Platypus and echidna genomes reveal mammalian biology and evolution.

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.

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.

Getting out of a mammalian egg: the egg tooth and caruncle of the echidna.

In the echidna, after development in utero, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. Using rare and unprecedented access to limited echidna pre- and post-hatching tissues, development of the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence. Unlike therian tooth germs that develop by placode invagination, the echidna egg tooth developed by evagination, similar to the first teeth in some reptiles and fish. The egg tooth ankylosed to the premaxilla, rather than forming a tooth root with ligamentous attachment found in other mammals, with loss of the egg tooth associated with high levels of activity odontoclasts and apoptosis. The caruncle formed as a separate mineralisation from the adjacent nasal capsule, and as observed in birds and turtles, the nasal region epithelium on top of the nose expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with typical reptilian teeth, suggesting that this tooth has been conserved from a common ancestor of mammals and reptiles.

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

The plant trans-Golgi network component ECHIDNA regulates defense, cell death, and endoplasmic reticulum stress.

The trans-Golgi network (TGN) acts as a central platform for sorting and secreting various cargoes to the cell surface, thus being essential for the full execution of plant immunity. However, the fine-tuned regulation of TGN components in plant defense and stress response has been not fully elucidated. Our study revealed that despite largely compromising penetration resistance, the loss-of-function mutation of the TGN component protein ECHIDNA (ECH) induced enhanced postinvasion resistance to powdery mildew in Arabidopsis thaliana. Genetic and transcriptome analyses and hormone profiling demonstrated that ECH loss resulted in salicylic acid (SA) hyperaccumulation via the ISOCHORISMATE SYNTHASE 1 biosynthesis pathway, thereby constitutively activating SA-dependent innate immunity that was largely responsible for the enhanced postinvasion resistance. Furthermore, the ech mutant displayed accelerated SA-independent spontaneous cell death and constitutive POWDERY MILDEW RESISTANCE 4-mediated callose depositions. In addition, ECH loss led to a chronically prolonged endoplasmic reticulum stress in the ech mutant. These results provide insights into understanding the role of TGN components in the regulation of plant immunity and stress responses.

An evaluation of DNA sample source and molecular markers to determine gender in the short-beaked echidna (Tachyglossus aculeatus).

The short-beaked echidna is sexually monomorphic such that gender identification without veterinary intervention is challenging. The aim of this study was to evaluate and compare the most optimal noninvasive genetic source by extracting echidna genomic DNA (gDNA) from fecal scats, plucked hair, and quills to perform genetic sex testing using a range of molecular markers. Sex determination of 14 captive short-beaked echidnas was determined by amplifying isolated DNA from noninvasive samples, targeting two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametologue [CRSPY] and anti-Müllerian hormone Y-gametologue [AMHY]), in addition to four confirmed sex-specific RADseq markers. Results of noninvasive samples were compared with blood samples and clinical records. Receiver operating characteristic curves were used to assess accuracy of sex determination of markers for each sample type. The gender of the echidnas was successfully identified on 75% of occasions using fecal samples, 90.6% occasions using hair, and 84.6% occasions with quills. Overall, the male-specific RADseq markers accurately identified the sex of echidnas with all sample types for 90% of animals; compared with 81.5% using CRSPY, and 82.0% using AMHY to identify sex. Collection of hair, quills, and feces provides a useful alternative to invasively collected samples, however, the accuracy of results depends on sample type and genetic marker selected. We found gender determination in the short-beaked echidna was most accurate using four male-specific RADseq markers on gDNA isolated from blood and hair. The noninvasive genetic sexing techniques documented here will inform and facilitate husbandry and genetic management of captive echidna populations.

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.

Functional Diversity and Evolution of Bitter Taste Receptors in Egg-Laying Mammals.

Egg-laying mammals (monotremes) are a sister clade of therians (placental mammals and marsupials) and a key clade to understand mammalian evolution. They are classified into platypus and echidna, which exhibit distinct ecological features such as habitats and diet. Chemosensory genes, which encode sensory receptors for taste and smell, are believed to adapt to the individual habitats and diet of each mammal. In this study, we focused on the molecular evolution of bitter taste receptors (TAS2Rs) in monotremes. The sense of bitter taste is important to detect potentially harmful substances. We comprehensively surveyed agonists of all TAS2Rs in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) and compared their functions with orthologous TAS2Rs of marsupial and placental mammals (i.e., therians). As results, the agonist screening revealed that the deorphanized monotreme receptors were functionally diversified. Platypus TAS2Rs had broader receptive ranges of agonists than those of echidna TAS2Rs. While platypus consumes a variety of aquatic invertebrates, echidna mainly consumes subterranean social insects (ants and termites) as well as other invertebrates. This result indicates that receptive ranges of TAS2Rs could be associated with feeding habits in monotremes. Furthermore, some orthologous receptors in monotremes and therians responded to ß-glucosides, which are feeding deterrents in plants and insects. These results suggest that the ability to detect ß-glucosides and other substances might be shared and ancestral among mammals.

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.

Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus).

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.

ECHIDNA (Emerging CHemIcals Database for National Awareness): a framework to prioritise contaminants of emerging concern in water.

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.

Optimizing captive short-beaked echidna (Tachyglossus aculeatus) fecal sample identification and hormonal analysis.

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.

Reproductive behaviour before and after oestrus and oviposition in the captive short-beaked echidna (Tachyglossus aculeatus).

CONTEXT: Most of our current knowledge regarding echidna reproductive behaviour is based on qualitative measurements; therefore, it is unclear if specific behavioural cues could be utilised in their captive reproductive management. AIMS: This study aimed to identify quantitative changes in general and reproductive behaviour of echidna breeding pairs and pregnant females that might facilitate the detection of oestrus and impending oviposition and provide a summary of reproductive behaviour observed in a captive colony over a three-year observation period. METHODS: Three echidna breeding pairs and two trios were monitored daily for seven reproductive and eight general behaviours during the 2020 breeding season. After confirmed copulation, females were monitored for four egg-laying and eight general behaviours until egg incubation. General observations of reproductive behaviours during the 2018-2020 breeding seasons were recorded as part of routine husbandry. KEY RESULTS: For breeding pairs, there was a significant rate of change over time before and after copulation for the behaviours 'urogenital sniffing', 'rolling' and 'copulation attempt'. For pregnant females, time engaged in 'pacing' significantly increased while 'time eating' and the 'quantity of food eaten' significantly decreased on the day of oviposition. We were not able to identify oestrus from specific behaviours, but our observations suggest that the female echidna's period of receptivity is less than 24h. CONCLUSIONS: The frequency that males express 'urogenital sniffing', 'rolling' and 'copulation attempt' toward the female can be used to alert zookeepers that copulation has likely occurred. Increased pacing, reduced feeding time and quantity of food eaten can aid zookeepers to identify impending oviposition. IMPLICATIONS: This study demonstrates that there are quantifiable changes in specific echidna behaviours that can be incorporated into zoo husbandry practices to improve the reproductive management of this species.

Can spines tell a story? Investigation of echidna spines as a novel sample type for hormone analysis in monotremes.

The short-beaked echidna (Tachyglossus aculeatus) is a monotreme endemic to Australia and New Guinea, and is the most widespread native mammal in Australia. Despite its abundance, there are considerable gaps in our understanding of echidna life history such as reproductive cycles in both sexes, patterns of stress physiology, and possible seasonal changes in metabolism. Slow-growing integumentary sample types comprised of keratin (hair, claw, etc.) have been used in other wildlife to assess these questions via analysis of longitudinal patterns in steroid and thyroid hormones that are deposited in these tissues as they grow. Hairs and spines comprise the pelage of echidnas, the spines being keratinized structures homologous to hair. Thus, echidna spines could be a viable sample type for hormone analysis contributing to a better understanding of the biology of echidnas. The aim of this work was to determine whether steroid hormones are detectable in echidna spines, to perform assay validations, and to establish a protocol for extracting and quantifying hormones in echidna spines using commercially available assay kits. We also inspected cross-sectioned spines using light and electron microscopy for any evidence of annual growth markers that might enable inferences about spine growth rate. Corticosterone, progesterone, estradiol, and testosterone were detectable in all samples, and echidna spine extract passed standard assay validations (parallelism and accuracy), indicating that commercially available assay kits can quantify hormones accurately in this sample type. No visible growth marks were identified in the spines and thus spine growth rate is currently unknown. Echidna spines show promise as a novel matrix from which hormones can be quantified; next steps should involve determination of spine annual growth rate, possible seasonal changes in growth rate, and persistence of spines over time in order to perform physiological validations, i.e., relationship between physiological status and hormone concentrations in spines.

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.

Plasma progesterone secretion during gestation of the captive short-beaked echidna.

This study describes the progesterone profile during pregnancy in sexually mature female captive short-beaked echidnas (Tachyglossus aculeatus aculeatus). Echidnas were monitored daily by video surveillance to confirm key reproductive behaviour. Plasma samples were collected and pouch morphology was assessed three times a week. The pouch of the female echidna only develops during gestation and it was possible to create a four-stage grading system using the most distinguishable characteristics of pouch development. Maximum pouch development was associated with declining progesterone concentrations, with the pouch closing in a drawstring-like manner at oviposition. Control of pouch development in pregnant echidnas is not yet clear but later pouch development is associated with a decrease in progesterone and pouch closure and may be under mechanical influences of the egg or young in the pouch. The length of pregnancy was 16.7 ± 0.2 days with a 15.1 ± 1.0 days luteal phase followed by an incubation period in the pouch. Eggs could be detected in utero at least 4 days before oviposition. Plasma progesterone peaked at 10.5 ± 0.9 ng/mL within 12 days of mating but then declined to basal levels within 1 day of oviposition and remained basal throughout egg incubation, confirming that progesterone is elevated throughout pregnancy and that gestation does not extend beyond the luteal phase. After the loss of an egg or pouch young, most females entered a second oestrous cycle and ovulated, suggesting echidnas are seasonally polyoestrous. The duration of the luteal phase in the echidna corresponds with that observed in other mammals.

Snowflake morays, Echidna nebulosa, exhibit similar feeding kinematics in terrestrial and aquatic treatments.

Some species of durophagous moray eels (Muraenidae) have been documented emerging from the marine environment to capture intertidal crabs but how they consume prey out of water is unknown. Here, we trained snowflake morays, Echidna nebulosa, to undulate out of the aquatic environment to feed on land. On land, snowflake morays remove prey from the substrate by biting and swallow prey using pharyngeal jaw enabled transport. Although snowflake morays exhibit smaller jaw rotation angles on land when apprehending their prey, transport kinematics involving dorsoventral flexion of the head to protract the pharyngeal jaws and overall feeding times did not differ between terrestrial and aquatic treatments. We suggest that their elongate body plan, ability to rotate their heads in the dorsoventral and lateral directions, and extreme pharyngeal movements all contribute to the ability of durophagous morays to feed in the terrestrial environment.

Arabidopsis ECHIDNA protein is involved in seed coloration, protein trafficking to vacuoles, and vacuolar biogenesis.

Flavonoids are a major group of plant-specific metabolites that determine flower and seed coloration. In plant cells, flavonoids are synthesized at the cytosolic surface of the endoplasmic reticulum and are sequestered in the vacuole. It is possible that membrane trafficking, including vesicle trafficking and organelle dynamics, contributes to flavonoid transport and accumulation. However, the underlying mechanism has yet to be fully elucidated. Here we show that the Arabidopsis ECHIDNA protein plays a role in flavonoid accumulation in the vacuole and protein trafficking to the vacuole. We found defective pigmentation patterns in echidna seed, possibly caused by reduced levels of proanthocyanidins, which determine seed coloration. The echidna mutant has defects in protein sorting to the protein storage vacuole as well as vacuole morphology. These findings indicate that ECHIDNA is involved in the vacuolar trafficking pathway as well as the previously described secretory pathway. In addition, we found a genetic interaction between echidna and green fluorescent seed 9 (gfs9), a membrane trafficking factor involved in flavonoid accumulation. Our findings suggest that vacuolar trafficking and/or vacuolar development, both of which are collectively regulated by ECHIDNA and GFS9, are required for flavonoid accumulation, resulting in seed coat pigmentation.

Description of five new species of Ixodes Latreille, 1795 (Acari: Ixodidae) and redescription of I. luxuriosus Schulze, 1935, I. steini Schulze, 1935 and I. zaglossi Kohls, 1960, parasites of marsupials, rodents and echidnas in New Guinea Island.

Ixodes acer n. sp., Ixodes giluwensis n. sp., Ixodes mirzai n. sp., Ixodes planiscutatus n. sp. and Ixodes stellae n. sp. (Acari: Ixodidae), are described based on females ex various marsupials (Dasyuromorphia: Dasyuridae, Diprotodontia: Petauridae, Peramelemorphia: Peramelidae), rodents (Rodentia: Muridae) and a bird (Galliformes: Megapodiidae) from Indonesia (Papua Province) and Papua New Guinea. Females of all new species are similar to those of I. luxuriosus Schulze, 1935, I. steini Schulze, 1935 and I. zaglossi Kohls, 1960, but can be distinguished from them and each other by the distinctness of lateral carinae and cervical grooves on the scutum, length of setae on the idiosoma, sculpture of the basis capituli and palpal segment I dorsally, size of the dorsal cornua, shape and sculpture of the palpal segment I ventrally, size of ventral spur on the palpal segment I, dental formula on the hypostome and measurements of various structures and their proportions. For comparative purposes the female of I. luxuriosus, I. steini and I. zaglossi is redescribed and lectotypes for I. luxuriosus and I. steini have been designated. Studied females of I. luxuriosus, I. steini and I. zaglossi were found on marsupials (Dasyuromorphia: Dasyuridae; Diprotodontia: Macropodidae, Petauridae, Phalangeridae, Pseudocheiridae; Peramelemorphia: Peramelidae), rodents (Rodentia: Muridae) and echidnas (Monotremata: Tachyglossidae) in Indonesia (Papua Province) and Papua New Guinea.