Validation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus).

The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic-pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.

Immune-endocrine interactions in marsupials and monotremes.

Interactions between the immune and endocrine systems are not well studied in marsupials and monotremes. One exception to this is the phenomenon of semelparity, which is well covered in the literature as this is an unusual reproductive strategy amongst mammals and is only observed in some dasyurid and didelphid marsupials. Thymus involution provides a direct link between the endocrine and immune systems and warrants further study in marsupials and monotremes. The thymus is a primary immune tissue which is essential for overall immune function. Whilst the organ is large in juvenile animals, it begins to involute around puberty due to the suppressive effects of sex steroids. Thymus involution has a significant effect on the immune system, as it signals the onset of immune aging and decline in function. The output of naïve T lymphocytes by the thymus decreases, increasing susceptibility of aged individuals to infection and cancers. Understanding the links between the immune and endocrine system in marsupials and monotremes may shed light on diseases such as devil facial tumour disease (DFTD) which threatens the future of the Tasmanian devil. We hypothesise that changes in sex hormones around puberty may drive changes in the immune system, such as thymus involution, which may make devils more susceptible to DFTD as they age. In addition, the Schwann cell origin of DFTD may enable tumours to respond to sex hormones, as occurs in similar cancers in humans.

Implications of monotreme and marsupial chromosome evolution on sex determination and differentiation.

Studies of chromosomes from monotremes and marsupials endemic to Australasia have provided important insight into the evolution of their genomes as well as uncovering fundamental differences in their sex determination/differentiation pathways. Great advances have been made this century into solving the mystery of the complicated sex chromosome system in monotremes. Monotremes possess multiple different X and Y chromosomes and a candidate sex determining gene has been identified. Even greater advancements have been made for marsupials, with reconstruction of the ancestral karyotype enabling the evolutionary history of marsupial chromosomes to be determined. Furthermore, the study of sex chromosomes in intersex marsupials has afforded insight into differences in the sexual differentiation pathway between marsupials and eutherians, together with experiments showing the insensitivity of the mammary glands, pouch and scrotum to exogenous hormones, led to the hypothesis that there is a gene (or genes) on the X chromosome responsible for the development of either pouch or scrotum. This review highlights the major advancements made towards understanding chromosome evolution and how this has impacted on our understanding of sex determination and differentiation in these interesting mammals.

Timing of mammalian peripheral trigeminal system development relative to body size: A comparison of metatherians with rodents and monotremes.

Specializations of the trigeminal sensory system are present in all three infraclasses of mammals (metatheria, eutheria, prototheria or monotremata). The trigeminal sensory system has been suggested as a critically important modality for sampling the path to the pouch and detecting the nipple or milk patch, but the degree to which that system may be required to function at birth varies significantly. Archived sections of the snout and brainstem of embryonic and postnatal mammals were used to test the relationship between structural maturity of the two ends of the trigeminal nerve pathway and the body size of mammalian young in metatherians, rodents and monotremes. A system for staging different levels of structural maturity of the vibrissae and trigeminal sensory was applied to embryos, pouch young and hatchlings and correlated with body length. Dasyurids are born at the most immature state with respect to vibrissal and trigeminal sensory nucleus development of any available metatherian, but these components of the trigeminal system are also developmentally advanced relative to body size when dasyurids are compared to other metatherians. Vibrissal and trigeminal sensory nucleus development is at a similar stage of development at birth and for a given body size in non-dasyurid metatherians; and trigeminal sensory nucleus development in monotremes is at a similar stage at birth to metatherians. Rodents reach a far more advanced stage of vibrissal and trigeminal sensory nucleus development at birth than do metatherians, and in the case of the mouse have a more developmentally advanced trigeminal system than all available metatherians at any given body length. Precocious development of the trigeminal sensory pathway relative to body size is evident in dasyurids, as might be expected given the small birth size of those metatherians. Nevertheless, the trigeminal sensory system in metatherians in general is not precocious relative to body size when these species are considered alongside the pace of trigeminal somatosensory development in rodents.

Bioactive Functions of Milk Proteins: a Comparative Genomics Approach.

The composition of milk includes factors required to provide appropriate nutrition for the growth of the neonate. However, it is now clear that milk has many functions and comprises bioactive molecules that play a central role in regulating developmental processes in the young while providing a protective function for both the suckled young and the mammary gland during the lactation cycle. Identifying these bioactives and their physiological function in eutherians can be difficult and requires extensive screening of milk components that may function to improve well-being and options for prevention and treatment of disease. New animal models with unique reproductive strategies are now becoming increasingly relevant to search for these factors.

The development of the olfactory organs in newly hatched monotremes and neonate marsupials.

Olfactory cues are thought to play a crucial role in the detection of the milk source at birth in mammals. It has been shown that a marsupial, the tammar wallaby, can detect olfactory cues from its mother's pouch at birth. This study investigates whether the main olfactory and accessory olfactory system are similarly well developed in other marsupials and monotremes at birth/hatching as in the tammar. Sections of the head of various marsupial and two monotreme species were investigated by light microscopy. Both olfactory systems were less well developed in the kowari and Eastern quoll. No olfactory or vomeronasal or terminal nerves could be observed; the main olfactory bulb (MOB) had only two layers while no accessory olfactory bulb or ganglion terminale were visible. All other investigated marsupials and monotremes showed further developed olfactory systems with olfactory, vomeronasal and terminal nerves, a three-layered MOB, and in the marsupials a prominent ganglion terminale. The main olfactory system was further developed than the accessory olfactory system in all species investigated. The olfactory systems were the least developed in species in which the mother's birth position removed most of the difficulty in reaching the teat, placing the neonate directly in the pouch. In monotremes they were the furthest developed as Bowman glands were found underlying the main olfactory epithelium. This may reflect the need to locate the milk field each time they drink as they cannot permanently attach to it, unlike therian mammals. While it still needs to be determined how an odour signal could be further processed in the brain, this study suggests that marsupials and monotremes possess well enough developed olfactory systems to be able to detect an odour cue from the mammary area at birth/hatching. It is therefore likely that neonate marsupials and newly hatched monotremes find their way to the milk source using olfactory cues, as has been previously suggested for the marsupial tammar wallaby, rabbits, rats and other eutherians.

Universal scaling of production rates across mammalian lineages.

Over many millions of years of independent evolution, placental, marsupial and monotreme mammals have diverged conspicuously in physiology, life history and reproductive ecology. The differences in life histories are particularly striking. Compared with placentals, marsupials exhibit shorter pregnancy, smaller size of offspring at birth and longer period of lactation in the pouch. Monotremes also exhibit short pregnancy, but incubate embryos in eggs, followed by a long period of post-hatching lactation. Using a large sample of mammalian species, we show that, remarkably, despite their very different life histories, the scaling of production rates is statistically indistinguishable across mammalian lineages. Apparently all mammals are subject to the same fundamental metabolic constraints on productivity, because they share similar body designs, vascular systems and costs of producing new tissue.

The nocturnal bottleneck and the evolution of mammalian vision.

Evidence from the early paleontological record of mammalian evolution has often been interpreted as supporting the idea that mammals were nocturnal for most of their early history. Multiple features of extant mammal sensory systems, such as evolutionary modifications to the light-regulated circadian system, photoreceptor complement, and retinal morphology, support this nocturnal hypothesis for mammalian evolution. Here, we synthesize data on eye shape and orbit orientation in mammals as these data compare to other amniotes. Most mammals differ from other amniotes in retaining an eye design optimized for high visual sensitivity, with the requisite reduction in acuity, which is typically restricted to scotopically (i.e. low light) adapted amniotes. Mammals also possess the more convergent (similarly facing) orbits and, on average, the largest binocular visual fields among amniotes. Based on our analyses, we propose that extant mammals retain a scotopic eye design as well as expanded binocular zones as a result of their nocturnal origin. Only anthropoid primates notably differ from general mammalian patterns, and possibly have evolved an eye shape more typical of the ancestral amniote condition.

Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes.

The present review examines whether monotremes may help to resolve three questions relating to sperm production in mammals: why the testes descend into a scrotum in most mammals, why spermatozoa are infertile when they leave the testes and require a period of maturation in the specific milieu provided by the epididymides, and why ejaculated spermatozoa cannot immediately fertilise an ovum until they undergo capacitation within the female reproductive tract. Comparisons of monotremes with other mammals indicate that there is a need for considerable work on monotremes. It is hypothesised that testicular descent should be related to epididymal differentiation. Spermatozoa and ova from both groups share many of the proteins that are thought to be involved in gamete interaction, and although epididymal sperm maturation is significant it is probably less complex in monotremes than in other mammals. However, the monotreme epididymis is unique in forming spermatozoa into bundles of 100 with greatly enhanced motility compared with individual spermatozoa. Bundle formation involves a highly organised interaction with epididymal proteins, and the bundles persist during incubation in vitro, except in specialised medium, in which spermatozoa separate after 2-3 h incubation. It is suggested that this represents an early form of capacitation.

Reproductive biology in egg-laying mammals.

The duck-billed platypus and short-beaked echidna are iconic species in Australia. Their morphology and physiology have puzzled scientists all over the world for more than 200 years. Recent genetic studies, particularly the platypus whole-genome sequencing project, have revealed the molecular basis of some of the extraordinary characteristics of monotremes. This and other works demonstrate the great value of research on our most distantly related mammalian relatives for comparative genomics and developmental biology. In this review we focus on the reproductive biology of monotremes and discuss works that unravel genes involved in lactation, testicular descent, gamete biology and fertilization, and early development. In addition we discuss works on the evolution of the complex sex chromosome system in platypus and echidna, which has also significant impact on our general understanding of mammalian sex chromosomes and sex determination.

The "minimal boundary curve for endothermy" as a predictor of heterothermy in mammals and birds: a review.

According to the concept of the "minimal boundary curve for endothermy", mammals and birds with a basal metabolic rate (BMR) that falls below the curve are obligate heterotherms and must enter torpor. We examined the reliability of the boundary curve (on a double log plot transformed to a line) for predicting torpor as a function of body mass and BMR for birds and several groups of mammals. The boundary line correctly predicted heterothermy in 87.5% of marsupials (n = 64), 94% of bats (n = 85) and 82.3% of rodents (n = 157). Our analysis shows that the boundary line is not a reliable predictor for use of torpor. A discriminate analysis using body mass and BMR had a similar predictive power as the boundary line. However, there are sufficient exceptions to both methods of analysis to suggest that the relationship between body mass, BMR and heterothermy is not a causal one. Some homeothermic birds (e.g. silvereyes) and rodents (e.g. hopping mice) fall below the boundary line, and there are many examples of heterothermic species that fall above the boundary line. For marsupials and bats, but not for rodents, there was a highly significant phylogenetic pattern for heterothermy, suggesting that taxonomic affiliation is the biggest determinant of heterothermy for these mammalian groups. For rodents, heterothermic species had lower BMRs than homeothermic species. Low BMR and use of torpor both contribute to reducing energy expenditure and both physiological traits appear to be a response to the same selective pressure of fluctuating food supply, increasing fitness in endothermic species that are constrained by limited energy availability. Both the minimal boundary line and discriminate analysis were of little value for predicting the use of daily torpor or hibernation in heterotherms, presumably as both daily torpor and hibernation are precisely controlled processes, not an inability to thermoregulate.

Mammalian diversity: gametes, embryos and reproduction.

The class Mammalia is composed of approximately 4800 extant species. These mammalian species are divided into three subclasses that include the monotremes, marsupials and eutherians. Monotremes are remarkable because these mammals are born from eggs laid outside of the mother's body. Marsupial mammals have relatively short gestation periods and give birth to highly altricial young that continue a significant amount of 'fetal' development after birth, supported by a highly sophisticated lactation. Less than 10% of mammalian species are monotremes or marsupials, so the great majority of mammals are grouped into the subclass Eutheria, including mouse and human. Mammals exhibit great variety in morphology, physiology and reproduction. In the present article, we highlight some of this remarkable diversity relative to the mouse, one of the most widely used mammalian model organisms, and human. This diversity creates challenges and opportunities for gamete and embryo collection, culture and transfer technologies.

Tactile neural mechanisms in monotremes.

Monotremes, perhaps more than any other order of mammals, display an enormous behavioural reliance upon the tactile senses. In the platypus, Ornithorhynchus anatinus, this is manifest most strikingly in the special importance of the bill as a peripheral sensory organ, an importance confirmed by electrophysiological mapping that reveals a vast area of the cerebral cortex allocated to the processing of tactile inputs from the bill. Although behavioural evidence in the echidna, Tachyglossus aculeatus, suggests a similar prominence for tactile inputs from the snout, there is also a great reliance upon the distal limbs for digging and burrowing activity, pointing to the importance of tactile information from these regions for the echidna. In recent studies, we have investigated the peripheral tactile neural mechanisms in the forepaw of the echidna to establish the extent of correspondence or divergence that has emerged over the widely different evolutionary paths taken by monotreme and placental mammals. Electrophysiological recordings were made from single tactile sensory nerve fibres isolated in fine strands of the median or ulnar nerves of the forearm. Controlled tactile stimuli applied to the forepaw glabrous skin permitted an initial classification of tactile sensory fibres into two broad divisions, according to their responses to static skin displacement. One displayed slowly adapting (SA) response properties, while the other showed a selective sensitivity to the dynamic components of the skin displacement. These purely dynamically-sensitive tactile fibres could be subdivided according to vibrotactile sensitivity and receptive field characteristics into a rapidly adapting (RA) class, sensitive to low frequency (

Body temperature in captive long-beaked echidnas (Zaglossus bartoni).

The routine occurrence of both short-term (daily) and long-term torpor (hibernation) in short-beaked echidnas, but not platypus, raises questions about the third monotreme genus, New Guinea's Zaglossus. We measured body temperatures (T(b)) with implanted data loggers over three and a half years in two captive Zaglossus bartoni at Taronga Zoo, Sydney. The modal T(b) of both long-beaks was 31 degrees C, similar to non-hibernating short-beaked echidnas, Tachyglossus aculeatus, in the wild (30-32 degrees C) and to platypus (32 degrees C), suggesting that this is characteristic of normothermic monotremes. T(b) cycled daily, usually over 2-4 degrees C. There were some departures from this pattern to suggest periods of inactivity but nothing to indicate the occurrence of long-term torpor. In contrast, two short-beaked echidnas monitored concurrently in the same pen showed extended periods of low T(b) in the cooler months (hibernation) and short periods of torpor at any time of the year, as they do in the wild. Whether torpor or hibernation occurs in Zaglossus in the wild or in juveniles remains unknown. However, given that the environment in this study was conducive to hibernation in short-beaks, which do not easily enter torpor in captivity, and their large size, we think that torpor in wild adult Zaglossus is unlikely.

Review of the monotreme fossil record and comparison of palaeontological and molecular data.

Monotremes have traditionally been considered a remnant group of mammals descended from archaic Mesozoic stock, surviving to the present day on the relatively isolated Australian continent. Challenges to this orthodoxy have been spurred by discoveries of 'advanced' Cretaceous monotremes (Steropodon galmani, Archer, M., et al., 1985. First Mesozoic mammal from Australia-an Early Cretaceous monotreme, Nature. 318, 363-366) as well as by results from molecular data linking monotremes to therian mammals (specifically to marsupials in some studies). This paper reviews the monotreme fossil record and briefly discusses significant new information from additional Cretaceous Australian material. Mesozoic monotremes (including S. galmani) were a diverse group as evidenced by new material from the Early Cretaceous of New South Wales and Victoria currently under study. Although most of these new finds are edentulous jaws (limiting dental comparisons and determination of dietary niches), a range of sizes and forms has been determined. Some of these Cretaceous jaws exhibit archaic features-in particular evidence for the presence of a splenial bone in S. galmani-not seen in therian mammals or in post-Mesozoic (Tertiary and Quaternary) monotreme taxa. Tertiary monotremes were either archaic ornithorhynchids (toothed platypuses in the genera Monotrematum and Obdurodon) or tachyglossids (large echidnas in the genera Megalibgwilia and Zaglossus). Quaternary ornithorhynchid material is referable to the sole living platypus species Ornithorhynchus anatinus. Quaternary echidnas, however, were moderately diverse and several forms are known (Megalibgwilia species; 'Zaglossus' hacketti; Zaglossus species and Tachyglossus aculeatus).