Karyotypic conservation in the mammalian order monotremata (subclass Prototheria)

The order Monotremata, comprising the platypus and two species of echidna (Australian and Nuigini) is the only extant representative of the mammalian subclass Prototheria, which diverged from subclass Theria (marsupials and placental mammals) 150–200 million years ago. The 2n=63, 64 karyotype (newly described here) of the Nuigini echidna is almost identical in morphology and G-band pattern to that of the Australian echidna, from which it diverged about a million years ago. The karyotype of the platypus (2n=52) has several features in common with those of the echidna species; six pairs of large autosomes, many pairs of small (but not micro-) chromosomes, and a series of small unpaired chromosomes which form a multivalent at meiosis. Comparison of the G-band patterns of platypus and echidna autosomes reveals considerable homology. Chromomycin banding demonstrates GC-rich heterochromatin at the centromeres of many platypus and echidna chromosomes, and at the nucleolar organizing regions; some of this heterochromatin C-bands weakly in platypus (but not echidna) spreads. Late replication banding patterns resemble G-banding patterns and confirm the homologies between the species. Striking heteromorphism between chromosomes of some of the large autosomal pairs can be accounted for in the echidna by differences in amount of chromomycin-bright, late replicating heterochromatin. The sex chromosomes in all three species also bear striking homology, despite the difference in sex determination mechanism between platypus (XX/XY) and the echidna species (X₁X₁X₂X₂/X₁X₂Y). The platypus X and echidna X₁ each represent about 5.8% of haploid chromosome length, and are G-band identical. Y chromosomes are similar between species, and are largely homologous to the X (or X₁).     

The anatomy of the cerebral cortex of the echidna (Tachyglossus aculeatus)

The cerebral cortex of the echidna is notable for its extensive folding and the positioning of major functional areas towards its caudal extremity. The gyrification of the echidna cortex is comparable in magnitude to prosimians and cortical thickness and neuronal density are similar to that seen in rodents and carnivores. On the other hand, many pyramidal neurons in the cerebral cortex of the echidna are atypical with inverted somata and short or branching apical dendrites. All other broad classes of neurons noted in therian cortex are also present in the echidna, suggesting that the major classes of cortical neurons evolved prior to the divergence of proto- and eutherian lineages. Dendritic spine density on dendrites of echidna pyramidal neurons in somatosensory cortex and apical dendrites of motor cortex pyramidal neurons is lower than that found in eutheria. On the other hand, synaptic morphology, density and distribution in somatosensory cortex are similar to that in eutheria. In summary, although the echidna cerebral cortex displays some structural features, which may limit its functional capacities (e.g. lower spine density on pyramidal neurons), in most structural parameters (e.g. gyrification, cortical area and thickness, neuronal density and types, synaptic morphology and density), it is comparable to eutheria.     

Calcium-binding and structural stability of echidna and canine milk lysozymes.

For echidna and canine milk lysozymes, which were presumed to be the calcium-binding lysozymes by their amino acid sequences, we have quantitated their calcium-binding strength and examined their guanidine unfolding profiles. The calcium-binding constants of echidna and canine lysozymes were determined to be 8.6 x 10(6) M(-1) and 8.9 x 10(6) M(-1) in 0.1 M KCl at pH 7.1 and 20 C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of alpha-lactalbumin, through a stable molten globule intermediate. However, neither calcium-bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg-white lysozyme, avian calcium-binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel-Boston-Berlin: Birkhauzer Verlag. pp 9-31) that the lineage of avian and echidna calcium-binding lysozymes and that of eutherian calcium-binding lysozymes diverged separately from that of conventional lysozymes.     

Histochemical,ultrastructural and hormonal studies on the pars distalis of the echidna (Tachyglossus aculeatus)

There were no consistent significant differences between the concentrations of luteinizing hormone (LH) and adrenocorticotrophin (ACTH) in the rostral compared with the caudal zone of the echidna pars distalis. This suggests that LH is secreted by cells containing S-type granules (probably corresponding to secretory vesicles 200-300 nm diameter) which are distributed throughout the gland. Some of the cells containing vesicles 100-200 nm diameter, seen in small numbers in both zones of the gland, may be responsible for the secretion of ACTH. The concentration of pituitary LH is in the range of that found in eutherian mammals, but the concentration of ACTH is lower than that reported for other vertebrates, and this may be linked causally with the remarkably low rate of corticosteroid secretion in the echidna. The absence of significantly increased levels of pituitary LH and ACTH in a chronically orchidectomized and adrenalectomized animal adds to other evidence which suggests that mechanisms involving a negative feedback of steroid hormones on the hypothalamo-hypophysial axis may not be fully developed in the echidna.     

Antibody response to sheep red blood cells in platypus and echidna

There is limited information regarding the kinetics of antibody responses exhibited by the platypus and the echidna in response to a T cell dependent antigen. In this preliminary study a platypus, an echidna and a rabbit were inoculated with sheep red blood cells to compare their antibody responses and kinetics. The antibody titres, produced by the platypus and echidna, were less than those elicited in the rabbit. Furthermore, the echidna and platypus exhibited a weak secondary response. This was most likely due to a failure of the platypus and echidna to undergo the characteristic IgM to IgG isotype switch following second antigen exposure. The conformational structure of these antibodies may differ from eutherian antibodies. This was further supported by a heat sensitivity experiment that indicated that these antibodies are more labile than rabbit immunoglobulins and therefore structurally less stable.     

Sperm maturation, fluid transport, and secretion and absorption of protein in the epididymis of the echidna, Tachyglossus aculeatus

Extratesticular sperm maturation in the echidna mainly occurs in the initial segment of the ductus epididymidis. The process involves the development of motility, migration and loss of the cytoplasmic droplet, a decrease in permeability to Congo red and the formation of sperm bundles. The spermatozoa are supported in the bundles by a matrix of electron-dense material; the bundles are very motile is undiluted samples of luminal fluids. Micropuncture studies of anaesthetized echidnas showed that the ductuli efferentes absorb 74% of the fluid and 46% of the soluble protein that enters them. The initial segment of the ductus epididymidis absorbs 83% of the fluid which enters it, and its secretions increase the concentration of protein in luminal fluid by 107%. Denatured, linear-gradient polyacrylamide gel electrophoresis of micropuncture samples showed that 1 protein (apparent M4 = 100 500) which is not present in blood plasma is present in rete testis fluid, and a glycoprotein which is present in rete testis fluid (apparent Mr = 78500) is absorbed by the ductuli efferentes. Six proteins which are not present in blood plasma are secreted into the initial segment of the ductus epididymidis; 5 are glycoproteins (apparent Mr = 48500, 39000, 32000, 20500 and 19000) and one (apparent Mr = 82500) is not. The most prominent electrophoresis bands corresponded to the glycoproteins with apparent molecular weights of 48500, 20500 and 19000.     

Cool Sex? Hibernation and Reproduction Overlap in the Echidna

During hibernation there is a slowing of all metabolic processes, and thus it is normally considered to be incompatible with reproduction. In Tasmania the egg-laying mammal, the echidna (Tachyglossus aculeatus) hibernates for several months before mating in mid-winter, and in previous studies we observed males with females that were still hibernating. We monitored the reproductive activity of radio-tracked echidnas by swabbing the reproductive tract for sperm while external temperature loggers provided information on the timing of hibernation. Additional information was provided by camera traps and ultrasound imaging. More than a third of the females found in mating groups were torpid, and the majority of these had mated. Some females re-entered deep torpor for extended periods after mating. Ultrasound examination showed a developing egg in the uterus of a female that had repeatedly re-entered torpor. The presence of fresh sperm in cloacal swabs taken from this female on three occasions after her presumed date of fertilization indicated she mated several times after being fertilized. The mating of males with torpid females is the result of extreme competition between promiscuous males, while re-entry into hibernation by pregnant females could improve the possibility of mating with a better quality male.     

Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.     

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 β-glucosides, which are feeding deterrents in plants and insects. These results suggest that the ability to detect β-glucosides and other substances might be shared and ancestral among mammals.     

Gene mapping studies confirm the homology between the platypus X and echidna X1 chromosomes and identify a conserved ancestral monotreme X chromosome

The identification of the sex chromosomes in the three extant species of Prototherian mammals (the monotremes) is complicated by their involvement in a multivalent translocation chain at the first division of male meiosis. The platypus X chromosome, identified by the presence of two copies in females and one in males, has been found to possess a suite of genes that have been mapped to the X chromosomes of all eutherian and metatherian mammals. We have extended gene mapping studies to a member of the only other extant monotreme family, the echidna, which has a G-band equivalent X₁ chromosome, as well as a smaller X₂. We find that the five human X-linked genes (G6PD, GDX, F9, AR and MCF2) map to the echidna X₁ chromosome in locations equivalent to those on the platypus X. These results confirm that the echidna X₁ is the original X chromosome in this species, and identify a conserved ancestral monotreme X chromosome.     

The gastric mucosa of two monotremes: the duck-billed platypus and echidna

The gastric mucosa of both the echidna and platypus is aglandular and the lining epithelium is stratified squamous. The latter exhibits three principle layers: stratum germinativum, stratum spinosum, and stratum corneum. The cytoplasm of cells composing the first two strata of both species shows bundles of tonofibrils and numerous free ribosomes. Cells of the stratum spinosum in the platypus also show numerous dense granules limited to the peripheral cytoplasm. The stratum spinosum of both species is comprised of fusiform-shaped cells whose adjacent cell membranes show extensive interlocking. The stratum spinosum of the echidna in addition shows numerous intercellular bridges. Cells of the stratum corneum become flattened and elongate and in the echidna nuclei near the surface appear to degenerate. Cells comprising the stratum corneum of the platypus exhibit well preserved nuclei and contain scattered large granules of varying electron density. Prior to sloughing, cells near the surface of both species show a separation of adjacent cell membranes. True keratinization is not found in the gastric lining epithelium of either species and the epithelium lining of the stomach of the echidna more closely represents a form of parakeratosis. Delicate papillae containing capillaries extend considerable distances into the overlying epithelium of both species and are thought to contribute to its nutrition.     

Topography and chemoarchitecture of the striatum and pallidum in a monotreme, the short-beaked echidna (Tachyglossus aculeatus)

The topography and chemoarchitecture of the striatum and pallidum in a monotreme, the short-beaked echidna (Tachyglossus aculeatus) have been studied using Nissl staining in conjunction with myelin staining, enzyme reactivity to acetylcholinesterase and NADPH diaphorase, and immunoreactivity to parvalbumin, calbindin, calretinin, tyrosine hydroxylase, neuropeptide Y, and neurofilament protein (SMI-32 antibody). All those components of the striatum and pallidum found in eutherian mammals could also be identified in the echidna's brain, with broad chemoarchitectural similarities to those regions in eutherian brains also apparent. There was a clear chemoarchitectural gradient visible with parvalbumin immunoreactivity of neurons and fibers, suggesting a subdivision of the echidna caudatoputamen into weakly reactive rostrodorsomedial and strongly reactive caudoventrolateral components. This may, in turn, relate to subdivision into associative versus sensorimotor CPu and reflect homology to the caudate and putamen of primates. Moreover, the chemoarchitecture of the echidna striatum suggested the presence of striosome-matrix architecture. The morphology of identified neuronal groups (i.e., parvalbumin, calbindin, and neuropeptide Y immunoreactive) in the echidna striatum and pallidum showed many similarities to those seen in eutherians, although the pattern of distribution of calbindin immunoreactive neurons was more uniform in the caudatoputamen of the echidna than in therians. These observations indicate that the same broad features of striatal and pallidal organization apply across all mammals and suggest that these common features may have arisen before the divergence of the monotreme and therian lineages.     

The complexity of expressed kappa light chains in egg-laying mammals

Complementary DNAs encoding immunoglobulin light chains were isolated from two monotreme species, Ornithorhynchus anatinus (duckbill platypus) and Tachyglossus aculeatus (echidna). The sequences of both the variable and constant regions of these clones had greater similarity to IGK than to other light chain classes and phylogenetic analyses place them squarely within the mammalian IGK group, establishing them as monotreme IGK homologues. The constant region sequences of all clones were essentially identical within each species and, along with Southern blot results, the data are consistent with a single IGKC in each species. The expressed IGKV repertoires from both platypus and echidna were randomly sampled and there appear to be at least four platypus and at least nine echidna IGKV subgroups. The IGKV subgroups are highly divergent within species, in some cases sharing as little as 57% nucleotide identity. Two of the IGKV subgroups are present in both species, so there is some degree of overlap in the germline repertoires of these two monotremes. Overall the complexity seen in platypus and echidna IGK light chains is comparable with that of other mammals considered to have high levels of germline diversity and is in contrast to what has been found so far for monotreme IGL.Electronic Supplementary Material Supplementary material is available for this article at .     

Assessment of the hemorheological profile of koala and echidna

Koala, a marsupial, and echidna, a monotreme, are mammals native to Australia. Blood viscosity (62.5–1250 s^?1), red blood cell (RBC) deformability, RBC aggregation, aggregability and surface charge, and hematological parameters were measured in blood samples from six koalas and six echidnas and compared to adult human blood. Koala had the largest RBC mean cell volume (107.7±2.6 fl) compared to echidna (81.3±2.6 fl) and humans (88.4±1.2 fl). Echidna blood exhibited the highest viscosity over the entire range of shear rates. Echidna RBC were significantly less deformable than koala RBC but more deformable than human RBC. Echidna RBC had significantly lower aggregability (i.e., aggregation in standardized dextran medium) than koala or human RBC, while aggregation in autologous plasma was similar for the three species. Erythrocyte surface charge as indexed by RBC electrophoretic mobility was similar for human and echidna cells but was 40% lower for koala RBC. Data obtained during this preliminary study indicate that koala and echidna have distinct hemorheological characteristics; investigation of these properties may reveal patterns relevant to specific behavioral and physiological features of these animals.     

Iron(III) binding proteins of echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus)

Iron (III) binding proteins are isolated from echidna (Tachyglossus aculeatus multiaculeatus) and platypus (Ornithorhynchus anatinus) milk and blood. On the basis of several criteria it is shown that the milk proteins are not lactoferrins, but are transferrins similar to the corresponding transferrins from the blood. The heterogeneity of the proteins, particularly the echidna milk transferrin, is, at least in part, due to different levels of sialic acid. Their N-terminal sequences (30 residues) are determined and compared with those of other transferrins and lactoferrins. The role of the proteins is discussed.     

Active cation transport and Na+K+Mg ATPase of the monotreme erythrocytes

The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb⁸⁶ influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb⁸⁶ influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes.     

The unique sex chromosome system in platypus and echidna

A striking example of the power of chromosome painting has been the resolution of the male platypus karyotype and the pairing relationships of the chain of ten sex chromosomes. We have extended our analysis to the nine sex chromosomes of the male echidna. Cross-species painting with platypus shows that the first five chromosomes in the chain are identical in both, but the order of the remainder are different and, in each species, a different autosome replaces one of the five X chromosomes. As the therian X is homologous mainly to platypus autosome 6 and echidna 16, and as SRY is absent in both, the sex determination mechanism in monotremes is currently unknown. Several of the X and Y chromosomes contain genes orthologous to those in the avian Z but the significance of this is also unknown. It seems likely that a novel testis determinant is carried by a Y chromosome common to platypus and echidna. We have searched for candidates for this determinant among the many genes known to be involved in vertebrate sex differentiation. So far fourteen such genes have been mapped, eleven are autosomal in platypus, two map to the differential regions of X chromosomes, and one maps to a pairing segment and is likewise excluded. Search for the platypus testis-determining gene continues, and the extension of comparative mapping between platypus and birds and reptiles may shed light on the ancestral origin of monotreme sex chromosomes.