Implications of heat exchange for a free-living endangered marsupial determined by non-invasive thermal imaging.

We used thermal imagining and heat balance modelling to examine the thermal ecology of wild mammals, using the diurnal marsupial numbat (Myrmecobius fasciatus) as a model. Body surface temperature was measured using infra-red thermography at environmental wet and dry bulb temperatures of 11.7-29°C and 16.4-49.3°C, respectively; surface temperature varied for different body parts and with environmental temperature. Radiative and convective heat exchange varied markedly with environmental conditions and for various body surfaces reflecting their shapes, surface areas and projected areas. Both the anterior and posterior dorsolateral body areas functioned as thermal windows. Numbats in the shade had lower rates of solar radiative heat gain but non-solar avenues for radiative heat gain were substantial. Radiative gain was higher for black and lower for white stripes, but overall, the stripes had no thermal role. Total heat gain was generally positive (<4 to >20 W) and often greatly exceeded metabolic heat production (3-6 W). Our heat balance model indicates that high environmental heat loads limit foraging in open areas to as little as 10 min and that climate change may extend periods of inactivity, with implications for future conservation and management. We conclude that non-invasive thermal imaging is informative for modelling heat balance of free-living mammals.

Modeling heterothermic fitness landscapes in a marsupial hibernator using changes in body composition.

Hibernation is an adaptive strategy that allows animals to enter a hypometabolic state, conserving energy and enhancing their fitness by surviving harsh environmental conditions. However, addressing the adaptive value of hibernation, at the individual level and in natural populations, has been challenging. Here, we applied a non-invasive technique, body composition analysis by quantitative magnetic resonance (qMR), to calculate energy savings by hibernation in a population of hibernating marsupials (Dromiciops gliroides). Using outdoor enclosures installed in a temperate rainforest, and measuring qMR periodically, we determined the amount of fat and lean mass consumed during a whole hibernation cycle. With this information, we estimated the daily energy expenditure of hibernation (DEEH) at the individual level and related to previous fat accumulation. Using model selection approaches and phenotypic selection analysis, we calculated linear (directional, ß), quadratic (stabilizing or disruptive, γ) and correlational (ρ) coefficients for DEEH and fat accumulation. We found significant, negative directional selection for DEEH (ßDEEH = - 0.58 ± 0.09), a positive value for fat accumulation (ßFAT = 0.34 ± 0.07), and positive correlational selection between both traits (ρDEEH × FAT = 0.24 ± 0.07). Then, individuals maximizing previous fat accumulation and minimizing DEEH were promoted by selection, which is visualized by a bi-variate selection surface estimated by generalized additive models. At the comparative level, results fall within the isometric allometry known for hibernation metabolic rate in mammals. Thus, by a combination of a non-invasive technique for body composition analysis and semi-natural enclosures, we were characterized the heterothermic fitness landscape in a semi-natural population of hibernators.

Extracellular vesicle proteomes of two transmissible cancers of Tasmanian devils reveal tenascin-C as a serum-based differential diagnostic biomarker.

The iconic Tasmanian devil (Sarcophilus harrisii) is endangered due to the transmissible cancer Devil Facial Tumour Disease (DFTD), of which there are two genetically independent subtypes (DFT1 and DFT2). While DFT1 and DFT2 can be differentially diagnosed using tumour biopsies, there is an urgent need to develop less-invasive biomarkers that can detect DFTD and distinguish between subtypes. Extracellular vesicles (EVs), the nano-sized membrane-enclosed vesicles present in most biofluids, represent a valuable resource for biomarker discovery. Here, we characterized the proteome of EVs from cultured DFTD cells using data-independent acquisition-mass spectrometry and an in-house spectral library of > 1500 proteins. EVs from both DFT1 and DFT2 cell lines expressed higher levels of proteins associated with focal adhesion functions. Furthermore, hallmark proteins of epithelial-mesenchymal transition were enriched in DFT2 EVs relative to DFT1 EVs. These findings were validated in EVs derived from serum samples, revealing that the mesenchymal marker tenascin-C was also enriched in EVs derived from the serum of devils infected with DFT2 relative to those infected with DFT1 and healthy controls. This first EV-based investigation of DFTD increases our understanding of the cancers' EVs and their possible involvement in DFTD progression, such as metastasis. Finally, we demonstrated the potential of EVs to differentiate between DFT1 and DFT2, highlighting their potential use as less-invasive liquid biopsies for the Tasmanian devil.

Nonlinear sequence similarity between the Xist and Rsx long noncoding RNAs suggests shared functions of tandem repeat domains.

The marsupial inactive X chromosome expresses a long noncoding RNA (lncRNA) called Rsx that has been proposed to be the functional analog of eutherian Xist Despite the possibility that Xist and Rsx encode related functions, the two lncRNAs harbor no linear sequence similarity. However, both lncRNAs harbor domains of tandemly repeated sequence. In Xist, these repeat domains are known to be critical for function. Using k-mer based comparison, we show that the repeat domains of Xist and Rsx unexpectedly partition into two major clusters that each harbor substantial levels of nonlinear sequence similarity. Xist Repeats B, C, and D were most similar to each other and to Rsx Repeat 1, whereas Xist Repeats A and E were most similar to each other and to Rsx Repeats 2, 3, and 4. Similarities at the level of k-mers corresponded to domain-specific enrichment of protein-binding motifs. Within individual domains, protein-binding motifs were often enriched to extreme levels. Our data support the hypothesis that Xist and Rsx encode similar functions through different spatial arrangements of functionally analogous protein-binding domains. We propose that the two clusters of repeat domains in Xist and Rsx function in part to cooperatively recruit PRC1 and PRC2 to chromatin. The physical manner in which these domains engage with protein cofactors may be just as critical to the function of the domains as the protein cofactors themselves. The general approaches we outline in this report should prove useful in the study of any set of RNAs.

Development of the skin in the eastern quoll (Dasyurus viverrinus) with focus on cutaneous gas exchange in the early postnatal period.

A morphological and morphometric study of the skin development in the eastern quoll (Dasyurus viverrinus) was conducted to follow the transition from cutaneous to pulmonary gas exchange in this extremely immature marsupial species. Additionally, the development of the cardiac and respiratory system was followed, to evaluate the systemic prerequisites allowing for cutaneous respiration. The skin in the newborn D. viverrinus was very thin (36 ± 3 µm) and undifferentiated (no hair follicles, no sebaceous and perspiratory glands). Numerous superficial cutaneous capillaries were encountered, closely associated with the epidermis, allowing for gaseous exchange. The capillary volume density was highest in the neonate (0.33 ± 0.04) and decreased markedly during the first 4 days (0.06 ± 0.01). In the same time period, the skin diffusion barrier increased from 9 ± 1 µm to 44 ± 6 µm. From this age on the skin development was characterized by thickening of the different cutaneous layers, formation of hair follicles (day 55) and the occurrence of subcutaneous fat (day 19). The heart of the neonate D. viverrinus had incomplete interatrial, inter-ventricular, and aortico-pulmonary septa, allowing for the possibility that oxygenated blood from the skin mixes with that of the systemic circulation. The fast-structural changes in the systemic circulations (closing all shunts) in the early postnatal period (3 days) necessitate the transition from cutaneous to pulmonary respiration despite the immaturity of the lungs. At this time, the lung was still at the canalicular stage of lung development, but had to be mature enough to meet the respiratory needs of the growing organism. The morphometric results for the skin development of D. viverrinus suggest that cutaneous respiration is most pronounced in neonates and decreases rapidly during the first 3 days of postnatal life. After this time a functional transition of the skin from cutaneous respiration to insulation and protection of the body takes place.

Sequence comparisons of cytochrome P450 aromatases from Australian animals predict differences in enzymatic activity and/or efficiency†.

Aromatase (P450arom, CYP19A1) is the terminal enzyme in the synthesis of the steroid hormone family of estrogens. Not surprisingly, this enzyme has structural similarities between the limited number of species studied thus far. This study examined the structure of aromatases from four diverse Australian species including a marsupial (tammar wallaby; Macropus eugenii), monotreme (platypus; Ornithorhynchus anatinus), ratite (emu; Dromaius novaehollandiae) and lizard (bearded dragon; Pogona vitticeps). We successfully built homology models for each species, using the only crystallographically determined structure available, human aromatase. The amino acid sequences showed high amino acid sequence identity to the human aromatase: wallaby 81%, platypus 73%, emu 75% and bearded dragon at 74%. The overall structure was highly conserved among the five species, although there were non-secondary structures (loops and bends) that were variable and flexible that may result in some differences in catalytic activity. At the N-terminal regions, there were deletions and variations that suggest that functional distinctions may be found. We found that the active sites of all these proteins were identical, except for a slight variation in the emu. The electrostatic potential across the surfaces of these aromatases highlighted likely variations to the protein-protein interactions of these enzymes with both redox partner cytochrome P450 reductase and possibly homodimerization in the case of the platypus, which has been postulated for the human aromatase enzyme. Given the high natural selection pressures on reproductive strategies, the relatively high degree of conservation of aromatase sequence and structure across species suggests that there is biochemically very little scope for changes to have evolved without the loss of enzyme activity.

Oncogenesis as a Selective Force: Adaptive Evolution in the Face of a Transmissible Cancer.

Similar to parasites, malignant cells exploit the host for energy, resources and protection, thereby impairing host health and fitness. Although cancer is widespread in the animal kingdom, its impact on life history traits and strategies have rarely been documented. Devil facial tumour disease (DFTD), a transmissible cancer, afflicting Tasmanian devils (Sarcophilus harrisii), provides an ideal model system to monitor the impact of cancer on host life-history, and to elucidate the evolutionary arms-race between malignant cells and their hosts. Here we provide an overview of parasite-induced host life history (LH) adaptations, then both phenotypic plasticity of LH responses and changes in allele frequencies that affect LH traits of Tasmanian devils in response to DFTD are discussed. We conclude that akin to parasites, cancer can directly and indirectly affect devil LH traits and trigger host evolutionary responses. Consequently, it is important to consider oncogenic processes as a selective force in wildlife.

Dynamic changes to claudins in the uterine epithelial cells of the marsupial Sminthopsis crassicaudata (Dasyuridae) during pregnancy.

The fluid that surrounds the embryo in the uterus contains important nourishing factors and secretions. To maintain the distinct microenvironment in the uterine lumen, the tight junctions between uterine epithelial cells are remodeled to decrease paracellular movement of molecules and solutes. Modifications to tight junctions between uterine epithelial cells is a common feature of pregnancy in eutherian mammals, regardless of placental type. Here we used immunofluorescence microscopy and western blot analysis to describe distributional changes to tight junctional proteins, claudin-1, -3, -4, and -5, in the uterine epithelial cells of a marsupial species, Sminthopsis crassicaudata. Immunofluorescence microscopy revealed claudin-1, -3, and -5 in the tight junctions of the uterine epithelium of S. crassicaudata during pregnancy. These specific claudins are associated with restricting passive movement of fluid between epithelial cells in eutherians. Hence, their function during pregnancy in S. crassicaudata may be to maintain the uterine luminal content surrounding developing embryos. Claudin-4 disappears from all uterine regions of S. crassicaudata at the time of implantation, in contrast with the distribution of this claudin in some eutherian mammals. We conclude that like eutherian mammals, distributional changes to claudins in the uterine epithelial cells of S. crassicaudata are necessary to support pregnancy. However, the combination of individual claudin isoforms in the tight junctions of the uterine epithelium of S. crassicaudata differs from that of eutherian mammals. Our findings suggest that the precise permeability of the paracellular pathway of the uterine epithelium is species-specific.

A functional transcriptomic analysis in the relict marsupial Dromiciops gliroides reveals adaptive regulation of protective functions during hibernation.

The small South American marsupial, Dromiciops gliroides, known as the missing link between the American and the Australian marsupials, is one of the few South American mammals known to hibernate. Expressing both daily torpor and seasonal hibernation, this species may provide crucial information about the mechanisms and the evolutionary origins of marsupial hibernation. Here, we compared torpid and active individuals, applying high-throughput sequencing technologies (RNA-seq) to profile gene expression in three D. gliroides tissues and determine whether hibernation induces tissue-specific differential gene expression. We found 566 transcripts that were significantly up-regulated during hibernation (369 in brain, 147 in liver and 50 in skeletal muscle) and 339 that were down-regulated (225 in brain, 79 in liver and 35 in muscle). The proteins encoded by these differentially expressed genes orchestrate multiple metabolic changes during hibernation, such as inhibition of angiogenesis, prevention of muscle disuse atrophy, fuel switch from carbohydrate to lipid metabolism, protection against reactive oxygen species and repair of damaged DNA. According to the global enrichment analysis, brain cells seem to differentially regulate a complex array of biological functions (e.g., cold sensitivity, circadian perception, stress response), whereas liver and muscle cells prioritize fuel switch and heat production for rewarming. Interestingly, transcripts of thioredoxin-interacting protein (TXNIP), a potent antioxidant, were significantly over-expressed during torpor in all three tissues. These results suggest that marsupial hibernation is a controlled process where selected metabolic pathways show adaptive modulation that can help to maintain homeostasis and enhance cytoprotection in the hypometabolic state.

A new research paradigm for bivariate allometry: combining ANOVA and non-linear regression.

A novel statistical routine is presented here for exploring and comparing patterns of allometric variation in two or more groups of subjects. The routine combines elements of the analysis of variance (ANOVA) with non-linear regression to achieve the equivalent of an analysis of covariance (ANCOVA) on curvilinear data. The starting point is a three-parameter power equation to which a categorical variable has been added to identify membership by each subject in a specific group or treatment. The protocol differs from earlier ones in that different assumptions can be made about the form for random error in the full statistical model (i.e. normal and homoscedastic, normal and heteroscedastic, lognormal and heteroscedastic). The general equation and several modifications thereof were used to study allometric variation in field metabolic rates of marsupial and placental mammals. The allometric equations for both marsupials and placentals have an explicit, non-zero intercept, but the allometric exponent is higher in the equation for placentals than in that for marsupials. The approach followed here is extraordinarily versatile, and it has wider application in allometry than standard ANCOVA performed on logarithmic transformations.

Epithelial cadherin disassociates from the lateral plasma membrane of uterine epithelial cells throughout pregnancy in a marsupial.

The uterine luminal epithelium is the first site of contact between fetal and maternal tissues during therian pregnancy and must undergo specialised changes for implantation of the blastocyst to be successful. These changes, collectively termed the plasma membrane transformation (PMT), allow the blastocyst to attach to the uterine epithelium preceding the formation of a placenta. There are similarities in the morphological and molecular changes occurring in live-bearing eutherian species during the PMT studied so far. Similar cellular remodelling occurs in a marsupial species, the fat-tailed dunnart (Sminthopsis crassicaudata), despite the divergence of marsupials from eutherian mammals over 130 mya, which resulted in the evolution of distinct reproductive strategies. Adhesion molecules along the lateral plasma membrane of uterine epithelium provide a barrier to invasion by the embryo. We thus characterised the presence and change in distribution of epithelial cadherin (E-cadherin) in uterine epithelium from non-pregnant fat-tailed dunnarts and compared it to dunnarts in early-, mid- and late-stage pregnancy. E-cadherin staining is localised to the lateral plasma membrane in uterine epithelium from non-pregnant and early-stage pregnant dunnarts. The E-cadherin staining is cytoplasmic in epithelium from uteri of mid- and late-stage pregnant dunnarts. This loss of localised staining suggests that the adherens junction dissociates from the lateral plasma membrane, allowing for invasion between the epithelial cells by the blastocyst. As the changes during pregnancy to cadherin were similar in the laboratory rat with highly invasive (haemochorial) placentation, a live-bearing lizard species with non-invasive (epitheliochorial) placentation and a marsupial, the fat-tailed dunnart, which has invasive (endotheliochorial) placentation, we suggest that the molecular mechanisms allowing for successful pregnancy are conserved among mammals during the early stages of pregnancy regardless of placental invasiveness.

Early cell lineage specification in a marsupial: a case for diverse mechanisms among mammals.

Early cell lineage specification in eutherian mammals results in the formation of a pluripotent inner cell mass (ICM) and trophoblast. By contrast, marsupials have no ICM. Here, we present the first molecular analysis of mechanisms of early cell lineage specification in a marsupial, the tammar wallaby. There was no overt differential localisation of key lineage-specific transcription factors in cleavage and early unilaminar blastocyst stages. Pluriblast cells (equivalent to the ICM) became distinguishable from trophoblast cells by differential expression of POU5F1 and, to a greater extent, POU2, a paralogue of POU5F1. Unlike in the mouse, pluriblast-trophoblast differentiation coincided with a global nuclear-to-cytoplasmic transition of CDX2 localisation. Also unlike in the mouse, Hippo pathway factors YAP and WWTR1 showed mutually distinct localisation patterns that suggest non-redundant roles. NANOG and GATA6 were conserved as markers of epiblast and hypoblast, respectively, but some differences to the mouse were found in their mode of differentiation. Our results suggest that there is considerable evolutionary plasticity in the mechanisms regulating early lineage specification in mammals.

Mitogen-activated Tasmanian devil blood mononuclear cells kill devil facial tumour disease cells.

Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo.

Chemical characterization of milk oligosaccharides of the tiger quoll (Dasyurus maculatus), a marsupial.

Milk oligosaccharides were separated from the carbohydrate fraction of milk of the tiger quoll a species of marsupial that is closely related to the eastern quoll, Dasyurus viverrinus. They were characterized by (1)H - nuclear magnetic resonance spectroscopy and matrix - assisted laser desorption/ionization time-of-flight mass spectrometry. The following oligosaccharides were identified; Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Neu5Ac(α2-3) Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc with an α(2-3)Neu5Ac linked to ß(1-4)Gal residue of either branch of Gal(ß1-4)GlcNAc(ß1-6) units, and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc with a ß(1-3) linked Gal and an α(2-3) linked Neu5Ac. In addition, larger oligosaccharides were characterized as follows; Gal(ß1-3){Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)GlcNAc(ß1-6)}Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3){Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)GlcNAc(ß1-6)}Gal(ß1-4)Glc and their α(2-3) linked Neu5Ac derivatives.

The functional requirements of mammalian hair: a compromise between crypsis and thermoregulation?

Mammalian fur often shows agouti banding with a proximal dark band near the skin and a lighter distal band. We examined the function of both bands in relation to camouflage, thermal properties of pelts, and thermal energetics of dunnarts (Sminthopsis crassicaudata), which are known to use torpor and basking. Although the distal band of dunnart fur darkened with increasing latitude, which is important for camouflage, it did not affect the thermal properties and the length of the dark band and total hair length were not correlated. In contrast, the length of the proximal dark band of preserved pelts exposed to sunlight was positively correlated (r (2) = 0.59) with the temperature underneath the pelt (T pelt). All dunnarts offered radiant heat basked by exposing the dark band of the hair during both rest and torpor. Basking dunnarts with longer dark bands had lower resting metabolism (r (2) = 0.69), warmed faster from torpor (r (2) = 0.77), required less energy to do so (r (2) = 0.32), and reached a higher subcutaneous temperature (T sub) at the end of rewarming (r (2) = 0.75). We provide the first experimental evidence on the possible dual function of the color banding of mammalian fur. The distal colored band appears to be important for camouflage, whereas the length of the dark proximal hair band facilitates heat gain for energy conservation and allows animals to rewarm quickly and economically from torpor.

Chemical characterization of milk oligosaccharides of the eastern quoll (Dasyurus viverrinus).

Structural characterizations of marsupial milk oligosaccharides have been performed in four species to date: the tammar wallaby (Macropus eugenii), the red kangaroo (Macropus rufus), the koala (Phascolarctos cinereus) and the common brushtail possum (Trichosurus vulpecula). To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, the oligosaccharides in the carbohydrate fraction of eastern quoll milk were characterized in this study. Neutral and acidic oligosaccharides were separated and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the neutral oligosaccharides were Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (3",3'-digalactosyllactose), Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I), Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose II), Gal(ß1-3)[Gal(ß1-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose III) and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novooctaose). The structures of the acidic oligosaccharides detected are Neu5Ac(α2-3)Gal(ß1-4)Glc (3'-sialyllactose), Gal(ß1-3)(O-3-sulfate)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate a), Gal(ß1-3)[Gal(ß1-4)(O-3-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose a), Gal(ß1-3)[Neu5Ac(α2-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose c), Neu5Ac(α2-3) Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc with an α(2-3) Neu5Ac linked to ß(1-4)Gal residue of either branch of Gal(ß1-4)GlcNAc(ß1-6) units. The most predominant oligosaccharides in the carbohydrate fraction of mid-lactation milk were found to be lacto-N-novopentaose I and lacto-N-novooctaose, i.e., branched oligosaccharides that contain N-acetylglucosamine. The predominance of these branched oligosaccharides, rather than of a series of linear ß(1-3) linked galacto oligosaccharides, appears to be the main feature of the eastern quoll milk oligosaccharides that differentiates them from those of the tammar wallaby and the brushtail possum.

The value of eutherian-marsupial comparisons for understanding the function of glucocorticoids in female mammal reproduction.

This article is part of a Special Issue "SBN 2014". Chronic stress is known to inhibit female reproductive function. Consequently, it is often assumed that glucocorticoid (GC) concentrations should be negatively correlated with reproductive success because of the role they play in stress physiology. In contrast, a growing body of evidence indicates that GCs play an active role in promoting reproductive function. It is precisely because GCs are so integral to the entire process that disruptions to adrenal activity have negative consequences for reproduction. The goal of this paper is to draw attention to the increasing evidence showing that increases in adrenal activity are important for healthy female reproduction. Furthermore, we outline several hypotheses about the functional role(s) that GCs may play in mediating reproduction and argue that comparative studies between eutherian and marsupial mammals, which exhibit some pronounced differences in reproductive physiology, may be particularly useful for testing different hypotheses about the functional role of GCs in reproduction. Much of our current thinking about GCs and reproduction comes from research involving stress-induced levels of GCs and has led to broad assumptions about the effects of GCs on reproduction. Unfortunately, this has left a gaping hole in our knowledge about basal GC levels and how they may influence reproductive function, thereby preventing a broader understanding of adrenal physiology and obscuring potential solutions for reproductive dysfunction.

The evolution of relative brain size in marsupials is energetically constrained but not driven by behavioral complexity.

Evolutionary increases in mammalian brain size relative to body size are energetically costly but are also thought to confer selective advantages by permitting the evolution of cognitively complex behaviors. However, many suggested associations between brain size and specific behaviors - particularly related to social complexity - are possibly confounded by the reproductive diversity of placental mammals, whose brain size evolution is the most frequently studied. Based on a phylogenetic generalized least squares analysis of a data set on the reproductively homogenous clade of marsupials, we provide the first quantitative comparison of two hypotheses based on energetic constraints (maternal investment and seasonality) with two hypotheses that posit behavioral selection on relative brain size (social complexity and environmental interactions). We show that the two behavioral hypotheses have far less support than the constraint hypotheses. The only unambiguous associates of brain size are the constraint variables of litter size and seasonality. We also found no association between brain size and specific behavioral complexity categories within kangaroos, dasyurids, and possums. The largest-brained marsupials after phylogenetic correction are from low-seasonality New Guinea, supporting the notion that low seasonality represents greater nutrition safety for brain maintenance. Alternatively, low seasonality might improve the maternal support of offspring brain growth. The lack of behavioral brain size associates, found here and elsewhere, supports the general 'cognitive buffer hypothesis' as the best explanatory framework of mammalian brain size evolution. However, it is possible that brain size alone simply does not provide sufficient resolution on the question of how brain morphology and cognitive capacities coevolve.

ARX/Arx is expressed in germ cells during spermatogenesis in both marsupial and mouse.

The X-linked aristaless gene, ARX, is essential for the development of the gonads, forebrain, olfactory bulb, pancreas, and skeletal muscle in mice and humans. Mutations cause neurological diseases, often accompanied by ambiguous genitalia. There are a disproportionately high number of testis and brain genes on the human and mouse X chromosomes. It is still unknown whether the X chromosome accrued these genes during its evolution or whether genes that find themselves on the X chromosome evolve such roles. ARX was originally autosomal in mammals and remains so in marsupials, whereas in eutherian mammals it translocated to the X chromosome. In this study, we examined autosomal ARX in tammars and compared it with the X-linked Arx in mice. We detected ARX mRNA in the neural cells of the forebrain, midbrain and hindbrain, and olfactory bulbs in developing tammars, consistent with the expression in mice. ARX was detected by RT-PCR and mRNA in situ hybridization in the developing tammar wallaby gonads of both sexes, suggestive of a role in sexual development as in mice. We also detected ARX/Arx mRNA in the adult testis in both tammars and mice, suggesting a potential novel role for ARX/Arx in spermiogenesis. ARX transcripts were predominantly observed in round spermatids. Arx mRNA localization distributions in the mouse adult testis suggest that it escaped meiotic sex chromosome inactivation during spermatogenesis. Our findings suggest that ARX in the therian mammal ancestor already played a role in male reproduction before it was recruited to the X chromosome in eutherians.

Reassessing the relationship between brain size, life history, and metabolism at the marsupial/placental dichotomy.

A vigorous discussion surrounds the question as to what enables some mammals--including primates and cetaceans--to evolve large brains. We recently published a study suggesting that the radiation of marsupial mammals is highly relevant to this question because of the unique reproductive and metabolic traits within this clade. In particular, we controversially suggested that marsupial brain sizes are not systematically smaller than those of placentals, and that elevated basal metabolic rates (BMR) are not linked to larger marsupial brains. As our dataset was found to contain some erroneous body size data, derived from a published source, we here use an updated and corrected dataset and employ standard as well as phylogenetically corrected analyses to re-assess and elaborate on our original conclusions. Our proposal that marsupials are not systematically smaller-brained than placentals remains supported, particularly when the unusually large-brained placental clade, Primates, is excluded. Use of the new dataset not only confirms that high metabolic rates are not associated with larger brain size in marsupials, but we additionally find some support for a striking negative correlation between BMR and brain size. The best supported correlates of large brain size remain the reproductive traits of weaning age and litter size. These results support our suggestion that mammalian brain sizes (including, by inference, those of monotremes) are predominantly constrained by the ability of females to fuel the growth of their offspring's large brains, rather than by the maintenance requirements of the adult brain.