between Eucalyptus species, ranging from 0 to 61 mg.g-1 DM (0 to 81% of total phenolics), in 175 samples representing 13 Eucalyptus species. Foliage from six individuals from each of ten species of Eucalyptus were then offered to batches of newly hatched P. atomaria larvae, and feeding, instar progression and mortality of the first and second instar larvae were recorded. Although feeding and survival parameters differed dramatically between individual plants, they were not influenced by the oxidizable phenolic concentration of leaves, suggesting that P. atomaria larvae may have effective mechanisms to deal with oxidizable phenolics. Larvae feeding on plants with higher nitrogen (N) concentrations had higher survival rates and reached third instar earlier, but N concentrations did not explain most of the variation in feeding and survival. The cause of variation in eucalypt herbivory by P. atomaria larvae is therefore still unknown, although oxidizable phenolics could potentially defend eucalypt foliage against other insect herbivores.

The Use of Polyethylene Glycol in Mammalian Herbivore Diet Studies: What Are We Measuring?

Polyethylene glycol (PEG) has been used to study the intake and digestion of tannin-rich plants by mammalian herbivores because it preferentially binds to tannins. However, it is not clear whether the responses of herbivores to dietary PEG is due to increased protein availability from the release of tannin-bound protein, amelioration of tannin effects, or whether PEG also may bind to other compounds and change their activity in the gut. We used three native New Zealand tree species to measure the effect of PEG on the amount of foliage eaten by invasive common brushtail possums (Trichosurus vulpecula) and on in vitro digestible nitrogen (available N). The addition of PEG increased the in vitro available N content of Weinmannia racemosa foliage, and possums ate significantly more PEG-treated foliage than untreated foliage. However, possums also ate more PEG-treated Fuchsia excorticata foliage, even though PEG did not increase in vitro available N in this species. Possums ate very little Melicytus ramiflorus, regardless of PEG treatment, even though M. ramiflorus contained the highest concentration of in vitro available N. These results prompted us to use PEG and a protein supplement, casein, to manipulate the available N concentration of diets containing ground eucalypt foliage, a well-studied food species for possums. Again, the response of possums to PEG was independent of changes in in vitro available N. In addition, altering the protein content of the diet via the addition of casein did not affect how much food the possums consumed. We conclude that the effects of PEG on dry matter intake by mammalian herbivores are not due solely to the release of tannin-bound protein. There is need for a better understanding of PEG-tannin interactions in order to ensure that the use of PEG in nutritional studies does not outstrip an understanding of its mechanisms of action.

From Leaf Metabolome to In Vivo Testing: Identifying Antifeedant Compounds for Ecological Studies of Marsupial Diets.

Identifying specific plant secondary metabolites that influence feeding behavior can be challenging, but a solid understanding of animal preferences can guide efforts. Common brushtail possums (Trichosurus vulpecula) predominantly eat Eucalyptus species belonging to the subgenus Symphyomyrtus, and avoid eating those belonging to the Monocalyptus subgenus (also called subgenus Eucalyptus). Using an unbiased (1)H NMR metabolomics approach, a previous study identified unsubstituted B ring flavanones in most species of monocalypts examined, whereas these compounds were absent from symphyomyrtles. We hypothesised that unsubstituted B ring flavanones act as feeding deterrents for common brushtail possums. In the current study, we tested this hypothesis by comparing how much possums ate of a basal diet, with diets containing one of four structurally related compounds; pinocembrin, flavanone (unsubstituted B ring flavanones), chrysin (the flavone analogue of pinocembrin), and naringenin (a flavanone with B ring substitution). We found that pinocembrin and flavanone deterred feeding relative to the basal diet, but that chrysin and naringenin did not at equivalent concentrations. Thus, unsubstituted B-ring flavanones may explain why brushtail possums avoid eating monocalypt species. Furthermore, small differences in the structure of secondary compounds can have a large impact on antifeedant properties. These results demonstrate that metabolomics can be a valuable tool for ecologists seeking to understand herbivore feeding preferences.

Extraction of pinocembrin from leaves of different species of Eucalyptus and its quantitative analysis by qNMR and HPTLC.

Pinocembrin, a flavanone with a variety of biological activities was isolated from Eucalyptus sieberi leaves and quantified in several other Eucalyptus species using qNMR and HPTLC densitometry. The effect of different extraction procedures on the extraction of the compound from Eucalyptus sieberi was also studied. The methods were validated in terms ofselectivity, specificity, linearity, recovery, precision and repeatability.

Four species of arboreal folivore show differential tolerance to a secondary metabolite.

The marsupials that eat Eucalyptus in south-eastern Australia provide an example of animals with similar niche requirements occurring sympatrically. They certainly differ in size, ranging from about 1 kg in the greater glider (Petauroides volans) and the closely related common ringtail possum (Pseudocheirus peregrinus), to 4 kg (common brushtail possum, Trichosurus vulpecula) and up to 15 kg in the koala (Phascolarctos cinereus). All species, however, may eat considerable amounts of eucalypt foliage, often favouring the same species, and thus appear to compete for food. In order to better understand the degree of competition for food, we measured feeding by the greater glider in response to increasing concentrations of a specific group of eucalypt plant secondary metabolites (PSM), the sideroxylonals, and then compared it to results published for the other species. The greater glider was more resilient than the other species to increasing concentrations of sideroxylonals. We suggest this allows gliders to feed on leaves from the eucalypt subgenus, Symphyomyrtus, while its small size and gliding ability allow it to feed where koalas cannot, on the young leaves on top of the canopy. In contrast, the common ringtail possum is well adapted to feeding from species of the subgenus Eucalyptus, which do not produce sideroxylonals but contain less available nitrogen (AvailN) than do the symphyomyrtles. These 'nutritional niches' segregate the forest and along with other factors, such as generalist and specialist feeding strategies and differences in body size and requirements for shelter, presumably minimise competition between the marsupial species.

Feeding rates of a mammalian browser confirm the predictions of a 'foodscape' model of its habitat.

Adequate nutrition is a fundamental requirement for the maintenance and growth of populations, but complex interactions between nutrients and plant toxins make it difficult to link variation in plant quality to the ecology of wild herbivores. We asked whether a 'foodscape' model of habitat that uses near-infrared spectroscopy to describe the palatability of individual trees in the landscape, predicted the foraging decisions of a mammalian browser, the koala (Phascolarctos cinereus). Specifically, we considered four behavioural decision points at which nutritional quality may influence an animal's decision. These were: which tree to enter, whether to feed from that tree, when to stop eating, and how long to remain in that tree. There were trends for koalas to feed in eucalypt trees that were more palatable than unvisited neighbouring conspecific trees, and than trees that they visited but did not eat. Koalas ate longer meals in more palatable trees, and stayed longer and spent more time feeding per visit to these trees. Using more traditional chemical analyses, we identified that an interaction between the concentrations of formylated phloroglucinol compounds (a group of plant secondary metabolites) and available N (an integrated measure of tannins, digestibility and N) influenced feeding. The study shows that foodscape models that combine spatial information with integrated measures of food quality are a powerful tool to predict the feeding behaviour of herbivores in a landscape.

Whole-body protein turnover reveals the cost of detoxification of secondary metabolites in a vertebrate browser.

The detoxification limitation hypothesis predicts that the metabolism and biotransformation of plant secondary metabolites (PSMs) elicit a cost to herbivores. There have been many attempts to estimate these costs to mammalian herbivores in terms of energy, but this ignores what may be a more important cost-increases in protein turnover and concomitant losses of amino acids. We measured the effect of varying dietary protein concentrations on the ingestion of two PSMs (1,8 cineole-a monoterpene, and benzoic acid-an aromatic carboxylic acid) by common brushtail possums (Trichosurus vulpecula). The dietary protein concentration had a small effect on how much cineole possums ingested. In contrast, protein had a large effect on how much benzoate they ingested, especially at high dietary concentrations of benzoate. This prompted us to measure the effects of dietary protein and benzoate on whole-body protein turnover using the end-product method following an oral dose of [(15)N] glycine. Increasing the concentration of dietary protein in diets without PSMs improved N balance but did not influence whole-body protein turnover. In contrast, feeding benzoate in a low-protein diet pushed animals into negative N balance. The concomitant increases in the rates of whole-body protein turnover in possums eating diets with more benzoate were indicative of a protein cost of detoxification. This was about 30 % of the dietary N intake and highlights the significant effects that PSMs can have on nutrient metabolism and retention.

A metabolomic approach to identifying chemical mediators of mammal-plant interactions.

Different folivorous marsupials select their food from different subgenera of Eucalyptus, but the choices cannot be explained by known antifeedants, such as formylated phloroglucinol compounds or tannins, or by nutritional quality. Eucalypts contain a wide variety of plant secondary metabolites so it is difficult to use traditional methods to identify the chemicals that determine food selection. Therefore, we used a metabolomic approach in which we employed (1)H nuclear magnetic resonance spectroscopy to compare chemical structures of representatives from the two subgenera and to identify chemicals that consistently differ between them. We found that dichloromethane extracts of leaves from most species in the subgenus Eucalyptus differ from those in Symphyomyrtus by the presence of free flavanones, having no substitution in Ring B. Although flavanoids are known to deter feeding by certain insects, their effects on marsupials have not been established and must be tested with controlled feeding studies.