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.

Hematologic and serum biochemical reference intervals for wild eastern quolls (Dasyurus viverrinus): Variation by age, sex, and season.

BACKGROUND: The eastern quoll (Dasyurus viverrinus) is an endangered carnivorous marsupial that has recently suffered significant population declines. Several small captive breeding populations have been established, with plans to translocate wild and captive individuals to areas of their former distribution. Accordingly, hematologic and serum biochemical reference intervals (RIs) established from wild eastern quoll populations are essential for monitoring the health and disease status of both captive and wild populations, and to evaluate the health of individuals before, during, and after translocation. OBJECTIVES: We aimed to establish hematologic and serum biochemical RIs for wild eastern quolls, and examine the effects of age, sex, and season. METHODS: We collected a total of 202 hematologic samples, 309 packed cell volume samples, and 335 serum biochemical samples from 168 individual quolls between May 2011 and November 2013. Species-level RIs were established, as well as RIs of groups separated by age (juvenile, adult) and sex (adult male, adult female) using nonparametric, robust, and parametric methods. Seasonal variation in age- and sex-specific reference values was also assessed. RESULTS: Strong age and seasonal variation were evident in many hematologic and serum biochemical analytes, with significant variation observed in serum biochemical analytes between the sexes. CONCLUSIONS: The observed age, sex, and seasonal variation reflect differences in the timing of growth and reproductive stressors, which interact with seasonal energetic demands. Our findings highlight the importance of using age-, sex-, and season-specific RIs for clinical evaluation of eastern quolls, as species-level RIs will inadvertently smooth and mask important seasonal fluctuations that reflect reproductive status at different times.

Energy Homeostasis in Monotremes.

In 1803, the French anatomist Étienne Geoffroy Saint-Hilaire decided that the newly described echidna and platypus should be placed in a separate order, the monotremes, intermediate between reptiles and mammals. The first physiological observations showed monotremes had low body temperatures and metabolic rates, and the consensus was that they were at a stage of physiological development intermediate between "higher mammals" and "lower vertebrates." Subsequent studies demonstrated that platypuses and echidnas are capable of close thermoregulation in the cold although less so under hot conditions. Because the short-beaked echidna Tachyglossus aculeatus, may show very large daily variations in body temperature, as well as seasonal hibernation, it has been suggested that it may provide a useful model of protoendotherm physiology. Such analysis is complicated by the very significant differences in thermal relations between echidnas from different climates. In all areas female echidnas regulate Tb within 1°C during egg incubation. The lactation period is considered to be the most energetically expensive time for most female mammals but lactating echidnas showed no measurable difference in field metabolic rate from non-lactating females, while the lactation period is more than 200 days for Kangaroo Island echidnas but only 150 days in Tasmania. In areas with mild winters echidnas show reduced activity and shallow torpor in autumn and early winter, but in areas with cold winters echidnas enter true hibernation with Tb falling as low as 4.5°C. Monotremes do not possess brown adipose tissue and maximum rates of rewarming from hibernation in echidnas were only half those of marmots of the same mass. Although echidnas show very large seasonal variations in fat stores associated with hibernation there is no relationship between plasma leptin and adiposity. Leptin levels are lowest during post-reproductive fattening, supporting suggestions that in evolutionary terms the anorectic effects of leptin preceded the adiposity signal. BMR of platypuses is twice that of echidnas although maximum metabolism is similar. High levels of thyroid hormones in platypuses may be driving metabolism limited by low body temperature. Monotremes show a mosaic of plesiomorphic and derived features but can still inform our understanding of the evolution of endothermy.

Frozen embryos? Torpor during pregnancy in the Tasmanian short-beaked echidna Tachyglossus aculeatus setosus.

We studied the interaction between torpor and reproduction in free-ranging female Tasmanian echidnas using a combination of techniques including urogenital smears, hormone analysis, ultrasonography, external temperature loggers and camera traps. Male echidnas initiated mating activity by locating hibernating females. All females that mated or were disturbed by males prior to July 27 re-entered hibernation, including many that were pregnant. Pregnant females only entered hibernation in early pregnancy when plasma progesterone concentrations were about twice basal and progesterone then remained constant during torpor. By re-entering hibernation pregnant females extended their gestation period and delayed egg-laying. Progesterone peaked 4-6days before egg-laying, then dropped rapidly.

Testing the Role of Climate Change in Species Decline: Is the Eastern Quoll a Victim of a Change in the Weather?

To conserve a declining species we first need to diagnose the causes of decline. This is one of the most challenging tasks faced by conservation practitioners. In this study, we used temporally explicit species distribution models (SDMs) to test whether shifting weather can explain the recent decline of a marsupial carnivore, the eastern quoll (Dasyurus viverrinus). We developed an SDM using weather variables matched to occurrence records of the eastern quoll over the last 60 years, and used the model to reconstruct variation through time in the distribution of climatically suitable range for the species. The weather model produced a meaningful prediction of the known distribution of the species. Abundance of quolls, indexed by transect counts, was positively related to the modelled area of suitable habitat between 1990 and 2004. In particular, a sharp decline in abundance from 2001 to 2003 coincided with a sustained period of unsuitable weather over much of the species' distribution. Since 2004, abundance has not recovered despite a return to suitable weather conditions, and abundance and area of suitable habitat have been uncorrelated. We suggest that fluctuations in weather account for the species' recent decline, but other unrelated factors have suppressed recovery.

Devil declines and catastrophic cascades: is mesopredator release of feral cats inhibiting recovery of the eastern quoll?

The eastern quoll (Dasyurus viverrinus) is a medium-sized Australian marsupial carnivore that has recently undergone a rapid and severe population decline over the 10 years to 2009, with no sign of recovery. This decline has been linked to a period of unfavourable weather, but subsequent improved weather conditions have not been matched by quoll recovery. A recent study suggested another mechanism: that declines in Tasmanian devil (Sarcophilus harrisii) populations, due to the spread of the fatal Devil Facial Tumour Disease, have released feral cats (Felis catus) from competitive suppression, with eastern quoll declines linked to a subsequent increase in cat sightings. Yet current evidence of intraguild suppression among devils, cats and quolls is scant and equivocal. We therefore assessed the influences of top-down effects on abundance and activity patterns among devils, feral cats and eastern quolls. Between 2011 and 2013, we monitored four carnivore populations using longitudinal trapping and camera surveys, and performed camera surveys at 12 additional sites throughout the eastern quoll's range. We did not find evidence of a negative relationship between devil and cat abundance, nor of higher cat abundance in areas where devil populations had declined the longest. Cats did not appear to avoid devils spatially; however, there was evidence of temporal separation of cat and devil activity, with reduced separation and increasing nocturnal activity observed in areas where devils had declined the longest. Cats and quolls used the same areas, and there was no evidence that cat and quoll abundances were negatively related. Temporal overlap in observed cat and quoll activity was higher in summer than in winter, but this seasonal difference was unrelated to devil declines. We suggest that cats did not cause the recent quoll decline, but that predation of juvenile quolls by cats could be inhibiting low density quoll populations from recovering their former abundance through a 'predator pit' effect following weather-induced decline. Predation intensity could increase further should cats become increasingly nocturnal in response to devil declines.

Fluctuations in oxygen influence facultative endothermy in bumblebees.

Bumblebees are facultative endotherms, having the ability to elevate thorax temperature above ambient temperature by elevating metabolism. Here, we investigated the influence of hypoxia on metabolic demands and thermoregulatory capabilities of the bumblebee Bombus terrestris. We measured thorax temperature, rates of oxygen consumption and carbon dioxide production, and abdominal pumping rates of bees randomly exposed to oxygen levels of 20, 15, 10 and 5 kPa at 26°C. Under normoxia, bumblebees maintained an elevated mean thorax temperature of 35.5°C. There was no significant change in thorax temperature at 15 kPa O2 (33.4°C). Mean thorax temperature decreased significantly at 10 kPa O2 (31.6°C) and 5 kPa O2 (27.3°C). Bees were able to maintain an elevated metabolic rate at 15 and 10 kPa O2. In normoxia, endothermic bees exhibited periods of rapid abdominal pumping (327 min(-1)) interspaced by periods of no abdominal pumping. At 10 kPa O2, abdominal pumping rate decreased (255 min(-1)) but became more continuous. Upon exposure to 5 kPa, metabolic rate and abdominal pumping rate (152 min(-1)) decreased, although the animals continued abdominal pumping at the reduced rate throughout the exposure period. Bumblebees are able to meet the energetic demands of endothermy at 15 kPa O2, but become compromised at levels of 10 kPa O2 and below.

Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection.

Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female's abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples.

Beyond the disease: Is Toxoplasma gondii infection causing population declines in the eastern quoll (Dasyurus viverrinus)?

Disease is often considered a key threat to species of conservation significance. For some, it has resulted in localised extinctions and declines in range and abundance. However, for some species, the assertion that a disease poses a significant threat of extinction is based solely on correlative or anecdotal evidence, often inferred from individual clinical case reports. While a species' susceptibility to a disease may be demonstrated in a number of individuals, investigations rarely extend to measuring the impact of disease at the population level and its contribution, if any, to population declines. The eastern quoll (Dasyurus viverrinus) is a medium-sized Australian marsupial carnivore that is undergoing severe and rapid decline in Tasmania, its last refuge. Reasons for the decline are currently not understood. Feral cats (Felis catus) may be undergoing competitive release following the ongoing decline of the Tasmanian devil (Sarcophilus harrisii), with cats suppressing eastern quolls through increased predation, competition, exclusion or exposure to diseases such as toxoplasmosis. To investigate the effects of Toxoplasma gondii infection, eastern quoll populations at four sites were regularly screened for the seroprevalence of T. gondii-specific IgG antibodies. Seroprevalence was approximately five times higher at sites with declining quoll populations, and there was a negative association between seroprevalence and quoll abundance. However, T. gondii infection did not reduce quoll survival or reproduction. Despite a high susceptibility to T. gondii infection, eastern quoll populations do not appear to be limited by the parasite or its resultant disease. Significantly higher seroprevalence is a signal of greater exposure to feral cats at sites where eastern quolls are declining, suggesting that increased predation, competition or exclusion by feral cats may be precipitating population declines.

Can an ancestral condition for milk oligosaccharides be determined? Evidence from the Tasmanian echidna (Tachyglossus aculeatus setosus).

The monotreme pattern of egg-incubation followed by extended lactation represents the ancestral mammalian reproductive condition, suggesting that monotreme milk may include saccharides of an ancestral type. Saccharides were characterized from milk of the Tasmanian echidna Tachyglossus aculeatus setosus. Oligosaccharides in pooled milk from late lactation were purified by gel filtration and high-performance liquid chromatography using a porous graphitized carbon column and characterized by (1)H NMR spectroscopy; oligosaccharides in smaller samples from early and mid-lactation were separated by ultra-performance liquid chromatography and characterized by negative electrospray ionization mass spectrometry (ESI-MS) and tandem collision mass spectroscopy (MS/MS) product ion patterns. Eight saccharides were identified by (1)H NMR: lactose, 2'-fucosyllactose, difucosyllactose (DFL), B-tetrasaccharide, B-pentasaccharide, lacto-N-fucopentaose III (LNFP3), 4-O-acetyl-3'-sialyllactose [Neu4,5Ac(α2-3)Gal(ß1-4)Glc] and 4-O-acetyl-3'-sialyl-3-fucosyllactose [Neu4,5Ac(α2-3)Gal(ß1-4)[Fuc(α1-3)]Glc]. Six of these (all except DFL and LNFP3) were present in early and mid-lactation per ESI-MS, although some at trace levels. Four additional oligosaccharides examined by ESI-MS and MS/MS are proposed to be 3'-sialyllactose [Neu5Ac(α2-3)Gal(ß1-4)Glc], di-O-acetyl-3'-sialyllactose [Neu4,5,UAc3(α2-3)Gal(ß1-4)Glc where U = 7, 8 or 9], 4-O-acetyl-3'-sialyllactose sulfate [Neu4,5Ac(α2-3)Gal(ß1-4)GlcS, where position of the sulfate (S) is unknown] and an unidentified 800 Da oligosaccharide containing a 4-O-acetyl-3'-sialyllactose core. 4-O-acetyl-3'-sialyllactose was the predominant saccharide at all lactation stages. 4-O-Acetylation is known to protect sialyllactose from bacterial sialidases and may be critical to prevent microbial degradation on the mammary areolae and/or in the hatchling digestive tract so that sialyllactose can be available for enterocyte uptake. The ability to defend against microbial invasion was probably of great functional importance in the early evolution of milk saccharides.

Identification and functional characterization of a novel monotreme- specific antibacterial protein expressed during lactation.

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother's body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.

Chemical composition of odorous secretions in the Tasmanian short-beaked echidna (Tachyglossus aculeatus setosus).

The short-beaked echidna is believed to use olfactory cues from a cloacal scent gland to attract and locate mates during the breeding season. We investigated the chemical composition of echidna secretions, including cloacal swabs and solid, "waxy" exudates from the cloaca and spurs. Scent samples from 37 individuals were collected over a 1-year period and analyzed using a range of different analytical techniques. A total of 186 compounds were identified, including volatile carboxylic acids, aldehydes, ketones, fatty acids, methyl esters, ethyl esters, terpenes, nitrogen- and sulphur-containing compounds, alcohols, and aromatics. Long chain and very long chain monounsaturated fatty acids, sterols, and sterol esters were identified as the major constituents of solid exudates, some of which have not previously been described from any animal skin gland. There was a high degree of composition overlap between male and female cloaca swabs; however, there is significant variation, which could mediate echidna mating behavior. Many of the volatile and nonvolatile chemicals detected are used for communication in other species, suggesting that chemical signals have important and diverse functions in echidna social interactions.

Does leptin signal adiposity in the egg-laying mammal, Tachyglossus aculeatus?

Leptin is a peptide hormone best known for its role in feedback regulation of adiposity in eutherian mammals. Normally an increase in adipose tissue mass leads to an increase in circulating leptin which increases energy expenditure and limits food intake, but in hibernating eutherian mammals this relationship may change to allow prehibernatory fattening. The echidna (Tachyglossus aculeatus) is a monotreme mammal which accumulates significant fat reserves before entering hibernation, and mates immediately at the end of hibernation. We hypothesised that echidnas would show a strong relationship between body mass and plasma leptin for most of the year which would change during the pre-hibernatory period. We measured plasma leptin and body mass in free-ranging echidnas over several reproductive and hibernation cycles. There were significant seasonal variations in plasma leptin in both sexes, with the highest levels occurring in hibernation and in mating females. The lowest levels were found in males when they were foraging maximally after the reproductive period. We used mass%, body mass at the time of sampling as a percentage of long term mean mass, as a proxy for adiposity. There was a weak negative relationship between mass% and plasma leptin, from which we infer a weak negative relationship between adiposity and plasma leptin as has been found in reptiles and birds, rather than the strong positive relationship found in other mammals.

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.

Rewarming rates and thermogenesis in hibernating echidnas.

We measured body temperatures (T(b)) in 14 free-ranging echidnas (Tachyglossus aculeatus) using implanted data-loggers. An average of 1020+/-744 days of T(b) data was recorded from each animal. The average maximum T(b) was 35.3+/-0.7 degrees C (n=14), and the lowest T(b) was 4.7 degrees C. Detailed analysis of rewarming events from four echidnas showed rewarming time to be dependent on initial T(b) (rewarming time in hours=15.6-0.41T(initial), n=31) with an average rewarming rate of 1.9+/-0.4 degrees C h(-1). Based on an hourly sampling rate, the peak rewarming rate was found to be 7.2+/-0.8 degrees C h(-1) (n=12), which was measured at a mean T(b) of 26.2+/-2.4 degrees C. This rate of heating was calculated to be equivalent to a peak oxygen consumption rate of 1.4+/-0.2 ml O2 g h(-1), approximately 9 times the basal metabolic rate. We found that a plot of rate of change of T(b) against T(b) for the entire data set from an individual echidna provided a useful summary and analytical tool.

Cooling rates and body temperature regulation of hibernating echidnas (Tachyglossus aculeatus).

Echidnas (Tachyglossus aculeatus) are amongst the largest deep hibernators, but it is difficult to get them to hibernate normally under laboratory conditions. We measured body temperature (Tb) in 14 free-ranging echidnas using implanted data-loggers. Cooling during entry into hibernation bouts followed a Newtonian cooling curve, and conductances calculated from cooling curves were identical to those observed in cold exposed euthermic echidnas. Comparison with a reference soil temperature demonstrated that echidnas showed behavioural thermoregulation during hibernation; early in the hibernation season echidnas preferred to hibernate in cool areas, while during the coldest months they moved to warmer hibernacula, giving a preferred Tb in the range 8-10 degrees C. Thermal buffering against excessive variation in Tb may be as important as maintaining a low Tb.

Field energetics of free-living, lactating and non-lactating echidnas (Tachyglossus aculeatus).

We measured daily energy expenditure (DEE) and water turnover rates in lactating and non-lactating short beaked echidnas (Tachyglossus aculeatus) using the doubly labelled water technique during the lactation period in spring. Reproductively inactive echidnas were on average significantly heavier (median: 3354 g; range: 2929-3780 g; N=4) than lactating females (median: 2695 g; range: 2690-2715 g; N=3) during the equivalent time period. The median water flux rate of lactating echidnas (152 ml day(-1); range: 120-198 ml day(-1)) did not differ significantly from that of non-lactating females (170 ml day(-1); range: 128-227 ml day(-1)). The median DEE of echidnas that were lactating was 645 kJ day(-1) (range: 581-850 kJ day(-1)), which was not different from the median DEE of non-reproductive control females (763 kJ day(-1); range: 720-766 kJ day(-1)). Lactating females somehow compensate for the energy costs of milk production, resulting in a daily energy budget that is not different from that of non-reproductive females. At least part of their energy minimising strategy could involve the use of moderate heterothermy, allowing a greater proportion of daily energy expenditure to diverted to milk production.