Immunoglobulin genetics of Ornithorhynchus anatinus (platypus) and Tachyglossus aculeatus (short-beaked echidna)

In this paper, we review data on the monotreme immune system focusing on the characterisation of lymphoid tissue and of antibody responses, as well the recent cloning of immunoglobulin genes. It is now known that monotremes utilise immunoglobulin isotypes that are structurally identical to those found in marsupials and eutherians, but which differ to those found in birds and reptiles. Monotremes utilise IgM, IgG, IgA and IgE. They do not use IgY. Their IgG and IgA constant regions contain three domains plus a hinge region. Preliminary analysis of monotreme heavy chain variable region diversity suggests that the platypus primarily uses a single VH clan, while the short-beaked echidna utilises at least 4 distinct VH families which segregate into all three mammalian VH clans. Phylogenetic analysis of the immunoglobulin heavy chain constant region gene sequences provides strong support for the Theria hypothesis. The constant region of IgM has proven to be a useful marker for estimating the time of divergence of mammalian lineages.     

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.     

Body temperature as an indicator of egg-laying in the echidna,Tachyglossus aculeatus

We investigated the usefulness of body temperature (T_b) as a guide to egg-laying in a monotreme mammal, the echidna, Tachyglossus aculeatus, and attempted to quantify changes in T_b and relate them to specific reproductive events. Six female echidnas were implanted with temperature loggers and then radio-tracked in the wild for up to 6 years. In reproductive years there was a significant reduction in T_b variability 21.7±2.5 days after final arousal from hibernation, which coincided with the time at which the female entered the nursery burrow. Egg-laying occurred within 2 days of this T_b change which lasted an average of 43±4 days.     

Temperature regulation in the echidna (Tachyglossus aculeatus)

The echidna (Tachyglossus aculeatus) maintained a body temperature of 30.7°C ± 1.03 s.d. (N = 23) at ambient temperatures (T_A) between 0 and 25°C. It may, however, also become hypothermic at low T_A. At T_A = 30°C or above the echidna became hyperthermic. The thermoneutral range was about 20–30°C. At low T_A the metabolic rate might be increased several fold. The thermal conductance was at a minimum at T_A = 20°C, and was not further reduced at lower T_A. At higher T_A the thermal conductance increased up to five-fold. The evaporation showed little change with increasing T_A. At the highest T_A we used (33°C) the evaporation on the average accounted for the dissipation of only about one-third of the metabolic heat produced. These findings suggest that the echidna, although it can maintain its body temperature at low ambient temperature, cannot rely upon evaporation as the major avenue for heat loss at high ambient temperature.     

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.     

Hibernation in a monotreme, the echidna (Tachyglossus aculeatus)

The body temperatures of five echidnas in Australia's Southern Alps were monitored by radio telemetry from February to December 1987. All five hibernated throughout the winter, showing very low body temperatures (4-9 degrees C, close to ambient) when torpid, compared with 28-33 degrees C in a typical day during the active season. Spontaneous arousals from hibernation occurred every 2-3 weeks, during which body temperatures rose rapidly to over 30 degrees C for several hours before dropping to be close to ambient again. The identification of "classical" hibernation in a monotreme, with a similar pattern to that seen in Eutheria and in an animal as large as the largest eutherian hibernator, has important implications for current ideas about the evolution of endothermy.     

Control of breathing in the echidna (Tachyglossus aculeatus) during hibernation

Resting non-hibernating echidnas are characterised by low metabolic rates, but also have a very low respiratory frequency and a variable respiratory minute volume, often resulting in low levels of arterial O(2) and high CO(2). As the echidna lies at one physiological extreme among the hibernators, in terms of its large size and low metabolism and ventilatory requirement when not hibernating, a study of control of breathing during hibernation in echidnas should provide a useful test of the generality of various models. We used non-invasive techniques to study breathing patterns and the control of ventilation in 6 echidnas. Hibernating echidnas (T(b) range 7-10 degrees C) showed episodic breathing with bursts of breaths (average 36+/-16 breaths in 24+/-5 min) followed by a period of apnea (76+/-17 min) then a series (8+/-4) of slow breaths at 14+/-1 min intervals leading up to the next burst. Increasing CO(2) levels in the inspired air increased the number of breaths in a burst, eventually leading to continuous breathing. Inter burst breaths were controlled by O(2): hypoxia increased inter burst breaths, and decreased burst length, while hyperoxia abolished inter burst breaths and increased the apneic period. Overall, while CO(2) was a strong respiratory stimulus in hibernating echidnas, O(2) had little effect on total ventilation, but did have a strong effect on the breathing pattern.     

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.     

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.     

Body mass, age and sexual maturity in short-beaked echidnas, Tachyglossus aculeatus

During the course of this 12 year field study body masses of 11 hatchling echidnas (Tachyglossus aculeatus multiaculeatus) and 25 pouch young between the ages of 5 and 60 days were recorded. Body mass increased from 0.3 to approximately 50 g in the first half of pouch life. It then quadrupled before young were placed in a burrow at 45 to 55 days of age. There was a positive correlation between the body mass of the female and that of her young at weaning. From 33 subadult echidnas located, tagged and radio tracked during this study, body masses of 10 were monitored to sexual maturity, i.e. when first encountered in a courtship train. Minimum age of sexual maturity ranged between 5 and 12 years. As subadults, there was no difference between mean body masses of males and females. At sexual maturity, mean body mass of females was significantly higher. No correlation was found between age at sexual maturity and body mass nor was there a significant difference in age of males and females at sexual maturity.     

The isolation and amino acid sequences of echidna (Tachyglossus aculeatus) milk lysozyme I and II.

Comparative studies of monotreme proteins are of particular value in gaining an understanding of the origin of mammals and their interrelationships. The presence of two lysozyme variants, echidna lysozyme I and II, has been confirmed in mature milk samples of Tachyglossus aculeatus multiaculeatus and Tachyglossus aculeatus aculeatus respectively. A simplified procedure is described for their isolation. Their amino acid sequences, the first determined for a monotreme secretory protein, are unusual. They are shown to be c-type lysozymes, each consisting of a single chain of 125 residues (terminating at Cys 125). The only other known c-type lysozyme with this termination is that of pigeon eggwhite. Echidna lysozyme is unique in having no Cys at position 6, but at position 9. It has precisely the residues relevant to the binding of Ca(II), and most of the residues implicated in the galactosyl transferase modifier action of alpha-lactalbumin. However, the weak modifier action previously observed for variant I, prepared by a different method, was not found for the present preparation. The evolutionary significance of the results is discussed.     

Morphology,histochemistry and physiology of the major salivary glands in the echidna Tachyglossus aculeatus (Monotremata)

The three major salivary glands of the monotreme echidna are described. The parotid is a typical serous gland with tubulo-acinar secretory endpieces and a well-developed system of striated ducts. The mandibular gland, although light microscopically resembling a mucous gland, secretes very little glycoprotein. Its cells are packed instead with serous granules, resembling in fine structure the “bull's eye” granules in the mandibular gland of the European hedgehog Erinaceus europaeus. The sublingual glands secrete an extremely viscous mucous saliva. Expulsion of this saliva through the narrow ducts is probably aided by contraction of the extensive myoepithelial sheaths surrounding the secretory tubules. Application of the glyoxylic acid induced fluorescence method failed to demonstrate adrenergic innervation in any of the glands.     

The effects of a saluretic (Lasix) on adrenal steroidogenesis in the echidna (Tachyglossus aculeatus)

1. Metabolic turnover studies during 7 days Lasix administration showed a water and sodium loss which sustained for 3 days, potassium was unaffected.2. Adrenal homogenates from treated echidnas converted ¹⁴C progesterone to cortisol, corticosterone, aldosterone, 11-deoxycortisol and 11-deoxycorticosterone in yields of ; 6 ± 0.2, 46 ± 4, 0.3 ± 0.1, 8 ± 2 and 13 ± 5% (mean ± SEM) respectively.3. Compared to controls, cortisol, corticosterone and aldosterone increasedby factors of 12, 7 and 15 respectively.4. The peripheral blood aldosterone was, in Lasix treated echidnas 44 ng/100 ml a 5-fold increase.5. The K_m and V for 11β-hydroxylation of 11-deoxycorticosterone were 35 μM and 9 ϱmol/min/mg. the V increase being 3-fold.