Lasiorhinus Research Articles

Estimating the probability of identity among genotypes in natural populations: cautions and guidelines

Individual identification using DNA fingerprinting methods is emerging as a critical tool in conservation genetics and molecular ecology. Statistical methods that estimate the probability of sampling identical genotypes using theoretical equations generally assume random associations between alleles within and among loci. These calculations are probably inaccurate for many animal and plant populations due to population substructure. We evaluated the accuracy of a probability of identity (P(ID)) estimation by comparing the observed and expected P(ID), using large nuclear DNA microsatellite data sets from three endangered species: the grey wolf (Canis lupus), the brown bear (Ursus arctos), and the Australian northern hairy-nosed wombat (Lasiorinyus krefftii). The theoretical estimates of P(ID) were consistently lower than the observed P(ID), and can differ by as much as three orders of magnitude. To help researchers and managers avoid potential problems associated with this bias, we introduce an equation for P(ID) between sibs. This equation provides an estimator that can be used as a conservative upper bound for the probability of observing identical multilocus genotypes between two individuals sampled from a population. We suggest computing the actual observed P(ID) when possible and give general guidelines for the number of codominant and dominant marker loci required to achieve a reasonably low P(ID) (e.g. 0.01-0.0001).

Highly reliable genetic identification of individual northern hairy‐nosed wombats from single remotely collected hairs: a feasible censusing method

The highly endangered northern hairy-nosed wombat (Lasiorhinus krefftii) is extremely difficult to study in the wild, and its numbers correspondingly difficult to estimate. Disturbance to the animals caused by trapping and radio-tracking may not only constitute an excessive risk to the population's viability, but may also yield biased data. The results of a pilot study are presented, which clearly show noninvasive genotyping to be a highly feasible and reliable alternative censusing method for L. krefftii. The protocol can identify individual wombats from single hairs collected remotely at burrow entrances, using: (i) a panel of microsatellite markers giving individual-specific genotypes; and (ii) a Y-linked sexing marker in combination with a single-copy X-linked amplification control. Using just the eight most variable microsatellites (of 20 available), only one in 200 pairs of full-sibs are predicted to share the same genotype. From 12 wombat hair samples collected on tape suspended over burrow entrances, three known female, two known male and an unknown wombat of each sex were identified. The approach will allow censusing of individuals that evade capture, and will also reveal some otherwise problematic aspects of the behaviour of this elusive animal.

Determination of seasonality in southern hairy-nosed wombats (Lasiorhinus latifrons) by analysis of fecal androgens.

Little is known about the reproductive biology of Australia's critically endangered northern hairy-nosed wombat (Lasiorhinus krefftii), largely due to its cryptic nature and the difficulty in accessing the small remaining population of about 70 animals. Using the noninvasive technique of fecal steroid analysis, we have examined the endocrinology of the more common yet closely related southern hairy-nosed wombat (Lasiorhinus latifrons). The aims of this study were to 1) develop and validate fecal androgen analysis in this species, 2) examine and compare seasonal differences in fecal and plasma androgens in male wombats, and 3) correlate seasonal differences in androgens with changes in male accessory glands (prostate and bulbourethral gland). Fecal androgens were extracted in ether; concentrated; separated by HPLC into testosterone (T), dihydrotestosterone (DHT), and 5 alpha-androstane-3 alpha,17 beta-diol (Adiol) fractions; and quantitated by RIA. The concentrations of androgens in fecal pellets from 14 wild southern hairy-nosed wombats as determined by RIA varied over the range 6.6-25.0 ng/g dry weight for T, 4.0-24.2 ng/g dry weight for DHT, and 0-34.8 ng/g dry weight for Adiol. For each androgen, a highly significant linear correlation was observed between plasma and fecal concentrations. When individuals were grouped into either breeding season (pellets collected between August-November) or nonbreeding season (collected between February-April), significant (P < 0.05) differences between seasons were observed for both plasma and fecal T, plasma DHT, and fecal Adiol. For all androgens, the mean fecal and plasma concentrations were higher during the breeding season than the nonbreeding season. A significant (P < 0.001) correlation was observed between fecal T and prostate weight, while DHT and Adiol correlations were nonsignificant. Significant correlations were observed, however, between all three fecal androgens and bulbourethral gland weight. These studies demonstrate that fecal T is a valid indicator of reproductive status in the male southern hairy-nosed wombat, with significant correlations observed between fecal T, plasma T, and prostate and bulbourethral gland weights. These findings have important implications for the study of the reproductive endocrinology of the critically endangered northern hairy-nosed wombat.

A set of microsatellite loci for the hairy-nosed wombats (Lasiorhinus krefftii and L. latifrons)

A set of microsatellite loci for the hairy-nosed wombats (Lasiorhinus krefftii and L. latifrons)

Assessment of the potential for competition between two sympatric herbivores - the northern hairy-nosed wombat, Lasiorhinus krefftii, and the eastern grey kangaroo, Macropus giganteus

The northern hairy-nosed wombat, Lasiorhinus krefftii, is a critically endangered grazing herbivore, restricted to a single population of just 65 individuals in the savanna of central Queensland. Lasiorhinus krefftii shares its habitat with another grazing herbivore of similar body size, the eastern grey kangaroo, Macropus giganteus. This study investigated the potential for M. giganteus to compete with L. krefftii for food. Analysis of faecal residues demonstrated that both herbivores consumed grass almost exclusively, with the exception of small quantities of sedges and dicotyledons. Dietary overlap between M. giganteus and L. krefftii was high, both in the consumption of plant species (90%) and plant parts (99%), suggesting that there is potential for dietary competition. Using the distribution of faecal deposits for both herbivores, analysis of habitat usage by multiple linear regression techniques indicated that M. giganteus exhibits clear associations with some habitat features but proved inconclusive for L. krefftii because of their use of defecation in social marking. However, an examination of the population dynamics of M. giganteus suggested that the densities reported in this study, although highly seasonal, are sufficiently low that competition with L. krefftii for food is currently negligible.

Parasites of the northern hairy-nosed wombat Lasiorhinus krefftii: Implications for conservation.

The northern hairy-nosed wombat Lasiorhinus krefftii is now restricted to a single population of less than 70 individuals at Epping Forest National Park, North Queensland, Australia, and is listed as critically endangered. We examined six trapped animals for ectoparasites, and 197 faecal samples for endoparasites. All ectoparasites (the tick Ambylomma triguttatum, the flea Echidnophaga cornuta and the louse Boopia dubia) were new host records. Nematode eggs and larvae were found in every faecal sample and the number of eggs varied significantly among months sampled. Cestode proglottids were also found. There was no indication that parasites were causing disease and few species were detected. This last remaining population of L. krefftii may be relatively immunologically na�ve, and we suggest that removing them from their natural environment to other areas as part of a captive breeding program should be attempted with caution.

Detecting population expansion and decline using microsatellites.

This article considers a demographic model where a population varies in size either linearly or exponentially. The genealogical history of microsatellite data sampled from this population can be described using coalescent theory. A method is presented whereby the posterior probability distribution of the genealogical and demographic parameters can be estimated using Markov chain Monte Carlo simulations. The likelihood surface for the demographic parameters is complicated and its general features are described. The method is then applied to published microsatellite data from two populations. Data from the northern hairy-nosed wombat show strong evidence of decline. Data from European humans show weak evidence of expansion.

Reproductive Strategies of Female Northern Hairy-Nosed Wombats

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Sarcoptic mange in wombats

Sarcoptic mange, commonly referred to as scabies, is a cosmopolitan disease affecting seven different orders within the class Mammalia. It is caused by the mite Sarcoptes scabiei (Linnaeus, 1758) Latreille, 1802 which is an obligate parasite of the skin. The mite is typically oval shaped, dorsally rounded and ventrally flattened. Its length varies between 200 and 500 μm depending on its life-cycle stage. Of the Australian marsupials, only wombats and the koala (Phascolarctos cinereus) (Vombatiformes) and the common ringtail possum (Pseudocheirus peregrinus) (Pseudocheiridae) have been reported with sarcoptic mange. In wombats, sarcoptic mange has been reported as the major infectious disease affecting the group, although it mainly occurs in only one of the three species, the common wombat (Vombatus ursinus). In this species sarcoptic mange can be debilitating. Several reports of severe sarcoptic mange in common wombats describe animals as emaciated and lacking hair with a thick dry crust, composed of keratin, many mites and their debris, bacteria and neutrophilic debris adherent to the skin (Figure 1). This crust is similar to that found in domestic animals with hyperkeratotic sarcoptic mange and humans with Norwegian scabies. The thick crust may be fissured by the movement of the wombat (Figure 2). The underlying skin may also crack resulting in haemorrhage, pyoderma and sometimes cutaneous myiasis. This may encompass the entire body with the head, neck, shoulders and limbs more commonly affected. Typically, the epidermis is thickened and there is a mild predominantly mononuclear inflammatory infiltrate in the dermis. In some animals hypersensitivity reactions occur towards Sarcoptes, with few mites present in the skin. Intense pruritus, a characteristic sign of hypersensitivity reactions to Sarcoptes in humans and animals has been reported in wombats with sarcoptic mange. Only occasional outbreaks of sarcoptic mange have been recorded in the southern hairy-nosed wombat (Lasiorhinus latifrons) and the disease has not been reported in the northern hairy-nosed wombat (Lasiorhinus krefftii).

Current distribution of sarcoptic mange in wombats.

To determine the distribution and prevalence of sarcoptic mange in wombats, particularly the common wombat (Vombatus ursinus). Questionnaire survey in two parts. Questionnaires were distributed to biologists, rangers, animal carers and naturalists. Part 1 of the questionnaire aimed to determine the present distribution of sarcoptic mange in wombats (103 responses). Part 2 invited respondents to assess the prevalence of sarcoptic mange in wombats over a 3 month period (four responses). Information on wombats from 66 localities was received. Each locality represented an area of about 2500 km2. Mange was observed at 93% of localities surveyed and Sarcoptes scabiei was present in common wombats at 52% of localities. Sarcoptic mange was highly prevalent (22%) in two common wombat populations in Victoria. Anecdotal evidence suggested that mange epizootics are sporadic, cause significant morbidity and mortality and have a substantial effect on local abundance. The respondents did not report sarcoptic mange in either northern hairy-nosed wombats (Lasiorhinus krefftii) or southern hairy-nosed wombats (Lasiorhinus latifrons). Sarcoptic mange occurs in common wombat populations throughout the range of the common wombat including Tasmania and Flinders Island. While mange epizootics are sporadic, they have the potential to threaten the long-term survival of small, remnant populations.

Combination of zolazepam and tiletamine as a sedative and anaesthetic for wombats

To assess the suitability of the combination of zolazepam and tiletamine for routine use as chemical restraint, sedative and anaesthetic in wild wombats. Sixty common wombats, 25 southern hairy-nosed wombats and 40 northern hairy-nosed wombats. Wombats caught in cage-traps in the wild were given injections of the anaesthetic combination either intramuscularly or intraperitoneally. Anaesthesia was maintained for up to 4 h in some cases, and for 12 h in one case. All wombats were released after anaesthesia. This drug combination is effective and apparently safe for the sedation or light anaesthesia of wombats. We did not observe adverse reactions or deaths and the dose range used (4 to 15 mg/kg) demonstrates a wide safety margin. The use of this anaesthetic combination was effective in reducing the stress to wombats from capture and handling.

The short-term effect of radio-packages on a free-ranging large herbivore, the northern hairy-nosed wombat

The short-term effect of radio-packages on the activity of the northern hairy-nosed wombat, Lasiorhinus krefftii, was investigated using activity data-loggers. Adjustment to the radio-package occurred between 10 and 18 days following capture and fitting of the radio-package. Following the adjustment period, activity levels were some 84–97% less than that of the initial activity level after the attachment of the radio-package. We believe that the adjustment period needs to be considered in studies involving the attachment of radio- packages.

Relatedness structure detected by microsatellite analysis and attempted pedigree reconstruction in an endangered marsupial, the northern hairy-nosed wombat Lasiorhinus krefftii.

The northern hairy-nosed (NHN) wombat is perhaps Australia's most endangered mammal. Being fossorial and nocturnal as well as rare, NHN wombats are difficult to observe in the wild. Hence little is known of their social biology, such as their mating and dispersal systems. A hypothesis has been advanced that adult females of the species disperse post-breeding, leaving their young to inhabit the natal burrow. Female-biased dispersal is expected to result in higher relatedness amongst males in a burrow cluster than amongst females in a burrow cluster. The usefulness of a panel of microsatellite markers in estimating the relatedness structure, and in reconstructing pedigrees for, the sole known population of NHN wombats was assessed. Microsatellite genotypes at eight or nine loci were obtained from 58 of the 85 known individuals, and used to estimate pairwise individual relatedness using Queller & Goodnight's (1989) RELATEDNESS 4.2. Our analysis gave the unexpected result that both males and females were significantly more closely related to their same-sex burrow cluster mates than random, while opposite-sex animals sharing burrows were only slightly (nonsignificantly) more related than random. This raises the possibility of dispersal patterns which lead to association of same-sex relatives. The observed relatedness structure is not expected to make likely a high incidence of inbred matings, as close relatives of the opposite sex are not significantly associated in space. Parentage analysis was attempted using genetic exclusion and LOD likelihood ratios, but proved difficult because of low genetic variation, incomplete sampling of potential parents, and paucity of ecological data such as known mother/offspring pairs and ages of individuals.

Evaluation of Techniques for Indirect Measurement of Body Composition in a Free-ranging Large Herbivore, the Southern Hairy-nosed Wombat

Several indirect methods for measuring body composition in a large herbivore, the southern hairy-nosed wombat (Lasiorhinus latifrons), were evaluated. Body composition was determined by whole-body chemical analysis of 15 wild-caught wombats, and compared with several indices of body fat: total body water measured by isotope dilution, bioelectrical impedance analysis (BIA), body-mass index, and a body- condition score. Total body water and total body fat (by soxhlet analysis) were highly correlated (r2 = 0.97, intercept s.e. = 1.00). Total body water measured by desiccation was highly correlated with isotope dilution space (r2 = 0.97, intercept s.e. = 0.43 for deuterium; r2 = 0.95, intercept s.e. = 0.44 for H218O). Percentage body fat by soxhlet analysis was highly correlated with total body water measured as deuterium dilution space (r2 = 0.83, intercept s.e. = 2.46). Multiple linear regression models using BIA plethysmograph measurements (resistance and impedance) and total body mass, were successful in predicting body fat (r2 = 0.90, s.e. = 1.99) and total body water (r2 = 0.90, s.e. = 1.64). Isotope-dilution techniques are the most accurate means of indirectly measuring total body water and total body fat, but at considerable expense of time and money. BIA offers reduced accuracy but at less cost and may be useful for measuring changes in body composition in populations of herbivores. Body-condition indices and scores correlate poorly with body fat, suggesting that their application as a means to predict body fat is limited.

Informative enzyme/probe combinations for the multilocus DNA fingerprinting of marsupials.

DNA fingerprinting has become an invaluable tool in the study of population genetics, paternity success, and individual identification; however, the species specificity of some methods has made the wide-range screening of many different species very time-consuming. In this study we describe the development and application of reliable and informative DNA fingerprinting techniques in a range of marsupial species using three different restriction enzyme and two oligonucleotide probe combinations. Six species from four marsupial families, the koala (Phascolarctidae), tammar wallaby (Macropodidae), southern hairy-nosed wombat (Vombatidae), kowari, and dusky and brown marsupial mice (Dasyuridae) were examined. Restriction enzymes HinfI AluI and HaeIII were used in combination with the digoxygenin (DIG)-labelled oligonucleotide probes (CAC)5 and (GGAT)4. The combinations of HinfI/(GGAT)4, AluI/ (CAC)5 and AluI/(GGAT)4 were the most informative, providing highly resolved bands, low background, and the lowest band sharing between individuals. The genetic diversity evident within the different species showed a clear relationship between the level of band sharing and population size. The greatest levels of band sharing were found in the kowaris (80%), which were part of a long-term captive colony originating from a few founders, and the lowest levels of band sharing were found in the marsupial mice (30-35%) and tammar wallaby (45%), which were caught from large outbred wild populations.

Adiaspiromycosis in bullfrogs (Rana catesbeiana).

Rare pulmonary infections associated with adiaspiromycosis have been reported in squirrels, armadillos, mice, rats, muskrats, beavers, rabbits, mink, martins, skunks, weasels, fox, Japanese pika, Australian hairy-nosed wombats, raccoons, goats, dogs, and humans. Pulmonary adiaspiromycosis is associated with inhaled conidia of soil fungi Chrysosporium (Emmonsia) parvum or Chrysosporium parvum var. crescens, with resultant pulmonary granulomas. In this report, we describes adiaspores of Chrysoporium sp. found in skeletal muscles of bullfrogs (Rana catesbeiana). Bullfrogs were obtained from a small farm pond, and rear legs were skinned in preparation for cooking. Discoloration of leg muscles was observed, and affected tissues were presented to the Clemson University Animal Diagnostic Laboratory for examination. Muscles of 6 rear legs contained randomly scattered flattened dark red to blue irregular lesions up to 1 cm in diameter. Affected muscle was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin (HE), and examined by light microscopy. Multiple thick-walled oval organisms up to 240 μm in diameter were randomly scattered throughout the leg muscles (Fig. 1). Cell walls of the organisms stained palely eosinophilic with periodic acid-Schiff (PAS) reaction and dark black with Grocott’s methenamine silver (GMS). Cytoplasm stained

Glucose-6-phosphate dehydrogenase and oxidant sensitivity of erythrocytes of three species of wombats

The investigations presented in this paper were undertaken to examine the antioxidant defence systems in the erythrocytes of three extant species of wombats: the common wombat (CW) (Vombatus ursinus), the southern hairy-nosed wombat (SHW) (Lasiorhinus latifrons) and the northern hairy-nosed wombat (NHW) (Lasiorhinus krefftii). Some of the important observations were: (1) the activity of glucose-6-phosphate dehydrogenase was 5.14 ± 0.96 enzyme units/g haemoglobin (EU/gHb) in CW, 35.69 ± 1.02 EU in SHW and 30.71 ± 1.49 EU in NHW and the levels of glucose phosphate isomerase were 40.1 ± 3.39, 93.2 ±1.96 and 74.9 ± 4.9 EU/gHb, respectively and (2) the erythrocytes of CW were found to be more sensitive to oxidant stress as measured by the degree of lipid peroxidation, haemolysis, number of Heinz bodies, and the methaemoglobin formation in the intact cells and/or haemolysates treated with three different oxidising agents — acetylphenylhydrazine, sodium nitrite and hydrogen peroxide. The CW lives in a temperate environment and the SHW, and NHW live in arid to semi-arid environments. The consequences in terms of antioxidant defence requirements are discussed.

Comparative study of the antioxidant defence systems in the erythrocytes of australian marsupials and monotremes

A comparison of the erythrocyte (RBC) antioxidant metabolites and enzymes in nine marsupial and two monotreme species was carried out. Reduced glutathione (GSH) concentrations were comparable with those reported for other marsupial and eutherian species. An important finding was that the erythrocytes of the southern hairy nosed wombat regenerated GSH faster than the erythrocytes from its close relative, the common wombat. The activities of glutathione- S-transferase, NADH-methaemoglobin reductase, Superoxide dismutase, and glutathione peroxidase (GSH-Px), showed similar levels and extents of variation as those observed in other marsupial and eutherian species. Catalase activities in the marsupials were lower than those measured in the two monotreme species and much lower than those reported in eutherian species. A negative correlation, significant at P < 0.05, was observed between GSH-Px and catalase activities in the RBC of the marsupials. Since both these enzymes “detoxify” H 2O 2, there appears to be a reciprocal relationship between the activities of these enzymes in marsupial RBC

Comparative erythrocyte metabolism in marsupials and monotremes

Concentrations of ATP and DPG, activities of 10 enzymes and the glycolytic rates were measured in the erythrocytes of 11 species of marsupials and two species of monotremes. Mean DPG concentrations were greater in the erythrocytes of marsupials than those of eutherian mammals. The opposite is true of ATP. Significant findings from the results of enzyme activities were: high activity of hexokinase (7.39 ± 0.82 EU/g Hb) in the short-beaked echidna, pyruvate kinase (37.49 ± 1.0 EU/g) Hb in bridled nailtail wallaby and glucose-6-phosphate dehydrogenase (G6PD; 41.66 ± 1.24 EU/g Hb) in black-striped wallaby. About 6- to 7-fold difference in the activity of G6PD levels between the two species of wombats was confirmed. Glucose phosphate isomerase activity was also shown to be twice as high in the red cells of the common wombat compared with those of the southern hairy nosed wombat. There were wide variations in the glycolytic rate among the species examined.

The Kidney Structure of the Common Wombat (Vombatus-Ursinus) and the Hairy-Nosed Wombat (Lasiorhinus-Latifrons)

The common wombat (Vombatus ursinus) lives in a mesic habitat whereas the hairy-nosed wombat (Lasiorhinus latifrons) inhabits a xeric habitat. The kidney structure of these two wombats was studied and it was found that kidney size, kidney weight, and kidney weight as a percentage of body weight, were larger in V. ursinus. However, relative medullary thickness, percentage medullary volume, and medullary thickness as a percentage of total thickness, were significantly greater in L. latifrons, suggesting that L. latifrons has a greater capacity to conserve water than does V. ursinus. The number of glomeruli and mean glomerular area were the same in both species whereas the mean diameter of the proximal and distal tubules was larger in V. ursinus. There were more proximal tubules per square millimetre in L. latifrons. The larger number of collecting ducts per square millimetre in the medullary region in L. latifrons indicates that the nephrons join the collecting ducts deep in the medulla, a feature that has been associated with increased urinary concentrating ability. Urinary pH was significantly lower, and urinary ammonia, creatinine and sodium levels were higher, in L. latifrons, as were plasma sodium and chloride levels. These data suggest that L. latifrons has a greater capacity to conserve water.