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Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four echidnas were provided with green food coloring powder mixed into 20 g of their regular feed with the dose adjusted weekly by 0.05 g. The proportion of organic (feces) versus inorganic matter (sand) in the fecal samples of three echidnas was determined by combustion of organic matter. Hormonal data was then expressed as metabolite concentration per total dry mass (with sand) of extracted sample versus metabolite concentration per total mass of organic material (without sand). The optimal dose of food coloring powder was 0.30 g: this was excreted in the feces of all echidnas within 24 h of consumption with color present for two consecutive days. Correction for inorganic content (sand) did not significantly affect variability of fecal progesterone metabolite levels (mean CV ± SE with sand: 142.3 ± 13.3%; without sand: 127.0 ± 14.4%; W = 6, p =.2500), or the magnitude of change from basal to elevated fecal progesterone metabolite concentrations (mean ± SE with sand: 8.4 ± 1.7; without sand: 6.6 ± 0.5, W = 10, p =.1250). Furthermore, progesterone metabolite concentrations before and after correction for sand contamination correlated strongly (r =.92, p = <.001). These methods will facilitate future reproductive endocrinology studies of echidna and other myrmecophagous species. Research highlights: The minimum dose of food coloring powder required to reliably impart color in echidna feces was 0.30 g.Correcting fecal progesterone data for sand resulted in significantly higher progesterone metabolite concentrations compared to the original data set but did not significantly change metabolite patterns or magnitude of change.Correcting echidna fecal progesterone metabolite data for sand contamination is unnecessary as it is unlikely to improve the detection of biologically relevant changes in fecal progesterone metabolites. [ABSTRACT FROM AUTHOR]

Monotremes are a group of egg-laying mammals, possessing a mosaic of ancestral and derived anatomical features. Despite much interest in monotremes from phylogenetic, morphological, and ecological perspectives, they have been the subject of relatively few biomechanical studies. In this study, we examined shoulder and proximal forelimb muscle anatomy and architecture in the short-beaked echidna, Tachyglossus aculeatus, through contrast-enhanced computed tomography and gross dissection. Muscle architecture is a major determinant of muscle function and can indicate specialized muscle roles, such as the capacity for generating large forces (through large physiological cross-sectional area, PCSA) or working ranges (through long fascicle lengths). We hypothesized that some muscles would exhibit architectural specializations convergent with other fossorial and/or sprawling animals, and that other muscles would reflect the echidna's unusual anatomy and locomotor style. Instead, we found the shoulder and proximal forelimb muscles in echidna to have little variation in their architecture. The muscles generally had long fascicles and small-to-intermediate PCSAs, consistent with force production over a wide working range. Further, muscles did not show overt differences in architecture that, in therian mammals, have been linked to increased forelimb mobility and the transition from sprawling to parasagittal posture. Our measures of architectural disparity placed the echidna closer to the tegu lizard than other sprawling fossorial mammals (e.g., mole). The low architectural diversity found in the echidna's shoulder and proximal forelimb muscles is interpreted as a lack of functional specialization into distinct roles. We hope our study will contribute to greater understanding of monotreme anatomy and biomechanical function, and to the reconstruction of musculoskeletal evolution in mammals. [ABSTRACT FROM AUTHOR]

Indigenous gut microbial communities (microbiota) play critical roles in health and may be especially important for the mother and fetus during pregnancy. Monotremes, such as the short-beaked echidna, have evolved to lay and incubate an egg, which hatches in their pouch where the young feeds. Since both feces and eggs pass through the cloaca, the fecal microbiota of female echidnas provides an opportunity for vertical transmission of microbes to their offspring. Here, we characterize the gut/fecal microbiome of female short-beaked echidnas and gain a better understanding of the changes that may occur in their microbiome as they go through pregnancy. Fecal samples from four female and five male echidnas were obtained from the Currumbin Wildlife Sanctuary in Queensland and sequenced to evaluate bacterial community structure. We identified 25 core bacteria, most of which were present in male and female samples. Genera such as Fusobacterium, Bacteroides, Escherichia-Shigella, and Lactobacillus were consistently abundant, regardless of sex or gestation stage, accounting for 58.00% and 56.14% of reads in male and female samples, respectively. The echidna microbiome remained stable across the different gestation stages, though there was a significant difference in microbiota composition between male and female echidnas. This study is the first to describe the microbiome composition of short-beaked echidnas across reproductive phases and allows the opportunity for this novel information to be used as a metric of health to aid in the detection of diseases triggered by microbiota dysbiosis., (© 2023 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Antibiotic resistance is a problem that necessitates the identification of new antimicrobial molecules. Milk is known to have molecules with antimicrobial properties (AMPs). Echidna Antimicrobial Protein (EchAMP) is one such lactation specific AMP exclusively found in the milk of Echidna, an egg-laying mammal geographically restricted to Australia and New Guinea. Previous studies established that EchAMP exhibits substantial bacteriostatic activity against multiple bacterial genera. However, the subsequent structural and functional studies were hindered due to the unavailability of pure protein. In this study, we expressed EchAMP protein using a heterologous expression system and successfully purified it to >95% homogeneity. The purified recombinant protein exhibits bacteriolytic activity against both Gram-positive and Gram-negative bacteria as confirmed by live-dead staining and scanning electron microscopy. Structurally, this AMP belongs to the family of intrinsically disordered proteins (IDPs) as deciphered by the circular-dichroism, tryptophan fluorescence, and NMR spectroscopy. Nonetheless, EchAMP has the propensity to acquire structure with amphipathic molecules, or membrane mimics like SDS, lipopolysaccharides, and liposomes as again observed through multiple spectroscopic techniques. Recombinant EchAMP exhibits broad-spectrum bacteriolytic activity by compromising the bacterial cell membrane integrity. Hence, we propose that this intrinsically disordered antimicrobial protein interact with the bacterial cell membrane and undergoes conformational changes to form channels in the membrane resulting in cell lysis. EchAMP, the evolutionarily conserved, lactation specific AMP from an oviparous mammal may find application as a broad-spectrum antimicrobial against pathogens that affect mammary gland or otherwise cause routine infections in humans and livestock. Unlabelled Image • Echidna milk protein, EchAMP was purified from the recombinant bacterial source • An intrinsically disordered protein with a broad-spectrum bacteriolytic activity • Transitions to alpha-helical conformation upon bacterial membrane interaction • Helical form causes bacterial membrane disruption, cytosol leakage, and cell death [ABSTRACT FROM AUTHOR]

The widespread presence of contaminants of emerging concern (CEC) in surface waters, treated wastewater and drinking water is an ongoing issue for the water industry. The absence of regulatory guidance and limited occurrence, toxicity and removal data are defining criteria of CEC and make it difficult to prioritise which CEC pose the greatest risk. The online Emerging CHemIcals Database for National Awareness (ECHIDNA) aims to classify and prioritise CEC based on their potential risk, with the information presented in an easily accessible and intuitive manner. A candidate list of almost 1,800 potential CEC, including pesticides, pharmaceuticals and industrial compounds, was compiled using both Australian and international resources. These were ranked based on in silico assessment of their persistent, bioaccumulative and toxic (PBT) properties, as well as potential chronic toxicity hazard, yielding 247 CEC for further prioritisation. Risk Quotients (RQ) identified between 5 and 87 CEC posing a risk to human and ecosystem health, respectively, across drinking water, surface water, treated wastewater and raw wastewater. While the ability of the water industry to effectively prioritise CEC is limited by candidate identification and data availability, ECHIDNA can provide valuable information for better decision-making surrounding CEC management.

The dietary preference of most crocodilians is generally thought to be fairly broad. However, the head morphology of slender-snouted crocodilians limits their ability to process large and complex prey. The slender-snouted Australian freshwater crocodile is known to be a dietary specialist consuming small aquatic prey, particularly aquatic arthropods and fish. Here, we report observations of predation events by Australian freshwater crocodiles on medium- and large-sized mammals in the Kimberley region of Western Australia including macropods, a large rodent and an echidna. We discuss the significance of our observations from an ecological and morphological perspective and propose that terrestrial mammalian prey may be a seasonally important prey item for some populations of freshwater crocodiles. [ABSTRACT FROM AUTHOR]

Case report Two cases of metastatic mineralisation caused by dietary excess of vitamin D are reported in hand-raised short-beaked echidna ( Tachyglossus aculeatus) puggles at the Australian Wildlife Health Centre. Oversupplementation was attributed to excessive levels of vitamin D in Wombaroo Echidna Milk Replacer fed to the puggles. No information exists regarding the naturally occurring vitamin D levels in echidna milk, but, given the low serum levels that have been observed in free-ranging animals, it is likely to be low. Conclusion The vitamin D concentration in Wombaroo Echidna Milk Replacer has been reduced. [ABSTRACT FROM AUTHOR]

The short-beaked echidna ( Tachyglossus aculeatus ) and the platypus ( Ornithorhynchus anatinus ) are iconic egg-laying monotremes (Mammalia: Monotremata) from Australasia. The aim of this study was to demonstrate the utility of diversity profiles in disease investigations of monotremes. Using small subunit (18S) rDNA amplicon deep-sequencing we demonstrated the presence of apicomplexan parasites and confirmed by direct and cloned amplicon gene sequencing Theileria ornithorhynchi , Theileria tachyglossi , Eimeria echidnae and Cryptosporidium fayeri . Using a combination of samples from healthy and diseased animals, we show a close evolutionary relationship between species of coccidia ( Eimeria ) and piroplasms ( Theileria ) from the echidna and platypus. The presence of E . echidnae was demonstrated in faeces and tissues affected by disseminated coccidiosis. Moreover, the presence of E . echidnae DNA in the blood of echidnas was associated with atoxoplasma-like stages in white blood cells, suggesting Hepatozoon tachyglossi blood stages are disseminated E . echidnae stages. These next-generation DNA sequencing technologies are suited to material and organisms that have not been previously characterised and for which the material is scarce. The deep sequencing approach supports traditional diagnostic methods, including microscopy, clinical pathology and histopathology, to better define the status quo . This approach is particularly suitable for wildlife disease investigation. [ABSTRACT FROM AUTHOR]

A single specimen of Echidna xanthospilos (Bleeker, 1859) was captured off Great Nicobar Island. This finding represents a new addition to the muraenid fauna of India. This is one of the data-deficient species hitherto known only from five countries in the Indo-Pacific. The specimen is described and illustrated. [ABSTRACT FROM AUTHOR]

In 1803, the French anatomist Étienne Geoffroy Saint-Hilaire decided that the newly described echidna and platypus should be placed in a separate order, the monotremes, intermediate between reptiles andmammals. The first physiological observations showed monotremes had low body temperatures and metabolic rates, and the consensus was that they were at a stage of physiological development intermediate between "higher mammals" and "lower vertebrates." Subsequent studies demonstrated that platypuses and echidnas are capable of close thermoregulation in the cold although less so under hot conditions. Because the short-beaked echidna Tachyglossus aculeatus, may show very large daily variations in body temperature, as well as seasonal hibernation, it has been suggested that it may provide a useful model of protoendotherm physiology. Such analysis is complicated by the very significant differences in thermal relations between echidnas from different climates. In all areas female echidnas regulate Tb within 1°C during egg incubation. The lactation period is considered to be the most energetically expensive time for most female mammals but lactating echidnas showed no measurable difference in field metabolic rate from non-lactating females, while the lactation period is more than 200 days for Kangaroo Island echidnas but only 150 days in Tasmania. In areas with mild winters echidnas show reduced activity and shallow torpor in autumn and early winter, but in areas with cold winters echidnas enter true hibernation with Tb falling as low as 4.5°C. Monotremes do not possess brown adipose tissue and maximum rates of rewarming from hibernation in echidnas were only half those of marmots of the same mass. Although echidnas show very large seasonal variations in fat stores associated with hibernation there is no relationship between plasma leptin and adiposity. Leptin levels are lowest during post-reproductive fattening, supporting suggestions that in evolutionary terms the anorectic effects of leptin preceded the adiposity signal. BMR of platypuses is twice that of echidnas although maximum metabolism is similar. High levels of thyroid hormones in platypuses may be driving metabolism limited by low body temperature. Monotremes show a mosaic of plesiomorphic and derived features but can still inform our understanding of the evolution of endothermy. [ABSTRACT FROM AUTHOR]

The short-beaked echidna (Tachyglossus aculeatus) is a monotreme and therefore provides a unique combination of phylogenetic history, morphological differentiation and ecological specialisation for a mammal. The echidna has a unique appendicular skeleton, a highly specialised myrmecophagous lifestyle and a mode of locomotion that is neither typically mammalian nor reptilian, but has aspects of both lineages. We therefore were interested in the interactions of locomotor biomechanics, ecology and movements for wild, free-living short-beaked echidnas. To assess locomotion in its complex natural environment, we attached both GPS and accelerometer loggers to the back of echidnas in both spring and summer. We found that the locomotor biomechanics of echidnas is unique, with lower stride length and stride frequency than reported for similar-sized mammals. Speed modulation is primarily accomplished through changes in stride frequency, with a mean of 1.39 Hz and a maximum of 2.31 Hz. Daily activity period was linked to ambient air temperature, which restricted daytime activity during the hotter summer months. Echidnas had longer activity periods and longer digging bouts in spring compared with summer. In summer, echidnas had higher walking speeds than in spring, perhaps because of the shorter time suitable for activity. Echidnas spent, on average, 12% of their time digging, which indicates their potential to excavate up to 204 m³ of soil a year. This information highlights the important contribution towards ecosystem health, via bioturbation, of this widespread Australian monotreme. [ABSTRACT FROM AUTHOR]

The motor neurons in the spinal cord of an echidna ( Tachyglossus aculeatus) have been mapped in Nissl-stained sections from spinal cord segments defined by spinal nerve anatomy. A medial motor column of motor neurons is found at all spinal cord levels, and a hypaxial column is found at most levels. The organization of the motor neuron clusters in the lateral motor column of the brachial (C5 to T3) and crural (L2 to S3) limb enlargements is very similar to the pattern previously revealed by retrograde tracing in placental mammals, and the motor neuron clusters have been tentatively identified according to the muscle groups they are likely to supply. The region separating the two limb enlargements (T4 to L1) contains preganglionic motor neurons that appear to represent the spinal sympathetic outflow. Immediately caudal to the crural limb enlargement is a short column of preganglionic motor neurons (S3 to S4), which it is believed represents the pelvic parasympathetic outflow. The rostral and caudal ends of the spinal cord contain neither a lateral motor column nor a preganglionic column. Branchial motor neurons (which are believed to supply the sternomastoid and trapezius muscles) are present at the lateral margin of the ventral horn in rostral cervical segments (C2-C4). These same segments contain the phrenic nucleus, which belongs to the hypaxial column. The presence or absence of the main spinal motor neuron columns in the different regions echidna spinal cord (and also in that of other amniote vertebrates) provides a basis for dividing the spinal cord into six main regions - prebrachial, brachial, postbrachial, crural, postcrural and caudal. The considerable biological and functional significance of this subdivision pattern is supported by recent studies on spinal cord hox gene expression in chicks and mice. On the other hand, the familiar 'segments' of the spinal cord are defined only by the anatomy of adjacent vertebrae, and are not demarcated by intrinsic gene expression. The recognition of segments defined by vertebrae (somites) is obviously of great value in defining topography, but the emphasis on such segments obscures the underlying evolutionary reality of a spinal cord comprised of six genetically defined regions. The six-region system can be usefully applied to the spinal cord of any amniote (and probably most anurans), independent of the number of vertebral segments in each part of the spinal column. [ABSTRACT FROM AUTHOR]

The paper is the first record of the occurrence of the Pink Lipped Moray Eel, Echidna rhodochilus (Bleeker 1863), from India. The current specimen was caught from the mangrove creeks of Carbyn’s Cove, South Andaman. This species is known so far from Western Pacific and southeastern Indian Ocean up to Australia and Indonesia. The present study indicates that there are more studies to be conducted on the ichthyofauna of these islands for a better understanding of the biodiversity of this area. [ABSTRACT FROM AUTHOR]

Background: To date, little has been documented about microorganisms harboured within Australian native ticks or their pathogenic potential. Recently, a Borrelia sp. related to the Relapsing Fever (RF) group was identified in a single tick removed from a wild echidna (Tachyglossus aculeatus). The present study investigated the presence of Borrelia in 97 Bothriocroton concolor ticks parasitizing echidnas in Queensland, New South Wales, and Victoria, Australia, using nested PCR with Borrelia-specific primers targeting the 16S rRNA (16S) and flaB genes. Results: Borrelia-specific PCR assays confirmed the presence of a novel Borrelia sp. related to the RF and reptile-associated (REP) spirochaetes in 38 (39 %) B. concolor ticks. This novel Borrelia sp. was identified in 41 % of the B. concolor ticks in Queensland and New South Wales, but not in any ticks from Victoria. The resulting flaB sequences (407 bp) were 88 and 86 % similar to the flaB sequences from Borrelia turcica and Borrelia hermsii, respectively. Of the ticks confirmed as Borrelia-positive following the flaB assay, 28 were positive with the 16S assay. Phylogenetic analysis of the 16S sequences (1097 bp) suggests that these sequences belong to a novel Borrelia sp., which forms a unique monophyletic clade that is similar to, but distinct from, RF Borrelia spp. and REP-associated Borrelia spp. Conclusions: We conclude that the novel Borrelia sp. identified in this study does not belong to the Borrelia burgdorferi (sensu lato) complex, and that the phylogenetic analysis of the partial 16S gene sequences suggests it forms a unique monophyletic cluster in the genus Borrelia, potentially forming a fourth major group in this genus associated with monotremes in Australia. However, a thorough molecular characterisation will be required to confirm the phylogenetic position of this unique Borrelia sp. The zoonotic potential and pathogenic consequences of this novel Borrelia sp. are unknown at the current time. [ABSTRACT FROM AUTHOR]

The early divergence of monotremes and therian mammals has resulted in considerable interest in the comparative physiology of the short-beaked echidna (Tachyglossus aculeatus), the most common and widespread living monotreme. However, there are many and varied interpretations of its physiology, reflecting the many and varied studies, limitations and uncertainties of aspects of some previous studies, and potential differences between the various subspecies. Consequently, we thoroughly examine here the standardized physiology of the most widely distributed subspecies of short-beaked echidna (T. aculeatus acanthion) over a wide range of ambient temperatures to definitively assess its physiology in a comparative context. We conclude that the low and variable body temperature of the short-beaked echidna is physiologically "primitive," but it also reflects adaptation to its myrmecophagous niche. Other aspects of its physiology are more typically mammalian. A low metabolic rate reflects its low body temperature, and ventilatory variables are matched to accommodate a modest gas exchange requirement. Thermal conductance is typical for a mammal of equivalent mass. In contrast to previous studies, we demonstrate that short-beaked echidnas can enhance evaporative water loss above thermoneutrality, like other mammals, with a similar capacity for evaporative heat loss. Cooling of their nasal blood sinus with nasal mucous may contribute to this enhanced evaporative cooling. Their capacity to evaporatively cool explains how their distribution can include habitats where ambient temperature, even in shelters, exceeds their supposed critical thermal limit. [ABSTRACT FROM AUTHOR]

Fires have occurred throughout history, including those associated with the meteoroid impact at the Cretaceous-Palaeogene (K-Pg) boundary that eliminated many vertebrate species. To evaluate the recent hypothesis that the survival of the K-Pg fires by ancestral mammals was dependent on their ability to use energy-conserving torpor, we studied body temperature fluctuations and activity of an egg-laying mammal, the echidna (Tachyglossus aculeatus), often considered to be a 'living fossil', before, during and after a prescribed burn. All but one study animal survived the fire in the prescribed burn area and echidnas remained inactive during the day(s) following the fire and substantially reduced body temperature during bouts of torpor. For weeks after the fire, all individuals remained in their original territories and compensated for changes in their habitat with a decrease in mean body temperature and activity. Our data suggest that heterothermy enables mammals to outlast the conditions during and after a fire by reducing energy expenditure, permitting periods of extended inactivity. Therefore, torpor facilitates survival in a fire-scorched landscape and consequently may have been of functional significance for mammalian survival at the K-Pg boundary. [ABSTRACT FROM AUTHOR]

Monotremata is a clade of egg-lying mammals, represented by the living platypus and echidnas, which is endemic to Australia, and adjacent islands. Occurrence of basal monotremes in the Early Cretaceous of Australia has led to the consensus that this clade originated on that continent, arriving later to South America. Here we report on the discovery of a Late Cretaceous monotreme from southern Argentina, demonstrating that monotremes were present in circumpolar regions by the end of the Mesozoic, and that their distinctive anatomical features were probably present in these ancient forms as well., (© 2023. The Author(s).)

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0-18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

For over two centuries, the platypus ( Ornithorhynchus anatinus) has been constructed and categorized in multiple ways. An unprecedented mélange of anatomical features and physiological functions, it long remained a systematic quandary. Nevertheless, since 1797, naturalists and biologists have pursued two recurring obsessions. Investigations into platypus reproduction and lactation have focused attention largely upon females of the species. Despite its apparent admixture of avian, reptilian and mammalian characters, the platypus was soon placed as a rudimentary mammal - primitive, naïve and harmless. This article pursues a different taxonomic trajectory, concentrating on a specifically male anatomical development: the crural spur and venom gland on the hind legs. Once the defining characteristic of both the platypus and echidna ( Tachyglossus aculeatus), by 1830 this sexed spur had been largely dismissed as inactive and irrelevant. For a creature regularly depicted as a biological outlier, the systematic and evolutionary implications of platypus poison have remained largely overlooked. In Australia, however, sporadic cases of 'spiking' led to consistent homologies being remarked between the platypus crural system and the venom glands of snakes. As with its reproductive reliance upon eggs, possession of an endogenous poison suggested significant reptilian affinities, yet the platypus has rarely been classed as an advanced reptile. Indeed, ongoing uncertainty regarding the biological purpose of the male's spur has ostensibly posed a directional puzzle. As with so many of its traits, however, platypus poison has been consistently described as a redundant remnant, rather than an emergent feature indicating evolutionary advance. [ABSTRACT FROM AUTHOR]

Captive breeding of the short-beaked echidna (Tachyglossus aculeatus) has proven a difficult challenge; as recently as 2009, there were fewer than 10 echidnas born in captivity. We present observations of captive reproductive behaviour following video surveillance and measurements of body temperature collected from six captive female echidnas over a six-year period. In the first series of observations (2009-10) we examined the efficacy of artificial burrow boxes as possible aids for reproductive success. Females with access to burrow boxes had significantly higher levels of reproductive activity (P = 0.001), there was coincidental improvement in the production of eggs or pouch young (two eggs, one unhatched and one offspring). During 2009-10, a range of reproductive behaviours (courtship, copulation and postcopulation) were documented and analysed, as were new observations of oestrous cycle activity. Female body temperature was characteristically stable during egg incubation during this study and has the potential to be used as a tool for the assessment of reproductive status. Following initial observations, burrow boxes and infrared lamps were implemented as standard husbandry in our echidna breeding facility and the effects on reproductive success were monitored, albeit less intensively, for a further four years (2011-14). Although no direct causal effect could be ascribed, the use of burrow boxes and heat lamps coincided with a total of 13 young being born to four females in the last four years (2011-14). These female echidnas were found to be receptive at intervals throughout the breeding season, both before and after presumed incubation phases, suggesting that captive animals exhibit polyoestry. In 2012 and 2014, the same female showed evidence of producing two young from one breeding event. Provision of burrow boxes to captive short-beaked echidnas coincided with a substantial increase in reproduction with 13 young being born to four females in the years 2011-14. Reproductive behaviours were documented as well as evidence for polyoestry and production of multiple young from one female from one breeding event. [ABSTRACT FROM AUTHOR]

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide ( PP)-cells were mainly found in the islet periphery. Distinct PP-rich ( PP-lobe) and PP-poor areas (non- PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution. [ABSTRACT FROM AUTHOR]

The monotreme pattern of egg-incubation followed by extended lactation represents the ancestral mammalian reproductive condition, suggesting that monotreme milk may include saccharides of an ancestral type. Saccharides were characterized from milk of the Tasmanian echidna Tachyglossus aculeatus setosus. Oligosaccharides in pooled milk from late lactation were purified by gel filtration and high-performance liquid chromatography using a porous graphitized carbon column and characterized by 1H NMR spectroscopy; oligosaccharides in smaller samples from early and mid-lactation were separated by ultra-performance liquid chromatography and characterized by negative electrospray ionization mass spectrometry (ESI-MS) and tandem collision mass spectroscopy (MS/MS) product ion patterns. Eight saccharides were identified by 1H NMR: lactose, 2′-fucosyllactose, difucosyllactose (DFL), B-tetrasaccharide, B-pentasaccharide, lacto-N-fucopentaose III (LNFP3), 4-O-acetyl-3′-sialyllactose [Neu4,5Ac(α2–3)Gal(β1–4)Glc] and 4-O-acetyl-3′-sialyl-3-fucosyllactose [Neu4,5Ac(α2–3)Gal(β1–4)[Fuc(α1–3)]Glc]. Six of these (all except DFL and LNFP3) were present in early and mid-lactation per ESI-MS, although some at trace levels. Four additional oligosaccharides examined by ESI-MS and MS/MS are proposed to be 3′-sialyllactose [Neu5Ac(α2–3)Gal(β1–4)Glc], di-O-acetyl-3′-sialyllactose [Neu4,5,UAc3(α2–3)Gal(β1–4)Glc where U = 7, 8 or 9], 4-O-acetyl-3′-sialyllactose sulfate [Neu4,5Ac(α2–3)Gal(β1–4)GlcS, where position of the sulfate (S) is unknown] and an unidentified 800 Da oligosaccharide containing a 4-O-acetyl-3′-sialyllactose core. 4-O-acetyl-3′-sialyllactose was the predominant saccharide at all lactation stages. 4-O-Acetylation is known to protect sialyllactose from bacterial sialidases and may be critical to prevent microbial degradation on the mammary areolae and/or in the hatchling digestive tract so that sialyllactose can be available for enterocyte uptake. The ability to defend against microbial invasion was probably of great functional importance in the early evolution of milk saccharides. [ABSTRACT FROM AUTHOR]