Respiration by buried echidnas Tachyglossus aculeatus

Short-beaked echidnas (Tachyglossus aculeatus) are famous for many unusual characteristics, among them the ability to avoid capture or predation by `sinking' into the soil until only the tips of the dorsal spines are visible (Burrell, 1926) and remaining there, holding fast against attempts to dislodge them, for long periods. This behaviour poses questions about how echidnas manage their respiratory gas exchange, cut off from a free flow of gases and threatened by the risk of inhaling particles of soil.

It is clear that echidnas are frequently exposed, at least for short periods, to increased hypoxia and hypercapnia, either when digging into soil or within their burrows (Augee et al.,1971). Previous studies have demonstrated that echidnas are physiologically well suited for burrowing. Augee et al.(Augee et al., 1971) covered echidnas with soil to simulate natural conditions and found them to be very tolerant of high carbon dioxide (CO₂) and low oxygen(O₂) under these conditions.

However, no studies have explored the processes by which O₂supply is maintained while an echidna is buried, completely surrounded by soil, without any tunnel to facilitate the convective movements of gas. Presumably echidnas re-breathe the interstitial gas around them while buried. The diffusive transport of O₂ to a buried mammal was explored in the Namib Desert golden mole Eremitalpa granti namibensis(Seymour and Seely, 1996),which survives long periods buried in sand. By comparing measurements of the P_O2 gradient away from the snout of a buried mole to the P_O2 gradient predicted from a mathematical model of gaseous diffusion through sand, Seymour and Seely(Seymour and Seely, 1996)explained that golden moles can survive being buried in sand by utilizing O₂ diffusing through sand into the tidal air space that surrounds the snout. However, their model predicted an upper size limit of approximately 200 g for resting mammals able to continue respiration in this manner. Echidnas are commonly 2–4 kg and can reach 7 kg, so the model used to explain sub-sand respiration by the golden mole cannot by itself explain the submergence capabilities observed in echidnas.

In this study we took a similar approach to that of Seymour and Seely(Seymour and Seely, 1996). We measured the P_O2 gradient away from the snouts of submerged echidnas and V̇_O2 in separate experiments on five individuals under similar conditions, in two media with differing porosity f_a values, with the hope of determining how echidnas can remained buried for long periods of time.

Five echidnas Tachyglossus aculeatus Shaw (2.34–4.18 kg)previously fitted intraperitoneally with calibrated temperature transmitters were used. Animals had been collected previously from Idalia National Park(latitude 24°53′S, longitude 144°46′E), 113 km WSW of Blackall in Australia's semi-arid zone(Brice et al., 2002), and the Texas area, 50 km SW of Stanthorpe in SE Queensland (latitude 28°43′S, longitude 151°28′E). The animals were held in a free-range enclosure at the University of Queensland's Pinjarra Hills Veterinary Farm.

To apply the model to echidnas, V̇_O2 values and P_O2 gradients away from buried animals were measured separately, and the assumption made that the V̇_O2 measured while submerged was indicative of the V̇_O2 during measurement of the P_O2 gradient, as was done previously with the golden mole (Seymour and Seely, 1996).

V̇_O2 was measured using a flow-through respirometry system, at an ambient temperature of 25°C. Each animal was placed in an air tight, 50 cm deep and 40 cm in diameter, cylindrical chamber ¾-filled with a test medium into which the echidnas could burrow. Gas entered the chamber into an air space above the burrowing medium and was extracted from below the burying medium through a wire mesh-covered hole at the base of the chamber. Two media with differing f_a values were used and each animal was exposed to each medium. The f_a of each of the two media was measured by filling a 1000 ml graduated cylinder with the medium and slowly adding it to 1000 ml of water in a 2000 ml cylinder, avoiding any bubbles(Seymour and Seely, 1996). The kitty litter, which would otherwise have absorbed water, was first sprayed with a water repellent spray (Motortech, Balchan International, Australia). While this method discounted any porosity of the kitty litter itself, we consider this to have been negligible.

The lid of the respirometry chamber was transparent, so lung ventilation movements could be directly observed during these experiments even when the animal was completely buried (the surface of the substrate moved slightly with each breath). The behaviour of an animal on introduction to the respirometry chamber, whether for V̇_O2 or P_O2 gradient measurements, was to burrow immediately vertically downward until submerged completely in the substrate, a behaviour identical to that of echidnas burying themselves in the wild. V̇_O2 was measured while the animal remained buried. Resting metabolic rate was considered to have been reached when the animal had been submerged and resting for 4 h and the fractional concentration of O₂ in the excurrent air was stable. Air from the chamber was passed through small diameter tubing to a CO₂ absorbent (Soda Lime) and then a desiccant (Drierite™)before passing into a mass flow meter (MFS-1; Sable Systems, Las Vegas, NV,USA). The mass flow meter pulled air though the chamber and its exhalent air was sampled via an oxygen analyser (Sable Systems PA-1B) calibrated with oxygen-free gas (0.00% O₂) and room air (20.95%O₂). The flow rate through the respirometry chamber was 750 ml min^–1 in all cases. Body temperature T_bwas monitored throughout the measurement period using the implanted temperature transmitter and a radio receiver connected to a pulse meter. The output voltage from the O₂ analyser and pulse meter were fed into PowerLab hardware (ADInstruments, Sydney, NSW, Australia) connected to a computer running Chart5 software (v5.0.1. ADInstruments).

Measurements of P_O2 within the substrate surrounding buried echidnas were performed in a large cylindrical plastic bin(measurements given above) at an ambient temperature of 25°C. Experiments were commenced by placing the animal on top of the substrate and, in all cases, echidnas burrowed immediately until they were completely submerged.

Gas samples from the snout region were collected through silicone tubing (1 mm i.d.) after flushing dead space from the tubing into 3 ml plastic syringes. The tip of this tube was secured onto the nose of the animal above the nostril using a combination of medical glue (collodian) and micropore tape. To measure the P_O2 gradient within the medium away from the snout region, further lengths of silicon tubing were attached at 2 cm intervals along the initial tubing, up to 10 cm away from the snout tip.

Gas samples (2 ml) were analysed for O₂ content with a thermally stabilised Clarke Oxygen Electrode (DOX, Analytical Sensors, Inc., Sugarland,TX, USA), connected to a Radiometer PHM73 gas analyser (Copenhagen, Denmark),calibrated with outside air (20.95% O₂) and oxygen-free gas.

Gas samples were taken immediately on submergence of the echidna, and further samples were taken at 15 min intervals for a 5 h period. Each animal was measured individually in each of the media. T_b was also monitored throughout these experiments using the implanted temperature transmitter.

During trial measurements of O₂ tensions around buried echidnas,the animals would move periodically. Such movements would be followed by a rise in P_O2 levels in the substrate, seemingly as a result of these movements. To determine if there was a causal relationship between the movements of echidnas in different substrates and the P_O2 measured in the snout region while submerged, a piezoelectric movement sensor (Sigma Delta technologies, Perth,Western Australia) was attached to the echidnas. This sensor was wrapped in electrical tape and glued to a dorsal spine on the shoulder region of the animal. The output voltage from the movement sensor was recorded,simultaneously with T_b and measured P_O2 samples, using PowerLab hardware connected to a computer running Chart5 software (v5.0.1. ADInstruments). Respiration rate and larger `flushing movements' were detected by the movement sensor(Fig. 1).

All results are presented as means ± s.d. Differences between empirical and theoretical values of P_O2 in substrate surrounding buried echidnas were tested using paired t-tests. Significance was assumed at P<0.05.

Data were collected on V̇_O2 of each echidna, the P_O2 in the snout region, and movement and activity level while submerged in two different substrates,coarse sand (f_a=0.42) and kitty litter(f_a=0.58).

V̇_O2measurements were taken at a chamber temperature of 25°C. As would be expected, V̇_O2 was greater during burrowing than resting, and substrate type did not influence resting rate of V̇_O2(Table 1). T_b and respiration rate always decreased during the course of V̇_O2measurements. T_b of echidnas at the start of the experiments ranged from 27.0 to 34.5°C and, on average, decreased 2.2±1.3°C (N=5) during a trial. Respiration decreased from 12.0±0.5 to 4.6±2.2 breaths min^–1 over the 5 h measurement period.

P_O2 measurements were taken at an ambient temperature of 25°C. The mean atmospheric P_O2 was 19.7 kPa (range 19.3–19.9 kPa) in water-saturated air. The P_O2 level in each substrate, without an echidna, was atmospheric. P_O2 levels of gas samples from near the snout of echidnas buried in the medium were always below the atmospheric level, even immediately after burial. The mean minimum P_O2values at the tip of the echidna's snout during burrowing in each substrate were 12.1±1.4 kPa in coarse sand (f_a=0.42, N=5) and 12.3±1.4 kPa in kitty litter(f_a=0.58, N=5). Gas samples taken at a series of distances away from the snout revealed a P_O2gradient within the substrate (Table 2).

T_b and respiration rate always decreased over time,independent of medium, during experiments that measured P_O2 gradients. T_b of echidnas at the start of the experiments ranged from 28.0 to 35.0°C and,on average, decreased 1.2±0.5°C (N=5) during a trial. Respiration decreased from 12.0±0.5 to 4.7±3.3 breaths min^–1 over the 5 h measurement period.

Measured V̇_O2data and published values for tidal volume(Bech et al., 1992) were used to generate theoretical diffusion gradients away from the snout for each of the substrates, using Eqn 1. These theoretical P_O2 values at distances away from the snout were compared with empirically measured P_O2values around buried echidnas (Figs 2, 3).

In kitty litter, which had the highest porosity(f_a=0.58), the theoretically calculated P_O2 values away from the snout were not significantly different from the measured values(Fig. 2), implying that diffusion into the tidal space was sufficient in this porous medium to accommodate the oxygen requirements of a resting, buried echidna.

In the natural substrate, coarse sand (f_a=0.42) the theoretical calculated values were significantly different from the measured values (Fig. 3). In coarse sand, supply of oxygen by diffusion alone was apparently insufficient to account for the measured rates of V̇_O2.

A behavioural pattern was observed in the experiments that measured P_O2 profiles in the substrate surrounding buried echidnas. P_O2 at any measurement distance from the snout did not remain constant, it fluctuated in a cyclic manner and mean values tended to increase as the experiment progressed(Fig. 4). When first introduced to the burrowing medium, echidnas immediately dug down until completely encased in the substrate. At this stage, animals had a relatively high V̇_O2, T_b and respiration rate, and the minimum P_O2 near the snout was always lowest during this early phase of an experiment. The minimum P_O2 rose over time as V̇_O2 fell to a resting rate (Fig. 4). Periodic movements of the anterior body were more common early in these experiments when oxygen consumption rates were higher, and these movements were followed soon after by an increase in P_O2 close to the snout and further away from the snout (Fig. 4) indicating that such `flushing movements' caused the convective movement of oxygen from the atmosphere to the snout area.

The frequency of flushing movements varied between kitty litter and course sand (Fig. 4). In kitty litter there were approximately two movements h^–1 for the first hour and then one movement h^–1 for the rest of the experiment(Fig. 4A), while in course sand there were on average 6.2 movements h^–1 for the first hour,4.6 movements h^–1 for the second hour, and two movements h^–1 for the rest of the trial(Fig. 4B).TBL3 

It appeared that echidnas were able to maintain their O₂ supply while encased in substrate, including while they were digging in, by a combination of diffusion through the substrate augmented by periodic movements, which flush the interstitial air space around the nose. Measured V̇_O2 and published values for tidal volume (Bech et al.,1992) were used to generate theoretical P_O2 profiles away from the snout region, with which measured P_O2 values could be compared. In kitty litter, at rest, there was no significant difference between the measured and the predicted gradients, suggesting that diffusion was sufficient to meet the O₂ requirements when buried in this high porosity medium, as for golden moles buried in fine sand(Seymour and Seely, 1996). Even in this high porosity medium, however, resting echidnas chose to make periodic movements, albeit infrequently.

In the natural substrate, empirically measured and theoretical P_O2 values were significantly different. The actual mean minimum P_O2 values at the snout region and along the gradient were significantly greater than predicted by the model, suggesting that diffusive oxygen transport through the sand alone was insufficient to account for the measured V̇_O2. The higher than predicted P_O2 values appear to result from the periodic flushing movements during submergence, which caused temporarily increased interstitial O₂ levels closer to the snout.

Periodic movements were more frequent in coarse sand, particularly in the earlier phase of an experiment when V̇_O2 was highest. The higher frequency of `flushing' movements at the beginning of the burying trials enabled the echidna to stay submerged while experiencing higher O₂ demand, but as O₂ demand decreased later in trials,the frequency of flushing movements decreased. Once a resting rate of V̇_O2 was achieved,a steady state was achieved and flushing movements were regular but less frequent and the P_O2 profiles became more or less constant.

Monotremes are characterized by metabolic rates and T_bthat are lower than those typical of eutherian mammals(Bech et al., 1992; Griffiths, 1978; Schmidt-Nielsen et al., 1966)and this was confirmed in our study. Basal metabolic rates of echidnas have been reported to be only 25–50% of that predicted for eutherian mammals(Bech et al., 1992; Dawson et al., 1978; Dawson et al., 1979; Schmidt-Nielsen et al., 1966). However, echidnas are notoriously difficult subjects in which to measure resting V̇_O2,usually being very restless and attempting to escape while in classical respirometry chambers. This is the first study to measure V̇_O2 in echidnas buried in substrate. Indeed, further work in our laboratory has shown that providing echidnas with even a small quantity of material in the respirometer,into which they can bury their head, makes it much easier to achieve measurements at apparently resting levels (P. H. Brice, G. C. Grigg and L. A. Beard, unpublished observations). Accordingly, we think that buried echidnas are more relaxed than echidnas in a respirometer without anywhere to `hide'and are more likely to provide good data on resting metabolic rates.

The V̇_O2measured from echidnas in this study were also somewhat more variable than might be expected for metabolic rate measured in a typical mammal at rest. The variability may be explained in terms of their heterothermy. Echidnas have the advantages of endothermy, including the capacity for impressive homeothermic endothermy during incubation (Grigg et al., 2004). The modal T_b of active echidnas is 32°C (Grigg et al., 2004). However, they are very relaxed about using thermoregulatory mechanisms to maintain a stable T_b and periods of rest are typically accompanied by a drop in T_b. Accordingly, cyclic changes in daily T_b of 3–6°C are routine. They also show both short- and long-term torpors (Grigg et al., 2004). In our study, T_b at the start of a trial differed between echidnas and this is likely to account for much of the variability in measured V̇_O2. The declines in T_b during each experimental trial reflect the expected drops, which occur in echidnas at rest after a period of activity.

The P_O2 of the immediate O₂environment of the burrowing echidna showed a decrease in ambient oxygen to about 11 kPa, substantially below atmospheric (21 kPa). This is a much greater drop than was found in golden moles buried in sand(Seymour and Seely, 1996),where ambient P_O2 values were about 20 kPa. Kuhnen (1986) summarized published data on the burrow O₂ and CO₂ levels for 13 species of burrowing mammals. Typically, these species were exposed to P_O2 values between 17–20 kPa, but values down to 10 kPa have occasionally been recorded(Kuhnen, 1986). The results from the present study showed that the immediate O₂ environment of buried echidna near the snout was 12.3±0.2 kPa while active and 14.9±0.2 kPa while resting. A P_O2 of 11 kPa was recorded (Augee et al.,1971) in an echidna encased in substrate for a period of 4 h at a depth of 20 cm, which is in good agreement with our study. Bentley et al.(Bentley et al., 1967) found a P_O2 of 13.9 kPa in an echidna completely encased in crushed corncobs at a depth of 30–60 cm. The O₂environment tolerated by buried echidnas seems to put them at the extreme end among fossorial and semi-fossorial mammals.

O₂ supply is one aspect of survival under soil, but the mechanical problem of breathing in loose material also needs to be explored. The porosity of coarse sand was low due to a discontinuity of pore spaces and the smaller particles filling in the void spaces between the larger particles. The echidna apparently used this discontinuity to stay submerged and was able to use its snout to create a small air space and prevent the inhaling of soil particles into the nose.

We thank Peter Brice and Clare Stawski for helpful discussions and ideas on the project, and other researchers in the Physiological Ecology Laboratory at the University of Queensland. Animal collection and experimentation were approved by the University of Queensland Animal Ethics Committee (AEC approval number: ZOO/ENT/122/04/URG), and Queensland Parks and Wildlife Service (Permit number: WISP02184504).