Long-term movements and activity patterns of platypus on regulated rivers.

The platypus is a semi-aquatic mammal, endemic to freshwater habitats of eastern Australia. There are gaps in the understanding of platypus movement behaviour within river systems, including spatial and temporal organization of individuals. We tracked movements of 12 platypuses on the regulated Snowy and Mitta Mitta Rivers for up to 12-months, the longest continuous tracking of platypus using acoustic telemetry. Platypuses remained relatively localized, occupying 0.73-8.45 km of river over 12 months, consistent with previous tracking studies over shorter periods. Males moved further than females, and larger males had higher cumulative movements, suggesting a possible relationship to metabolic requirements. Platypuses moved greater distances on the Mitta Mitta River, possibly associated with impacts of altered flow regimes to their macroinvertebrate diet. Increased movements and diurnal activity during winter were primarily driven by males, possibly attributable to breeding behaviours, rather than increased costs of winter foraging. Evidence for relatively small movements has implications for declining populations, given areas of localised declines are unlikely to be supplemented by migrating platypuses, especially when dispersal is restricted by dam walls. Understanding platypus movement behaviour is pertinent for their conservation, as water resource development and habitat modification continue to reduce connectivity between populations across their distribution.

Platypus predation has differential effects on aquatic invertebrates in contrasting stream and lake ecosystems.

Predators can have strong impacts on prey populations, with cascading effects on lower trophic levels. Although such effects are well known in aquatic ecosystems, few studies have explored the influence of predatory aquatic mammals, or whether the same predator has similar effects in contrasting systems. We investigated the effects of platypus (Monotremata: Ornithorhynchus anatinus) on its benthic invertebrate prey, and tested predictions that this voracious forager would more strongly affect invertebrates-and indirectly, epilithic algae-in a mesotrophic lake than in a dynamic stream ecosystem. Hypotheses were tested using novel manipulative experiments involving platypus-exclusion cages. Platypuses had strongly suppressive effects on invertebrate prey populations, especially detritivores and omnivores, but weaker or inconsistent effects on invertebrate taxon richness and composition. Contrary to expectation, predation effects were stronger in the stream than the lake; no effects were found on algae in either ecosystem due to weak effects of platypuses on herbivorous invertebrates. Platypuses did not cause redistribution of sediment via their foraging activities. Platypuses can clearly have both strong and subtle effects on aquatic food webs that may vary widely between ecosystems and locations, but further research is needed to replicate our experiments and understand the contextual drivers of this variation.

Molecular basis for heat desensitization of TRPV1 ion channels.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a prototypical molecular sensor for noxious heat in mammals. Its role in sustained heat response remains poorly understood, because rapid heat-induced desensitization (Dh) follows tightly heat-induced activation (Ah). To understand the physiological role and structural basis of Dh, we carried out a comparative study of TRPV1 channels in mouse (mV1) and those in platypus (pV1), which naturally lacks Dh. Here we show that a temperature-sensitive interaction between the N- and C-terminal domains of mV1 but not pV1 drives a conformational rearrangement in the pore leading to Dh. We further show that knock-in mice expressing pV1 sensed heat normally but suffered scald damages in a hot environment. Our findings suggest that Dh evolved late during evolution as a protective mechanism and a delicate balance between Ah and Dh is crucial for mammals to sense and respond to noxious heat.

Use of implanted acoustic tags to assess platypus movement behaviour across spatial and temporal scales.

The platypus (Ornithorhynchus anatinus) is an evolutionarily distinct mammal, endemic to Australian freshwaters. Many aspects of its ecology and life-history, including detailed understanding of movements, are poorly known, hampered by its cryptic and mainly nocturnal habits and small numbers. We effectively trialled intraperitoneal implanted acoustic transmitters in nine platypuses in the Severn River (NSW), Australia, as a potential approach for studying movements in this challenging species. We tracked platypus movements over six months, at fine and broad spatial scales, using an array of acoustic sensors. Over six months (March-August 2016), four of five adult platypuses (two females\three males) maintained localized movements (average monthly maximums 0.37 km ± 0.03 sd), while one adult, one sub-adult, and one juvenile (males) moved further: average monthly maxima 1.2 km ± 2.0 sd, 0.9 km ± 0.6 sd, 4.5 km ± 5.9 sd, respectively. The longest recorded movement was by a male adult, covering 11.1 km in three days and travelling a maximum distance of about 13 km between records. Only one implanted animal was not detected immediately after release, indicative of transmission failure rather than an adverse event. High cumulative daily movements (daily 1.9 km ± 0.8 sd) indicated high metabolic requirements, with implications for previous estimates of platypus abundances and carrying capacities, essential for effective conservation. This novel approach offers new avenues to investigate relating to mating, nesting, and intraspecific competition behaviours and their temporal and spatial variation.

A NEED FOR DYNAMIC HEMATOLOGY AND SERUM BIOCHEMISTRY REFERENCE TOOLS: NOVEL USE OF SINE WAVE FUNCTIONS TO PRODUCE SEASONALLY VARYING REFERENCE CURVES IN PLATYPUSES (ORNITHORHYNCHUS ANATINUS).

Seasonal changes in hematology and serum biochemistry results, described by separate reference intervals for different seasons, have been reported in many animals. We developed a novel method to investigate seasonal variation in values and a reference tool (the reference curve) based on sine wave functions that, for suitable variables, represents data more appropriately than a fixed reference interval. We applied these techniques to values observed in blood samples from 126 adult wild platypuses ( Ornithorhynchus anatinus ; 58 females and 68 males). Samples were collected under isoflurane anesthesia from animals captured in the Inglis Catchment in northwest Tasmania. In general, packed cell volume (PCV), red cell count (RCC), and hemoglobin (Hb) values appeared to be lower than those in two studies that previously reported platypus hematology reference intervals. This likely resulted from reduced stress-related splenic contraction or isoflurane-associated splenic sequestration of red blood cells in our study. Reference curves were described for five variables (PCV, RCC, Hb, albumin, and magnesium). We found evidence that this seasonal variation may result from metabolic changes associated with seasonal variations in environmental temperature. These observations suggest that it is important for researchers reporting platypus hematology and serum biochemistry to look for seasonal changes in their data to ensure it is appropriately interpreted.

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.

Comparative cranial morphology in living and extinct platypuses: Feeding behavior, electroreception, and loss of teeth.

The modern platypus, Ornithorhynchus anatinus, has an eye structure similar to aquatic mammals; however, platypuses also have a "sixth sense" associated with the bill electro- and mechanoreception that they use without opening their eyes underwater. We hypothesize that Ornithorhynchus and the Miocene taxon Obdurodon have different sensory capacities, which may have resulted from differences in foraging behavior. To estimate differences in foraging, sensory systems, and anatomical divergence between these monotremes, we compared their skull morphologies. Results indicate that the bill of Obdurodon is more dorsally deflected than that of Ornithorhynchus, suggesting a pelagic foraging behavior in Obdurodon compared to the bottom-feeding behavior in Ornithorhynchus. The infraorbital foramen of Obdurodon, through which the maxillary nerve passes sensory data from the bill to the brain, is relatively less developed than that of Ornithorhynchus. Whereas bill-focused sensory perception was likely shared among Mesozoic monotremes, the highly developed electrosensory system of Ornithorhynchus may represent an adaptation to foraging in cloudy water. Computed tomography imagery indicates that the enlarged infraorbital canal of Ornithorhynchus restricts the space available for maxillary tooth roots. Hence, loss of functional teeth in Ornithorhynchus may possibly have resulted from a shift in foraging behavior and coordinate elaboration of the electroreceptive sensory system. Well-developed electroreceptivity in monotremes is known at least as far back as the early Cretaceous; however, there are differences in the extent of elaboration of the feature among members of the ornithorhynchid lineage.

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.

Internally coupled ears in living mammals.

It is generally held that the right and left middle ears of mammals are acoustically isolated from each other, such that mammals must rely on neural computation to derive sound localisation cues. There are, however, some unusual species in which the middle ear cavities intercommunicate, in which case each ear might be able to act as a pressure-difference receiver. This could improve sound localisation at lower frequencies. The platypus Ornithorhynchus is apparently unique among mammals in that its tympanic cavities are widely open to the pharynx, a morphology resembling that of some non-mammalian tetrapods. The right and left middle ear cavities of certain talpid and golden moles are connected through air passages within the basicranium; one experimental study on Talpa has shown that the middle ears are indeed acoustically coupled by these means. Having a basisphenoid component to the middle ear cavity walls could be an important prerequisite for the development of this form of interaural communication. Little is known about the hearing abilities of platypus, talpid and golden moles, but their audition may well be limited to relatively low frequencies. If so, these mammals could, in principle, benefit from the sound localisation cues available to them through internally coupled ears. Whether or not they actually do remains to be established experimentally.

Life history and dynamics of a platypus (Ornithorhynchus anatinus) population: four decades of mark-recapture surveys.

Knowledge of the life-history and population dynamics of Australia's iconic and evolutionarily distinct platypus (Ornithorhynchus anatinus) remains poor. We marked-recaptured 812 unique platypuses (total 1,622 captures), over four decades (1973-2014) in the Shoalhaven River, Australia. Strong sex-age differences were observed in life-history, including morphology and longevity. Apparent survival of adult females (Φ = 0.76) were higher than adult males (Φ = 0.57), as in juveniles: females Φ = 0.27, males Φ = 0.13. Females were highly likely to remain in the same pool (adult: P = 0.85, juvenile: P = 0.88), while residency rates were lower for males (adult: P = 0.74, juvenile: P = 0.46). We combined survival, movement and life-histories to develop population viability models and test the impact of a range of life-history parameters. While using estimated apparent survival produced unviable populations (mean population growth rate r = -0.23, extinction within 20 years), considering residency rates to adjust survival estimates, indicated more stable populations (r = 0.004, p = 0.04 of 100-year extinction). Further sensitivity analyses highlighted adult female survival and overall success of dispersal as most affecting viability. Findings provide robust life-history and viability estimates for a difficult study species. These could support developing large-scale population dynamics models required to underpin a much needed national risk assessment for the platypus, already declining in parts of its current distribution.

Independent evolution of transcriptional inactivation on sex chromosomes in birds and mammals.

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.

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.

Thermal fluctuations of haemoglobin from different species: adaptation to temperature via conformational dynamics.

Thermodynamic stability, configurational motions and internal forces of haemoglobin (Hb) of three endotherms (platypus, Ornithorhynchus anatinus; domestic chicken, Gallus gallus domesticus and human, Homo sapiens) and an ectotherm (salt water crocodile, Crocodylus porosus) were investigated using circular dichroism, incoherent elastic neutron scattering and coarse-grained Brownian dynamics simulations. The experimental results from Hb solutions revealed a direct correlation between protein resilience, melting temperature and average body temperature of the different species on the 0.1 ns time scale. Molecular forces appeared to be adapted to permit conformational fluctuations with a root mean square displacement close to 1.2 Å at the corresponding average body temperature of the endotherms. Strong forces within crocodile Hb maintain the amplitudes of motion within a narrow limit over the entire temperature range in which the animal lives. In fully hydrated powder samples of human and chicken, Hb mean square displacements and effective force constants on the 1 ns time scale showed no differences over the whole temperature range from 10 to 300 K, in contrast to the solution case. A complementary result of the study, therefore, is that one hydration layer is not sufficient to activate all conformational fluctuations of Hb in the pico- to nanosecond time scale which might be relevant for biological function. Coarse-grained Brownian dynamics simulations permitted to explore residue-specific effects. They indicated that temperature sensing of human and chicken Hb occurs mainly at residues lining internal cavities in the ß-subunits.

Three-dimensional limb joint mobility in the early tetrapod Ichthyostega.

The origin of tetrapods and the transition from swimming to walking was a pivotal step in the evolution and diversification of terrestrial vertebrates. During this time, modifications of the limbs­particularly the specialization of joints and the structures that guide their motions­fundamentally changed the ways in which early tetrapods could move. Nonetheless, little is known about the functional consequences of limb anatomy in early tetrapods and how that anatomy influenced locomotion capabilities at this very critical stage in vertebrate evolution. Here we present a three-dimensional reconstruction of the iconic Devonian tetrapod Ichthyostega and a quantitative and comparative analysis of limb mobility in this early tetrapod. We show that Ichthyostega could not have employed typical tetrapod locomotory behaviours, such as lateral sequence walking. In particular, it lacked the necessary rotary motions in its limbs to push the body off the ground and move the limbs in an alternating sequence. Given that long-axis rotation was present in the fins of tetrapodomorph fishes, it seems that either early tetrapods evolved through an initial stage of restricted shoulder and hip joint mobility or that Ichthyostega was unique in this respect. We conclude that early tetrapods with the skeletal morphology and limb mobility of Ichthyostega were unlikely to have made some of the recently described Middle Devonian trackways.

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.

Ostriches sleep like platypuses.

Mammals and birds engage in two distinct states of sleep, slow wave sleep (SWS) and rapid eye movement (REM) sleep. SWS is characterized by slow, high amplitude brain waves, while REM sleep is characterized by fast, low amplitude waves, known as activation, occurring with rapid eye movements and reduced muscle tone. However, monotremes (platypuses and echidnas), the most basal (or 'ancient') group of living mammals, show only a single sleep state that combines elements of SWS and REM sleep, suggesting that these states became temporally segregated in the common ancestor to marsupial and eutherian mammals. Whether sleep in basal birds resembles that of monotremes or other mammals and birds is unknown. Here, we provide the first description of brain activity during sleep in ostriches (Struthio camelus), a member of the most basal group of living birds. We found that the brain activity of sleeping ostriches is unique. Episodes of REM sleep were delineated by rapid eye movements, reduced muscle tone, and head movements, similar to those observed in other birds and mammals engaged in REM sleep; however, during REM sleep in ostriches, forebrain activity would flip between REM sleep-like activation and SWS-like slow waves, the latter reminiscent of sleep in the platypus. Moreover, the amount of REM sleep in ostriches is greater than in any other bird, just as in platypuses, which have more REM sleep than other mammals. These findings reveal a recurring sequence of steps in the evolution of sleep in which SWS and REM sleep arose from a single heterogeneous state that became temporally segregated into two distinct states. This common trajectory suggests that forebrain activation during REM sleep is an evolutionarily new feature, presumably involved in performing new sleep functions not found in more basal animals.

DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes.

DDX4 (VASA) is an RNA helicase expressed in the germ cells of all animals. To gain greater insight into the role of this gene in mammalian germ cell development, we characterized DDX4 in both a marsupial (the tammar wallaby) and a monotreme (the platypus). DDX4 is highly conserved between eutherian, marsupial, and monotreme mammals. DDX4 protein is absent from tammar fetal germ cells but is present from Day 1 postpartum in both sexes. The distribution of DDX4 protein during oogenesis and spermatogenesis in the tammar is similar to eutherians. Female tammar germ cells contain DDX4 protein throughout all stages of postnatal oogenesis. In males, DDX4 is in gonocytes, and during spermatogenesis it is present in spermatocytes and round spermatids. A similar distribution of DDX4 occurs in the platypus during spermatogenesis. There are several DDX4 isoforms in the tammar, resulting from both pre- and posttranslational modifications. DDX4 in marsupials and monotremes has multiple splice variants and polyadenylation motifs. Using in silico analyses of genomic databases, we found that these previously unreported splice variants also occur in eutherians. In addition, several elements implicated in the control of Ddx4 expression in the mouse, including RGG (arginine-glycine-glycine) and dimethylation of arginine motifs and CpG islands within the Ddx4 promoter, are also highly conserved. Collectively these data suggest that DDX4 is essential for the regulation of germ cell proliferation and differentiation across all three extant mammalian groups-eutherians, marsupials, and monotremes.

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.

Monotremes provide a key to understanding the evolutionary significance of epididymal sperm maturation.

It has been widely accepted that mammalian spermatozoa are infertile when they leave the testes and require a period of maturation in both the epididymis and the female reproductive tract before acquiring the ability to fertilize an oocyte. However, the necessity for such a complex process of posttesticular sperm maturation appears to be unique to mammals because it is well established that these processes do not directly influence the fertilizing ability of the spermatozoa of birds, reptiles, and other lower vertebrates. Because of their key evolutionary position and form of reproduction, we contend that monotremes (platypus and echidna) provide a unique model for resolving why these processes are necessary. In the present review, we examine evidence that the epididymal maturation of monotreme spermatozoa is far less complex than in other mammals. However, a unique feature of the monotreme epididymis lies in its ability to promote the formation of elaborate sperm bundles that serve to greatly enhance the cells' motility. It is suggested that this intriguing cooperative strategy used by monotreme sperm represents an early form of epididymal maturation that appears to have been elaborated upon during the evolution of higher mammals, possibly as an adaptation for sperm competition.