ADVANCED DIAGNOSTIC IMAGING AND PATHOLOGIC FINDINGS OF THYROID LESIONS IN TWO SHORT-BEAKED ECHIDNAS (TACHYGLOSSUS ACULEATUS).

Two zoo-maintained short-beaked echidnas (Tachyglossus aculeatus) had long histories of intermittent anorexia and lethargy. Case 1 presented with a recurrence of these signs after transfer to another facility and died shortly after arrival. A focal area of hyperattenuation within the paratracheal tissue of the cranial mediastinum was noted antemortem on CT. Postmortem, this corresponded with severe thyroid follicular hyperplasia with lymphoplasmacytic thyroiditis. Additional findings included a systemic fungal infection without an inflammatory response, suggesting underlying factors such as torpor or immunosuppression. In Case 2, an intrathoracic mass was identified during a preshipment examination. CT confirmed a contrast-enhanced mass compressing the cranial vena cava and right atrium, and the animal was euthanized. The mass was diagnosed histologically as thyroid adenocarcinoma. These cases report thyroiditis and thyroid adenocarcinoma in echidna and describe the use of IV contrast and CT as a diagnostic aid in this species.

Comparative genomics of monotremes provides insights into the early evolution of mammalian epidermal differentiation genes.

The function of the skin as a barrier against the environment depends on the differentiation of epidermal keratinocytes into highly resilient corneocytes that form the outermost skin layer. Many genes encoding structural components of corneocytes are clustered in the epidermal differentiation complex (EDC), which has been described in placental and marsupial mammals as well as non-mammalian tetrapods. Here, we analyzed the genomes of the platypus (Ornithorhynchus anatinus) and the echidna (Tachyglossus aculeatus) to determine the gene composition of the EDC in the basal clade of mammals, the monotremes. We report that mammal-specific subfamilies of EDC genes encoding small proline-rich proteins (SPRRs) and late cornified envelope proteins as well as single-copy EDC genes such as involucrin are conserved in monotremes, suggesting that they have originated in stem mammals. Monotremes have at least one gene homologous to the group of filaggrin (FLG), FLG2 and hornerin (HRNR) in placental mammals, but no clear one-to-one pairwise ortholog of either FLG, FLG2 or HRNR. Caspase-14, a keratinocyte differentiation-associated protease implicated in the processing of filaggrin, is encoded by at least 3 gene copies in the echidna. Our results reveal evolutionarily conserved and clade-specific features of the genetic regulation of epidermal differentiation in monotremes.

Postural, pilo-erective and evaporative thermal windows of the short-beaked echidna (Tachyglossus aculeatus).

We identify for wild, free-living short-beaked echidnas (Tachyglossus aculeatus) a novel evaporative window, along with thermal windows, and demonstrate the insulating properties of the spines, using infrared thermography. The moist tip of their beak, with an underlying blood sinus, functions as a wet bulb globe thermometer, maximizing evaporative heat loss via an evaporative window. The ventral surface and insides of the legs are poorly insulated sites that act as postural thermal windows, while the spines provide flexible insulation (depending on piloerection). These avenues of heat exchange likely contribute to the higher-than-expected thermal tolerance of this species. Our study highlights how technological advances that allow for non-contact measurement of thermal variables allow us to better understand the physiological capacity of animals in their natural environment.

EchidnaCSI: Engaging the public in research and conservation of the short-beaked echidna.

The short-beaked echidna is an iconic Australian animal and the most-widespread native mammal, inhabiting diverse environments. The cryptic nature of echidnas has limited research into their ecology in most areas; however, from the well-researched and endangered Kangaroo Island echidna population, we understand that the threats include habitat loss, roads, and invasive species. To obtain more information about echidnas Australia-wide, we established the Echidna Conservation Science Initiative (EchidnaCSI) citizen science project. EchidnaCSI calls on members of the public to submit photographs of wild echidnas and learn to identify and collect echidna scats for molecular analysis. To facilitate participation, we developed a smartphone application as well as ongoing social and traditional media activities and community events. In 3 y, more than 9,000 members of the public have downloaded the EchidnaCSI app, collecting 400 scats and submitting over 8,000 sightings of echidnas from across Australia. A subset of submitted scat samples were subjected to DNA extraction and PCR, which validated the approach of using citizen science for scat collection and viability for molecular analysis. To assess the impact of the project through public participation, we surveyed our participants (n = 944) to understand their demographics and motivations for engagement. Survey results also revealed that EchidnaCSI served as a gateway into citizen science more generally for many participants. EchidnaCSI demonstrates the potential for using citizen science approaches to collect high-quality data and material from a cryptic species over a very large geographic area and the considerable engagement value of citizen science research.

Short-beaked echidna (Tachyglossus aculeatus) home range at Fowlers Gap Arid Zone Research Station, NSW.

Echidnas (Tachyglossus aculeatus) are found Australia-wide and appear to be remarkably well-adapted to the arid zone, yet nearly all echidna research has been conducted in temperate, tropical and alpine zones. This study investigated the home range and movement of echidnas in western New South Wales. Radio telemetry tracking was used to locate the echidnas daily during the study period (March-May 2018, November 2018, March-May 2019 and August 2019); the observed home range was 1.47± 1.21km2. This is over twice the reported home range of temperate environments (<0.65km2), suggesting that echidnas exhibit larger home ranges in arid zones. The home range of individual echidnas ranged from 0.02km2 to 3.56km2. Echidnas exhibited a small degree of overlap (6.6%± 19.8%) but this varied considerably between individuals (between 0 to 84.2% overlap.) Four out of the thirteen echidnas died during this study, likely due to the severe drought that occurred during the study. This study provides insight into the movement and home range of echidnas in arid zones, revealing that desert echidnas have large home ranges, probably dependent on the availability of resources.

Novel detection of provenance in the illegal wildlife trade using elemental data.

Despite being the fourth largest criminal market in the world, no forensic tools have been sufficiently developed to accurately determine the legal status of seized animals and their parts. Although legal trading is permissible for farmed or captive-bred animals, many animals are illegally removed from the wild and laundered by masquerading them as captive bred. Here we present high-resolution x-ray fluorescence (XRF) as a non-invasive and cost-effective tool for forensic classification. We tested the efficacy of this technique by using machine learning on a training set of zoo specimens and wild-caught individuals of short-beaked echidnas (Tachyglossus aculeatus), a small insectivorous monotreme in Australia. XRF outperformed stable isotope analysis (δ13C, δ15N), reducing overall classification error below 4%. XRF has the added advantage of providing samples every 200 µm on a single quill, enabling 100% classification accuracy by taking the consensus of votes per quill. This accurate and cost-effective forensic technique could provide a much needed in situ solution for combating the illegal laundering of wildlife, and conversely, assist with certification of legally bred animals.

Digestibility of a new diet for captive short-beaked echidnas (Tachyglossus aculeatus).

Short-beaked echidnas (Tachyglossus aculeatus) are myrmecophages, or ant and termite insectivore specialists, and replicating their exact diet in captivity is problematic. Diets for captive animals often incorporate raw meat, eggs and cat food mixed together with water, and vitamin and mineral supplements. These diets have promoted a number of health problems in captive echidnas, such as gastritis, cystitis, gut impaction, obesity, and diarrhea. A manufactured diet was designed and three echidnas from two zoos were transitioned onto this diet to assess the acceptability and digestibility of this diet for echidnas. The new "test" diet was readily accepted by the echidnas with a 1 week transition period. Daily digestible energy intake was 280 kJ kg-0.75 d-1 , similar to another myrmecophagous species. Digestibility values were above 74% for all macronutrients. It was determined that this diet was an acceptable replacement for the previous diets and it was decided that the remaining echidnas at both institutions would be transitioned to the new diet. The diet will also be used for wild echidnas being rehabilitated in the zoo hospitals prior to release and commercially available within Australia. Further data are being collected to assess the use of this diet for seasonal weight management, transitioning hand-reared puggles and effects on gastrointestinal tract health. Zoo Biol. 36:56-61, 2017. © 2017 Wiley Periodicals, Inc.

Frozen embryos? Torpor during pregnancy in the Tasmanian short-beaked echidna Tachyglossus aculeatus setosus.

We studied the interaction between torpor and reproduction in free-ranging female Tasmanian echidnas using a combination of techniques including urogenital smears, hormone analysis, ultrasonography, external temperature loggers and camera traps. Male echidnas initiated mating activity by locating hibernating females. All females that mated or were disturbed by males prior to July 27 re-entered hibernation, including many that were pregnant. Pregnant females only entered hibernation in early pregnancy when plasma progesterone concentrations were about twice basal and progesterone then remained constant during torpor. By re-entering hibernation pregnant females extended their gestation period and delayed egg-laying. Progesterone peaked 4-6days before egg-laying, then dropped rapidly.

The private life of echidnas: using accelerometry and GPS to examine field biomechanics and assess the ecological impact of a widespread, semi-fossorial monotreme.

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.

Formation and Dissociation of Sperm Bundles in Monotremes.

Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

Reexamining Echidna Physiology: The Big Picture for Tachyglossus aculeatus acanthion.

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.

Cool echidnas survive the fire.

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.

Observations on fur development in echidna (Monotremata, Mammalia) indicate that spines precede hairs in ontogeny.

In the primitive mammal echidna, the initial 2-3 generations of skin appendages produced from birth forms spines and only later true hairs appear. Microscopy on preserved museum specimens reveals that the morphogenesis of spines and hairs is similar but that a larger dermal papilla is formed in spines. The growing shaft comprises a medulla surrounded by a cortex and by an external cuticle. A thick inner root sheath made of cornified cells surrounds the growing shaft inside the spine canal that eventually exits with a pointed tip. Hairs develop later with the same modality of spines but have a smaller papilla and give rise to a fur coat among spines. Therefore the integument of developing echidnas initially produces spines from large dermal papillae but the reduction in size of the papillae later determines the formation of hairs. Although the morphogenesis of spines and hairs can represent a case of specialization in this species, the primitive mammalian characteristics of echidnas has also inspired new speculations on the evolution of the mammalian hair from mammalian-like reptiles with a spiny coat. The resemblance in the morphogenesis between spines and hairs has suggested some hypothesis on hair evolution, in particular that hairs might be derived from the reduction of protective large spines present in ancient mammalian-like reptiles possibly derived from the reduction of pre-existing pointed scales. The hypothesis suggests that spines became reduced and internalized in the skin forming hairs.

Brain and behaviour of living and extinct echidnas.

The Tachyglossidae (long- and short-beaked echidnas) are a family of monotremes, confined to Australia and New Guinea, that exhibit striking trigeminal, olfactory and cortical specialisations. Several species of long-beaked echidna (Zaglossus robusta, Zaglossus hacketti, Megalibgwilia ramsayi) were part of the large-bodied (10 kg or more) fauna of Pleistocene Australasia, but only the diminutive (2-7 kg) Tachyglossus aculeatus is widespread today on the Australian mainland. We used high-resolution CT scanning and other osteological techniques to determine whether the remarkable neurological specialisations of modern echidnas were also present in Pleistocene forms or have undergone modification as the Australian climate changed in the transition from the Pleistocene to the Holocene. All the living and extinct echidnas studied have a similar pattern of cortical gyrification that suggests comparable functional topography to the modern short-beaked form. Osteological features related to olfactory, trigeminal, auditory and vestibular specialisation (e.g., foramina and cribriform plate area, osseous labyrinth topography) are also similar in living and extinct species. Our findings indicate that despite differences in diet, habitat and body size, the suite of neurological specialisations in the Tachyglossidae has been remarkably constant: encephalisation, sensory anatomy and specialisation (olfactory, trigeminal, auditory and vestibular), hypoglossal nerve size and cortical topography have all been stable neurological features of the group for at least 300,000 years.

Passive electroreception in aquatic mammals.

Passive electroreception is a sensory modality in many aquatic vertebrates, predominantly fishes. Using passive electroreception, the animal can detect and analyze electric fields in its environment. Most electric fields in the environment are of biogenic origin, often produced by prey items. These electric fields can be relatively strong and can be a highly valuable source of information for a predator, as underlined by the fact that electroreception has evolved multiple times independently. The only mammals that possess electroreception are the platypus (Ornithorhynchus anatinus) and the echidnas (Tachyglossidae) from the monotreme order, and, recently discovered, the Guiana dolphin (Sotalia guianensis) from the cetacean order. Here we review the morphology, function and origin of the electroreceptors in the two aquatic species, the platypus and the Guiana dolphin. The morphology shows certain similarities, also similar to ampullary electroreceptors in fishes, that provide cues for the search for electroreceptors in more vertebrate and invertebrate species. The function of these organs appears to be very similar. Both species search for prey animals in low-visibility conditions or while digging in the substrate, and sensory thresholds are within one order of magnitude. The electroreceptors in both species are innervated by the trigeminal nerve. The origin of the accessory structures, however, is completely different; electroreceptors in the platypus have developed from skin glands, in the Guiana dolphin, from the vibrissal system.

Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal.

Monotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λ(max) of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min-1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min-1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

Does leptin signal adiposity in the egg-laying mammal, Tachyglossus aculeatus?

Leptin is a peptide hormone best known for its role in feedback regulation of adiposity in eutherian mammals. Normally an increase in adipose tissue mass leads to an increase in circulating leptin which increases energy expenditure and limits food intake, but in hibernating eutherian mammals this relationship may change to allow prehibernatory fattening. The echidna (Tachyglossus aculeatus) is a monotreme mammal which accumulates significant fat reserves before entering hibernation, and mates immediately at the end of hibernation. We hypothesised that echidnas would show a strong relationship between body mass and plasma leptin for most of the year which would change during the pre-hibernatory period. We measured plasma leptin and body mass in free-ranging echidnas over several reproductive and hibernation cycles. There were significant seasonal variations in plasma leptin in both sexes, with the highest levels occurring in hibernation and in mating females. The lowest levels were found in males when they were foraging maximally after the reproductive period. We used mass%, body mass at the time of sampling as a percentage of long term mean mass, as a proxy for adiposity. There was a weak negative relationship between mass% and plasma leptin, from which we infer a weak negative relationship between adiposity and plasma leptin as has been found in reptiles and birds, rather than the strong positive relationship found in other mammals.

Distinct development of peripheral trigeminal pathways in the platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus).

The extant monotremes (platypus and echidnas) are believed to all be capable of electroreception in the trigeminal pathways, although they differ significantly in the number and distribution of electroreceptors. It has been argued by some authors that electroreception was first developed in an aquatic environment and that echidnas are descended from a platypus-like ancestor that invaded an available terrestrial habitat. If this were the case, one would expect the developmental trajectories of the trigeminal pathways to be similar in the early stages of platypus and short-beaked echidna development, with structural divergence occurring later. We examined the development of the peripheral trigeminal pathway from snout skin to trigeminal ganglion in sectioned material in the Hill and Hubrecht collections to test for similarities and differences between the two during the development from egg to adulthood. Each monotreme showed a characteristic and different pattern of distribution of developing epidermal sensory gland specializations (electroreceptor primordia) from the time of hatching. The cross-sectional areas of the trigeminal divisions and the volume of the trigeminal ganglion itself were also very different between the two species at embryonic ages, and remained consistently different throughout post-hatching development. Our findings indicate that the trigeminal pathways in the short-beaked echidna and the platypus follow very different developmental trajectories from the earliest ages. These findings are more consistent with the notion that the platypus and echidna have both diverged from an ancestor with rudimentary electroreception and/or trigeminal specialization, rather than the contention that the echidna is derived from a platypus-like ancestor.

Monotreme ossification sequences and the riddle of mammalian skeletal development.

The developmental differences between marsupials, placentals, and monotremes are thought to be reflected in differing patterns of postcranial development and diversity. However, developmental polarities remain obscured by the rarity of monotreme data. Here, I present the first postcranial ossification sequences of the monotreme echidna and platypus, and compare these with published data from other mammals and amniotes. Strikingly, monotreme stylopodia (humerus, femur) ossify after the more distal zeugopodia (radius/ulna, tibia/fibula), resembling only the European mole among all amniotes assessed. European moles also share extreme humeral adaptations to rotation digging and/or swimming with monotremes, suggesting a causal relationship between adaptation and ossification heterochrony. Late femoral ossification with respect to tibia/fibula in monotremes and moles points toward developmental integration of the serially homologous fore- and hindlimb bones. Monotreme cervical ribs and coracoids ossify later than in most amniotes but are similarly timed as homologous ossifications in therians, where they are lost as independent bones. This loss may have been facilitated by a developmental delay of coracoids and cervical ribs at the base of mammals. The monotreme sequence, although highly derived, resembles placentals more than marsupials. Thus, marsupial postcranial development, and potentially related diversity constraints, may not represent the ancestral mammalian condition.